PRECLINICAL)DETECTION)OF)HYPOADRENOCORTICISM)IN)DOGS) ) ) # # # # # # # # # By# Markus#Rick# # # # # # # # # # # # # # # # # # # # A#DISSERTATION# Submitted#to# Michigan#State#University# in#partial#fulfillment#of#the#requirements# for#the#degree#of# DOCTOR#OF#PHILOSOPHY# Pathology# # # 2011 ABSTRACT# PRECLINICAL#DETECTION#OF#HYPOADRENOCORTICISM#IN#DOGS By# Markus#Rick# # # Approximately#70P90%#of#hypoadrenocorticism#cases#in#human#medicine#result#from# immunePmediated#adrenalitis.#Although#clinical#signs#are#not#present#until#~90%#of#the#adrenal# cortex#is#destroyed,#several#different#antiPadrenal#autoantibodies#can#be#identified#in#serum.#In# human#medicine,#presence#of#these#antibodies#constitutes#the#primary#criteria#for#an#early# diagnosis#of#adrenalitis.#Such#a#diagnostic#tool#is#not#currently#available#in#veterinary#medicine.# However,#from#preliminary#studies,#we#hypothesized#that#as#in#human#hypoadrenocorticism,# anti#21Phydroxylase#antibody#production#occurs#in#canine#hypoadrenocorticism.#The#antibody# production#precedes#clinical#disease,#and#is#more#prevalent#in#dog#breeds#susceptible#to# developing#hypoadrenocorticism.#The#specific#aims#of#the#proposed#research#are#to:#1)# determine#whether#antiPadrenal#autoantibodies#are#present#in#dogs#with#hypoadrenocorticism,# 2)#establish#a#diagnostic#test#to#detect#canine#antiPadrenal#autoantibodies,#and#3)#determine# whether#development#of#antiPadrenal#autoantibodies#has#breed,#sex,#and#age#based# predispositions.## The#canine#21Phydroxylase#was#expressed#in#E.#coli,#using#standard#techniques.#The# protein#was#purified#and#two#rabbits#and#two#dogs#were#immunized#with#the#purified#protein.# The#obtained#positive#control#sera#from#these#animals#were#used#to#establish#an#enzymePlinked# immunosorbent#assay#(ELISA)#for#the#detection#of#21Phydroxylase#autoantibodies#in#dogs.#The# preliminary#data#obtained#with#this#ELISA#showed#that#approximately#30#%#of#dogs#with# naturally#occurring#primary#Addison’s#disease#produce#antibodies#against#21Phydroxylase.#What# remains#unknown#is#whether#autoantibody#production#precedes#clinical#disease,#implying#a#role# in#the#pathogenesis#of#the#disease;#if#it#is#then#the#presence#of#21Phydroxylase#autoantibodies# would#be#expected#to#be#more#prevalent#in#dog#breeds#susceptible#to#developing# hypoadrenocorticism.#Further#development#of#the#ELISA#will#enable#epidemiologic#studies#to# address#specific#aim#three.## # # Copyright) by) MARKUS)RICK) 2011 DEDICATION ) ) ) ) ) ) To)my)parents,)Erhard)and)Brigitte)Rick, for#their#support#throughout#this#lengthy#study.# # To)Dr.)Tina)Andrea)Mueller,) for#her#dedication#to#help#me#succeed.# # # # “Everything#should#be#made#as#simple#as#possible,#but#not#simpler.”# Albert#Einstein#(1879P1955)# # v# # ACKNOWLEDGEMENTS# # Special#thanks#to#the#distinguished#faculty#members#who#served#on#my#committee:### Drs.#Kurt#J.#Williams#(Advisor),#Katheryn#Meek#(CoPAdvisor),#Steve#R.#Bolin,#John#C.#Fyfe,# Raymond#F.#Nachreiner,#and#Kent#R.#Refsal.#Thanks#to#all#my#committee#members#for#their# support,#patience,#encouragement,#and#useful#suggestions.# I#am#especially#grateful#for#samples#from#Portuguese#Water#Dogs,#provided#by#Dr.# Gordon#Lark,#Utah,#samples#from#Great#Pyrenees,#provided#with#the#help#of#Brigitte#Doxtator,# Michigan,#and#samples#from#Nova#Scotia#Duck#Tolling#Retrievers,#provided#by#Dr.#Sherry#Seibel,# North#Carolina.## My#thanks#go#to#the#American#Kennel#Club,#Canine#Health#Foundation#(grant###2273),# North#Carolina,#Oxford#Biomedical#Research,#Michigan,#and#Dr.#Raymond#F.#Nachreiner,# Michigan,#for#financial#support.## Thanks#to#personnel#at#the#Diagnostic#Center#for#Population#and#Animal#Health,#the# College#of#Veterinary#Medicine,#and#others#at#Michigan#State#University,#Michigan,#with#special# thanks#to#Drs.#Laura#Nelson,#Fernando#Garcia,#George#Bohart,#Steve#Mehler,#Robert#Hausinger,# Joe#Leykham,#and#Simon#Petersen#Jones;#and#Dr.#Katheryn#Meek’s#laboratory,#the# Endocrinology#Laboratory,#staff#at#the#Vivarium,#the#RATTS#group,#Kelli#S.#Cicinelli,#Patricia#A.# Schultz,#Thomas#J.#Wood,#and#Rose#Wahl.## vi# I#would#like#to#thank#my#German#family,#the#Ricks,#as#well#as#my#American#‘families’,#the# Dombroskis,#Griffins,#Maxons,#and#Pattersons,#who#have#given#me#so#much#support#and#love.## Lastly,#I#would#like#to#thank#all#my#friends#who#have#supported#me#on#this#endeavor,# especially#Rebecca#A.#Tremble,#with#Evelyn#O.,#and#Roman#A.#for#letting#me#stay#with#them# during#the#last#six#months,#Emily#Lawler#for#editing#parts#of#this#dissertation,#and#Dr.#Tina#A.# Mueller#for#her#amazing#support.## # # vii# TABLE#OF#CONTENTS# List#of#Tables## ########### xi# List#of#Figures### xiii# List#of#Abbreviations# xix# Introduction# # 1# References# # 6# Chapter#I## Literature#review# # Physiology#of#the#adrenal#glands#and#their#hormones# # Addison’s#disease#in#human#medicine# ## Introduction# ## Pathophysiology# ## Symptoms#of#adrenal#insufficiency##### ## Diagnosis#of#adrenal#insufficiency### ## Treatment#of#adrenal#insufficiency### ## LongPterm#outcome#of#adrenal#insufficiency## # Addison’s#disease#in#dogs## ## Introduction## ## Pathophysiology### ## Symptoms#of#adrenal#insufficiency## ## Diagnosis#of#adrenal#insufficiency## ## Treatment#of#adrenal#insufficiency## ## LongPterm#outcome#of#adrenal#insufficiency## # Addison’s#disease#in#human#and#veterinary#medicine## # Appendix## # References## 11 11 15# 15# 21# 24# 26# 28# 31 32# 32# 37# 39# 40# 48# 55 55 57 71# Chapter#II## Pathogenesis#of#autoimmune#diseases## # Introduction## # Immunologic#tolerance### # Genetic#susceptibility## # Environmental#triggers,#internal#triggers#and#changes#in#pathologic## # # processes#during#autoimmunePdisease#progression### 85# 85 87 91 # 93 viii# # # # # # Autoimmune#mechanisms#of#tissue#injury## Th17#cells## Conclusions## Appendix## References### 98 99 100 102 106# Chapter#III## # # # # # # # # # # # # Preliminary#research#reading#to#my#hypothesis## # Retrospective#study:#histology#and#histopathology#of#normal#canine## # # adrenal#glands#and#adrenal#glands#from#Addison’s#disease#dogs### # Detection#of#antiPadrenal#autoantibodies#using#Western#blot#analysis## # Detection#of#21Phydroxylase#antibodies#with#a#commercially#available# # # radioimmunoassay#for#the#detection#of#human#21Phydroxylase# # # antibodies#### # Detection#of#21Phydroxylase#antibodies#with#radioactively#labeled## # # amino#acid#capture#probe## # Detection#of#adrenal#autoantibodies#with#indirect## # # immunofluorescence#technique## # Appendix## # References## 115# # 115# 116# # # 117# # 118# # 119# 122# 129# Chapter#IV## # # Hypothesis#and#specific#aims# # Hypothesis## # Specific#aims## 131# 131# 131# # Chapter#V## # # # # # # The#expression#of#canine#21Phydroxylase## # Introduction## # Materials#and#methods## # Results## # Discussion## # Appendix## # References## 132# 132# 133# 138# 139# 143# 150# Chapter#VI## # # # # # # # # Purification#of#fusion#proteins#21PhydroxylasePglutathione#SPtransferase## # (21PhydroxylasePGST)#and#21PhydroxylaseP6xhistidine## # (21PhydroxylasePHis)## # # Introduction# # # Materials#and#methods## # # Results## # # Discussion## # # Appendix## # # References## # # 153# 153# 154# 165# 169# 174# 195# ix# Chapter#VII## # # # # # # Creation#of#positive#control#sera#for#assay#development## # # Introduction## # # Materials#and#methods## # # Results## # # Discussion## # # Appendix## # # References## 198# 198# 199# 204# 207# 210# 217# Chapter#VIII## # # # # # # # Evaluation#of#immunoreactivity#of#canine#and#rabbit#sera#with#the## # purified#21PhydroxylasePHis## # # Introduction## # # Materials#and#methods## # # Results## # # Discussion## # # Appendix## # # References## # 220# 220# 220# 224# 225# 228# 235# Chapter#IX:## Discussion#and#future#research## 238# ## # # # x# LIST#OF#TABLES# # Table#I.1:## Classification#of#autoimmune#polyendocrine#syndrome#(APS)#in#humans## (adapted#from#Betterle#Endocr#Dev#2011;20:161P72).## Table#I.2:## Breeds#that#are#found#to#be#at#increased#risk#of#developing## # hypoadrenocorticism#(CI=confidence#interval,#N/A=data#not#available).#### 63# Table#I.3:## # Breeds#that#are#found#to#be#at#decreased#risk#to#develop## # # hypoadrenocorticism#(CI=confidence#interval;#N/A=data#not#available).#### 65# Table#I.4:## Mean#age#and#body#weight,#female#to#male#ratio#in#dogs#with# hypoadrenocorticism#(female#intact#(FI),#male#intact#(MI),#female#spayed## (FS),#male#neutered#(MN),#SD=standard#deviation,#SEM=standard#error## of#the#mean,#N/A=data#not#available).#### 67# Table#I.5:## Hypoadrenocorticism#and#age#(CI=confidence#interval).### 68# Table#I.6:## Hypoadrenocorticism#incidence#and#gender.### 69# Table#I.7:## Odds#ratios#for#hypoadrenocorticism#and#gender.## 70# Table#III.1:## # Results#with#a#commercially#available#radioimmunoassay#for#the#detection## # # of#21Phydroxylase#autoantibodies#in#human#serum.## 127# Table#III.2:## Results#with#radioactively#labeled#amino#acid#capture#probe.## 127# Table#V.1:## Overview#of#the#final#expression#systems.## 145# Table#VII.1:# Immunization#timeline.## 211# Table#VII.2:## ACTH#stimulation#test,#cortisol#concentrations.# 213# xi# 61# Table#VII.3:## ACTH#stimulation#test,#aldosterone#concentrations.## 214# Table#VIII.1:## Comparison#of#the#21PhydroxylasePHis#concentrations#in#the#four## # # # different#preparations,#clearly#showing#that#the#highest#concentration## # # # was#found#in#TBS,#containing#4#M#guanidine,#and#250#mM#imidazole.## 229# Table#VIII.2:## Information#on#the#Addisonian#dogs#that#were#tested#using#the#newly## # # # developed#ELISA.#A#German#Shepherd#Mix,#a#Rottweiler,#and#a#Jack## # # # Russell#tested#positive#for#the#presence#of#21Phydroxylase#antibodies.### 234# # # # xii# LIST#OF#FIGURES# # Figure#I.1:### Schematic#showing#the#cellular#zonation#of#the#adrenal#cortex#and#blood#flow# through#the#cortex#to#the#collecting#veins#in#the#medulla#(retrieved#on# 07/02/2011#from#http://www.hakeemsy.com/main/files/adrenal.jpg).#(For# interpretation#of#the#references#to#color#in#this#and#all#other#figures,#the# reader#is#referred#to#the#electronic#version#of#this#dissertation).## 58# Figure#I.2:## Steroidogenesis#(from:#Annual#Rev#Physiology#(2001)#63:193);#Cholesterol#is# either#obtained#from#the#diet#or#synthesized#from#acetate#by#a#CoA## reductase#enzyme.#The#adrenal#cortex#(also#ovaries#and#testes)#use# cholesterol#to#produce#a#range#of#steroid#hormones,#including## aldosterone,#cortisol#and#testosterone/estradiol.### 59# Figure#I.3:## # The#hypothalamicPpituitaryPadrenal#axis#(from:#Addison’s#Disease#in#the## # # # Dog,#Catherine#ScottPMoncrieff,#05/15/2011,#retrieved#from:# http://www.vetgrad.co.uk/show10MinuteTopUp.php?type=&Entity=# 10MinuteTopUps&Entity=10MinuteTopUps&ID=51#on#09/09/2011).## #####60# # Figure#I.4:## # Figure#II.1:## Main#features#of#autoimmune#Addison’s#disease#and#their#subtypes#in## # humans#(adapted#from#Betterle#Endocr#Dev#2011;20:161P72).### # 62# Heterogeneity#in#helper#T#cell#fates.#The#helper#T#cell#differentiation#process## is#initiated#by#signaling#from#dendritic#cell#to#T#cell#in#the#lymph#node,# resulting#in#division#and#differentiation.#The#mature#helper#T#cells#and## their#signature#transcription#factors#are#illustrated.#Cytokines#play#a## critical#role#in#the#induction#or#repression#of#the#lineages.#The#different## helper#T#cell#subsets#have#distinct#protective#and#pathological#roles.#Host# defense#is#orchestrated#by#the#three#major#fates,#Th1,#Th2,#and#Th17.# Adaptive#regulatory#T#(aTreg)#cells#can#downregulate#immune#responses,# although#a#physiological#role#in#vivo#is#yet#uncertain.#The#mature#helper## T#cell#progeny#must#eventually#exit#the#lymph#node#and#migrate#to## infected#tissue#to#exert#their#function#in#host#defense.#Some#of#the## mature#progeny#may,#instead,#migrate#to#B#cell#follicles#to#promote## antibody#subclasses#that#will#suit#the#particular#immune#response#(from:# Reiner,#S.#L.,#Development#in#Motion:#Helper#T#Cells#at#Work,#Cell#129,## April#6,#2007,#pages#33#–#36).# 103# xiii# Figure#II.2:## Th17#differentiation#in#mice#and#man#(from:#Gut#2009;58:1152P1167).#### Figure#II.3:## Requirements#for#the#development#of#an#autoimmune#disease.## # The#immune#response#of#a#genetically#predisposed#individual#to#an# environmental#pathogen,#in#association#with#defects#in## immunoregulatory#mechanisms,#can#lead#to#the#development#of#an# autoimmune#disease.#The#importance#of#the#single#components## represented#in#this#Venn#diagram#may#vary#between#individuals#and## diseases.#However,#the#appearance#of#an#autoimmune#disease#requires## the#convergence#of#all#three#components.#T,#T#cell;#B,#B#cell;## DC,#dendritic#cell#(from:#Focus#on#Autoimmunity,#Nature#Immunology,## Sept#2001#(Vol.#2)).#### 105# Figure#III.1:## Adrenal#gland#histology,#2.5x#magnification#hematoxylin#and#eosin#stain.### 123# Figure#III.2:## Adrenal#gland#histology,#20x#magnification,#hematoxylin#and#eosin#stain.## 124# Figure#III.3:## # Adrenal#gland#histology,#20x#magnification,#CD3#and#CD79a# # immunohistochemistry.# # 125# Figure#III.4:## Detection#of#anti#adrenal#antibodies#with#Western#blotting.## 126# Figure#III.5:## Detection#of#adrenal#autoantibodies#with#indirect#immunofluorescence# technique.## 128# Figure#V.1:## The#21PhydroxylasePGST#fusion#protein#is#primarily#found#in#the#insoluble# fraction.#(P):#uninduced;#(+):#induced#with#IPTG.## 144# Figure#V.2:## The#soluble#proteins#expressed#in#pET42b/#pGKJPE8,#clearly#showing#that## # hardly#any#21PhydroxylasePHis#is#in#this#fraction.#Different#conditions## # were#tested#(IPTG:#1#mM#IPTG;#AA:#1#mM#δPaminolevulinic#acid;## # Neg:#no#IPTG;#Tetra:#5#ng/#mL#Tetracycline;#Arab:#4#mg/#mL#of## # Arabinose),#which#enabled#me#to#identify#the#21Phydroxylase#and## # the#chaperone#proteins,#and#determine#their#location.# 146# Figure#V.3:## Inclusion#body#proteins#expressed#in#pET42b/#pGKJPE8,#showing#that#the## # 21PhydroxylasePHis#is#primarily#in#this#fraction.#Different#conditions## # were#tested#(IPTG:#1#mM#IPTG;#AA:#1#mM#δPaminolevulinic#acid;## xiv# 104# # # # Neg:#no#IPTG;#Tetra:#5#ng/#mL#Tetracycline;#Arab:#4#mg/#mL#of## Arabinose),#which#enabled#me#to#identify#the#21Phydroxylase## and#the#chaperone#proteins,#and#determine#their#location.# 147# Figure#V.4:## # # Fractionation#of#pET42b/#pGKJPE8,#demonstrating#that#the## # 21PhydroxylasePHis#fusion#protein#is#expressed#into#inclusion## # bodies#(induced#with#IPTG#to#the#left,#without#IPTG#to#the#right).### # # 148# Figure#V.5:## # # Fractionation#of#pGEXP5XP3/#pGKJPE8,#demonstrating#that#the## # 21PhydroxylasePGST#fusion#protein#is#expressed#into#inclusion#bodies## # (induced#with#IPTG#to#the#left,#without#IPTG#to#the#right).# # # 149# Figure#VI.1:## BioRad’s#Protean#II#XL#system## 175# Figure#VI.2:## Electrophoretic#elution#tank.#(a)#Top#view;#(b)#side#view;#(c)#end#view;## (d)#port#connector#for#pump#tubing.#A,#terminal#lug;#B,#platinum#wire# electrode;#C,#baffle#plate;#D,#separation#plate#for#electrode#chambers;## E,#slot#for#elution#cell;#F,#drain#trough;#G,#mirrored#surface;#H,## to#pump#tubing;#I,#to#tank#chamber;#J,#set#screw.#Plexiglas#covers#for## tank#chambers#are#not#shown.#(from:#Methods#in#Enzymology,## Volume#91,#Enzyme#Structure#Part#1,#1983,#page#229).### 176# Figure#VI.3:## Electrophoretic#elution#cell.#A,#gel#loading#well;#B,#sample#collection#well;## C,#Spectra/Por#disk;#D,#silicon#rubber#washer;#E.#screw#cap#with#open#top;# # F,#peg#for#holding#slot#in#elution#tank;#G,#cross#passage.#(from:#Methods## in#Enzymology,#Volume#91,#Enzyme#Structure#Part#1,#1983,#page#229).## 177# Figure#VI.4:## SDSPPAGE,#showing#the#21PhydroxylasePGST#prior#to#gelPpurification#and## # # small#scale#passive#elution.### # 178# Figure#VI.5:## SDSPPAGE,#showing#the#purified#21PhydroxylasePHis#and## # # 21PhydroxylasePGST#post#gel#purification#and#passive#elution#out#of## # # the#gelPmatrix.### # # 179# Figure#VI.6:## SDSPPAGE#maxi#gel,#after#the#21PhydroxylasePGST#was#excised.# Using#Coomassie#stain,#it#was#possible#to#accurately#identify#the#band## of#interest.### 180# xv# Figure#VI.7:## SDSPPAGE,#showing#the#gelPpurified#and#electroPeluted#21PhydroxylasePHis## in#comparison#to#BSA.#The#known#concentrations#of#BSA#were#used#to# estimate#the#concentration#of#the#electroPeluted#21PhydroxylasePHis.## 181# Figure#VI.8:## SDSPPAGE,#showing#the#gelPpurified#and#electroPeluted#21PhydroxylasePGST## in#comparison#to#BSA.#The#known#concentrations#of#BSA#were#used#to# estimate#the#concentration#of#the#electroPeluted#21PhydroxylasePGST.## 182# Figure#VI.9:## # # # WB#of#the#electroPeluted#21PhydroxylasePHis,#showing#that#the#purified## # # protein#reacts#strongly#with#an#antibody#against#His.## # # (From#left#to#right:#lane#1:#marker,#lanes#2#through#5:#21PhydroxylasePHis## # # from#different#electroPelution#runs).### 183# Figure#VI.10:# # # # #WB#of#the#electroPeluted#21PhydroxylasePGST,#showing#that#the#purified## # # protein#reacts#strongly#with#an#antibody#against#GST.#(From#left#to#right:## # # lane#1:#Marker,#lanes#2#through#4:#21PhydroxylasePGST#from#different## # # electroPelution#runs).## 184# Figure#VI.11:## MALDI#spectra#of#the#21PhydroxylasePGST.#The#peaks#are#similar#to#the## # # ones#of#the#theoretical#trypsin#digest#of#the#21PhydroxylasePGST.### # 185# Figure#VI.12:## SDSPPAGE,#showing#the#gelPpurified#and#degraded#electroPeluted## # # 21PhydroxylasePGST,#after#increasing#the#electroPpotential#during## # # electroPelution#from#50#V#to#75#V.## # # 186# Figure#VI.13:## # # # Solubilization#of#21PhydroxylasePHis#inclusion#bodies#with#TBS#containing## # # 8#M#guanidine,#and#5#mM#DTT#over#24#hours.#Some#of#the## # # 21PhydroxylasePHis#is#found#in#the#supernatant.## # # (SN:#supernatant;#P:#pellet).## 187# Figure#VI.14:## Solubilization#of#21PhydroxylasePHis#inclusion#bodies#with#TBS#containing## # # # 8#M#urea,#5#mM#DTT,#and#5%#Triton#XP100#over#24#hours.#The## # # # 21PhydroxylasePHis#remains#in#the#pellet.#(SN:#supernatant;#P#=#pellet).## 188# Figure#VI.15:## Solubilization#of#21PhydroxylasePHis#inclusion#bodies#with#TBS#containing## 8#M#guanidine#versus#TBS#containing#8#M#guanidine,#and#4#mM#DTT## after#2#h#20#min.#The#yield#of#21PhydroxylasePHis#in#the#supernatant#is# increased#with#DTT.#(SN:#supernatant;#P:#pellet).## 189# xvi# Figure#VI.16:## Pulldown#of#solubilized#21PhydroxylasePHis#in#TBS#containing#8#M## guanidine#with#HisPur#cobalt#resin,#without#the#addition#of#additional## # DTT.#The#21PhydroxylasePHis#was#solubilized#in#TBS,#containing#8#M## # guanidine#and#4#mM#DTT.#The#sample#was#diluted#1:2#with#TBS#prior#to# pulldown,#decreasing#the#final#DTT#concentration#to#2#mM.## (SN:#supernatant#(unbound#protein#post#incubation#with#HisPur#resin);## W:#wash#fractions;#E:#elution#fractions).### 190# Figure#VI.17:## FPLC#of#21PhydroxylasePHis,#loading#and#wash#fractions.#Contaminating## proteins#do#not#bind#to#the#HisPur#cobalt#resin#and#are#washed#off#during# washing.#Some#of#the#21PhydroxylasePHis#however#is#lost#during#loading## and#washing.#(SN,#induced:#induced#bacteria#culture#post#sonification## and#ultracentrifugation,#supernatant;#P,#uninduced:#uninduced#bacteria# culture#post#sonification#and#ultracentrifugation,#pellet#(negative#control);## P,#induced:#induced#bacteria#culture#post#sonification#and## ultracentrifugation,#pellet#(positive#control);#L:#loading#fraction,#number# corresponds#to#the#collected#fraction#number;#W:#wash#fraction,#number# corresponds#to#the#collected#fraction#number).### 191# Figure#VI.18:## FPLC#of#21PhydroxylasePHis,#wash#and#elution#fractions.#Some#of#the#21P hydroxylasePHis#was#lost#during#washing,#but#most#was#eluted#with#high# imidazole#concentration.#(W:#wash#fraction,#number#corresponds#to#the# collected#fraction#number;#E:#elution#fraction,#number#corresponds#to## the#collected#fraction#number).### 192# Figure#VI.19:## Localization#of#the#purified#21PhydroxylasePHis#post#dialysis#in#7#different# conditions.#Most#of#the#21PhydroxylasePhis#remains#in#the#pellet,#except## for#TBS#containing#4#M#guanidine,#TBS#containing#4#M#urea,#and#TBS# containing#2#%#SDS,#1#%#glycerol,#and#1#%#2Pmercaptoethanol.#‘Pool’#is## the#pool#of#fractions#that#were#used#for#the#dialysis.#(P:#pellet#post# centrifugation#of#the#dialysates).## 193# Figure#VI.20:## Localization#of#the#purified#21PhydroxylasePHis#post#dialysis#in#7#different# conditions.#Most#of#the#21PhydroxylasePhis#remains#in#the#pellet,#except## for#TBS#containing#4#M#guanidine,#TBS#containing#4#M#urea,#and#TBS# containing#2#%#SDS,#1#%#glycerol,#and#1#%#2Pmercaptoethanol,#in#which# # # the#majority#was#found#in#the#SN.#‘Pool’#is#the#pool#of#fractions#that#were## used#for#the#dialysis.#(SN:#supernatant#post#centrifugation#of## the#dialysates).### 194# Figure#VII.1:## Cortisol#concentrations#post#ACTH#[%#of#baseline].## xvii# 215# Figure#VII.2:## Cortisol#concentrations#post#ACTH.## 215# Figure#VII.3:## Aldosterone#concentrations#post#ACTH#[%#of#baseline].## 216# Figure#VII.4:## Aldosterone#concentrations#post#ACTH.## 216# Figure#VIII.1:## Western#blotting#of#the#rabbit#sera,#clearly#showing#immunoreactivity## # # with#the#expressed#21POHPhydroxylase#after#immunization,#but#not## # # before.## # # 230# Figure#VIII.2:## # # # # Western#blotting#of#the#dog#sera,#clearly#showing#immunoreactivity## # between#the#expressed#21PhydroxylasePHis#and#21PhydroxylasePGST# # and#the#two#study#dogs#post#immunization,#but#not#before.#Lucy’s## # serum#does#not#show#immunoreactivity.### # # # # # # ###231# Figure#VIII.3:## Comparison#of#the#three#21PhydroxylasePHis#preparations#in#ELISA,#using## # sera#from#the#study#dogs,#two#healthy#dogs,#and#buffer#alone.#All## # three#preparations#gave#similar#results.#The#optical#density#is## # significantly#increased#in#the#two#immunized#dogs#postPimmunization## # (2#to#more#than#3#fold),#demonstrating#immunoreactivity#between#the# purified#21PhydroxylasePHis#and#the#sera#from#these#animals.### 232# Figure#VIII.4:## Results#of#the#second#ELISA#experiment,#testing#canine#serum#from#healthy## dogs,#dogs#diagnosed#with#Addison’s#disease,#and#from#our#two#positive# control#study#and#dogs.#(1:#Blanket#pre#immunization;#2:#Blanket#post# immunization;#3:#Lola#pre#immunization;#4:#Lola#post#immunization;## 5#to#14:#healthy#dogs;#15#to#26:#dogs#diagnosed#with## hypoadrenocorticism#(see#table#VIII.2#for#details)).#Three#out#of#the## 12#dogs#that#have#been#diagnosed#with#hypoadrenocorticism#show#a## high#optical#density,#as#well#as#Blanket#and#Lola#post#immunization.### 233# # # xviii# LIST#OF#ABBREVIATIONS# ACN# Acetonitrile## ACTH## Adrenocorticotropic#hormone,#corticotropin,#adrenocorticotrophic#hormone# AHDL## Animal#Health#Diagnostic#Laboratory# AIDS# Acquired#immune#deficiency#syndrome# AIRE## Autoimmune#suppressor#or#autoimmune#regulator#gene## APS# Autoimmune#polyendocrine#syndrome## BCA# Bicinchoninic#acid# BV# BedPvolume# CAPS# 3P[cyclohexylamino]P1#propane#sulfonic#acid# CD4## Cluster#of#differentiation#4# CHAPS## (3P[(3PCholamidopropyl)#dimethylammonio]P1Ppropanesulfonate#)# CI## Confidence#interval# CO2## Carbon#dioxide# cpm# Counts#per#minutes# CRH## Corticotropin#releasing#hormone# DCPAH# Diagnostic#Center#for#Population#and#Animal#Health## DMSO# Dimethyl#sulfoxide# DOCA## Desoxycorticosterone#acetate# DOCP# Desoxycorticosterone#pivalate## DTT## Dithiothreitol# xix# EB# Equilibration#buffer# ECG# Electrocardiogram## EDTA## Ethylenediaminetetraacetic#acid# EIA# Enzyme#immunoassay# ELB# Elution#buffer# ELISA## EnzymePlinked#immunosorbent#assay# Foxp3# Forkhead#box#P3# FPLC# FastPprotein#liquid#chromatography## FR# Flowrate## GI## GastroPintestinal# GST## Glutathione#SPtransferase# H&E# Hematoxylin#and#eosin#stain# His## Hexa#histidinePtag# HIV## Human#immunodeficiency#virus# HLA## Human#leukocyte#antigen# HR# High#risk#dog#group## HRP# Horseradish#peroxidase# IFNPγ## InterferonPgamma# IgG## Immunoglobulin#gamma# IgGFc## Immunoglobulin#gamma#Fc#region# IHC# Immunohistochemistry# iIFA# Indirect#immunofluorescence#technique# xx# ILP2## InterleukinP2# IPEX## Immunodysregulation#polyendocrinopathy#enteropathy#XPlinked#syndrome# IPTG## Isopropyl#βPDP1Pthiogalactopyranoside# LB# Lysogeny#broth# LR# Low#risk#god#group/#negative#(healthy)#control#samples## MALDI# MatrixPassisted#laser#desorption/#ionization# MES### 2P(N#Pmorpholine)Pethanesulfonic#acid# MHC## Major#histocompatibility#complex# MICA## MHC#class#I#chain#related#A# MSU## Michigan#State#University# NaCl## Sodium#chloride# NADPH# Nicotinamide#adenine#dinucleotide#phosphate# NSB# NonPspecific#binding# PBS## Phosphate#buffered#saline# PBSPT## Phosphate#buffered#saline,#containing#1#%#Tween# PCR## Polymerase#chain#reaction# PDS## Polydioxanone# PMSF# Phenylmethylsulfonyl#fluoride# PVDF# Polyvinylidene#fluoride# RT# Room#temperature# SD# Sick#dog#group## SDSPPAGE## Sodium#dodecyl#sulfate#polyacrylamide#gel#electrophoresis# xxi# SN## Supernatant# TBS## Tris#buffered#saline# TCR# T#cell#receptor# TFA# Trifluoroacetic#acid# TgAA# Thyroglobulin#autoantibodies# TGFPβ## Transforming#growth#factor#beta# Th17## T#helper#17# TMB## 3,3u,5,5uPtetramethylbenzidine# TNFPα## Tumor#necrosis#factor#alpha# TSH## Thyroid#stimulating#hormone# WB# Wash#buffer# WB# Western#blot## xxii# INTRODUCTION# # The#clinical#characteristics#of#primary#hypoadrenocorticism#in#the#dog#have#been#well# 1P6 documented#in#the#veterinary#literature .#However,#clinical#signs#in#a#dog#are#nonspecific#and# 7 can#mimic#those#of#other#conditions .#The#clinical#signs#are#caused#by#deficiencies#of# glucocorticoids#and#mineralocorticoids#and#are#very#similar#in#dogs#and#humans.#In#detail,#the# lack#of#glucocorticoids#causes#anorexia,#vomiting,#weakness,#abdominal#pain,#diarrhea,#lethargy,# fasting#hypoglycemia#from#impaired#energy#metabolism,#and#makes#it#difficult#for#an#individual# to#handle#stress.#Mineralocorticoid#insufficiency#on#the#other#hand#might#not#cause#clinical# signs,#if#the#electrolyte#balance#is#not#disturbed.#However,#if#hyperkalemia#and#hyponatremia# occur,#above#mentioned#clinical#signs#worsen#and#cardiac#abnormalities#occur#due#to# hyperkalemia.#Hyponatremia#causes#hypovolemia,#hypotension,#and#reduces#cardiac#output.# Hypoperfusion#exacerbates#hyperkalemia#and#leads#to#many#other#clinical#features#including# elevation#of#liver#and#kidney#enzymes,#azotemia,#low#urine#specific#gravity,#shaking,#trembling# and#muscle#weakness.# More#than#onePthird#of#dogs#that#are#affected#with#Addison’s#disease#are#presented#in#a# lifePthreatening#condition#because#the#disease#goes#unrecognized#until#there#is#an#acute# adrenal#crisis 8,9 .#Many#dogs#die#during#such#an#acute#crisis.#If#treatment#is#possible,#emergency# 8 treatment#in#these#situations#is#intensive,#expensive#and#timePconsuming .#An#inexpensive# screening#tool#for#preclinical#adrenalitis#used#in#predisposed#dogs#and#in#animals#displaying# 1# vague#clinical#signs,#therefore,#has#the#potential#to#be#both#lifeP#and#moneyP#saving,#especially# since#maintenance#therapy#in#a#dog#with#hypoadrenocorticism#with#an#early#diagnosis#is# 8 relatively#straight#forward .##In#human#medicine,#the#importance#of#such#a#diagnostic#tool#is# already#recognized#because#clinical#signs#and#changes#in#laboratory#values#are#not#observed# 10 before#about#90#%#of#the#adrenal#cortex#is#destroyed .#We#suspect#the#same#to#be#true#in#dogs# and#expect#an#antiPadrenal#antibody#test#to#be#valuable#in#the#detection#of#early#immuneP mediated#adrenalitis#in#dogs.## So#far,#several#reports#describe#characteristic#bilateral#atrophy#of#the#adrenal#cortices# with#mononuclear#cell#infiltration#and#fibrosis#of#the#capsule#in#dogs#with# 11,12 hypoadrenocorticism .#AntiPadrenal#autoantibodies#have#been#isolated#from#two#dogs#with# naturally#occurring#hypoadrenocorticism#and#from#two#of#six#beagles#that#developed# 11,13 hypoadrenocorticism#after#inhalation#of#aerosols#of#plutoniumP238#dioxide .#In#a# retrospective#study,#24#adrenal#gland#tissue#slides#obtained#during#diagnostic#necropsies# performed#at#AHDL#(Animal#Health#Diagnostic#Laboratory)/#DCPAH#(Diagnostic#Center#for# Population#and#Animal#Health)#were#examined#(Rick,#Williams,#unpublished#data).#Normal# adrenal#glands#were#used#as#negative#controls.#The#severity#of#inflammation#was#subjectively# graded#as#mild#(1),#moderate#(2),#or#severe#(3)#by#two#independent#observers#depending#on#the# amount#of#inflammation#observed#(Rick#and#Williams).#Ten#cases#were#classified#as#grade#1,# eight#as#grade#2,#and#five#as#grade#3.#One#adrenal#gland#did#not#show#inflammation.#However,# in#this#case#the#adrenal#cortex#was#completely#collapsed#and#fibrotic.#The#number#of# lymphocytes#seemed#to#be#inversely#correlated#to#the#number#of#macrophages.#It#also# 2# appeared#that#sections#with#lesser#adrenal#cortical#atrophy#contained#higher#numbers#of# lymphocytes#and#lesser#macrophages.#Sections#with#more#cortical#atrophy#on#the#other#hand# contained#more#macrophages#and#fewer#lymphocytes.#None#of#the#12#control#adrenal#glands# showed#inflammation.#Immunohistochemistry#identified#both#T#(CD3)#(cluster#of# differentiation)#and#B#(CD79a)#cells#in#the#infiltrate,#although#the#majority#of#cells#were#T#cells.# Before#the#midP1950’s,#the#primary#causes#of#hypoadrenocorticism#in#human#beings# 14,15 were#tuberculosis#or#cases#were#classified#as#idiopathic .#Approximately#70P90%#of#recent# hypoadrenocorticism#cases#in#human#medicine#are#known#to#be#the#result#of#immunePmediated# adrenalitis 14P19 .#Evidence#supporting#such#a#pathogenesis#in#humans#includes#1)#lymphocytic# 20P22 infiltration#of#the#adrenal#cortex ,#2)#the#presence#of#circulating#autoantibodies#reactive#to# 18,23P26 the#adrenal#cortex#and#to#21Phydroxylase genes#in#the#HLAPDR#region 27,28 ,#3)#an#association#with#immunePresponse# ,#4)#the#frequent#occurrence#of#other#putative#autoimmune# 29P32 diseases#in#the#same#individual#or#in#family#members ,#5)#the#presence#of#IaPpositive#T# 33 lymphocytes#(“activated”#T#lymphocytes)#at#diagnosis ,#and#6)#other#cellPmediated# 34 abnormalities .#The#measurement#of#several#different#antiPadrenal#autoantibodies,#especially# the#measurement#of#21Phydroxylase#autoantibodies,#the#major#antigen#in#humanPimmuneP 18,23P26 mediated#hypoadrenocorticism ,#is#used#by#physicians#for#the#laboratory#diagnosis#of# adrenalitis#prior#to#the#onset#of#clinical#hypoadrenocorticism.## 3# Since#there#are#so#many#similarities#between#these#two#species#in#regards#to#Addison’s# disease,#we#therefore#hypothesized#that#the#same#is#true#in#dogs:#“As#in#human# hypoadrenocorticism,#antiP21Phydroxylase#antibody#production#occurs#in#naturally#occurring# primary#canine#hypoadrenocorticism.#The#antibody#production#precedes#clinical#disease,#and#is# more#prevalent#in#dog#breeds#susceptible#to#developing#hypoadrenocorticism.”## Hypoadrenocorticism#is#relatively#common#in#the#dog.#The#prevalence#has#been# 35 estimated#to#range#from#0.6#to#3.1#per#1000#dogs .#Affected#dogs#may#be#of#all#ages,#breeds# 8 8 and#sexes .#Recent#studies#indicate#a#higher#incidence#for#young#to#middlePaged#female#dogs .# Some#breeds#have#been#reported#to#have#a#10P50#%#higherPthanPaverage#risk#for# 36 hypoadrenocorticism .#Reported#breeds#are#Bearded#Collie,#West#Highland#White#Terrier,# Standard#Poodle,#Portuguese#Water#Dog,#Leonberger,#Great#Dane,#Airedale#Terrier,#Basset# Hound,#Wheaten#Terrier#and#Rottweiler.#Breeds#at#lower#risk#include#Boston#Terriers,#Lhasa# Apsos,#Yorkshire#Terriers#and#mixedPbreed#dogs 7,8,37P39 .#We#therefore#believe,#that#the# outcomes#of#the#proposed#study#will#improve#the#health#and#welfare#of#dogs#with#adrenal# disease#by#improving#the#understanding#of#its#pathogenesis#and#the#value#of#early#detection.# Hypoadrenocorticism#is#perceived#as#a#significant#problem,#especially#in#the#purebred#dog,#with# a#large#genetic#component#in#several#breeds,#but#progress#towards#understanding#and# elimination#of#the#disorder#has#been#hampered#by#lack#of#diagnostics#for#the#condition#before# onset#of#sometimes#catastrophic#signs.#The#techniques#arising#of#this#study#could#therefore#also# assist#in#breeding#programs#and#could#provide#a#strong#basis#for#future#molecular#genetic# research#of#the#pathogenesis#and#inheritance#of#canine#hypoadrenocorticism.# 4# In#this#dissertation,#the#preliminary#research#that#was#done#to#derive#this#hypothesis#is# described.#Then,#the#expression,#purification,#and#evaluation#of#recombinant#canine#21P hydroxylase#as#an#antigen#during#diagnostic#testing#are#explained#in#detail.#The#specific#aims#of# this#study#are#1)#to#establish#a#diagnostic#test#to#detect#canine#antiPadrenal#autoantibodies;#2)# to#determine#whether#antiPadrenal#autoantibodies#are#present#in#dogs#with# hypoadrenocorticism;#3)#to#determine#whether#development#of#antiPadrenal#autoantibodies# has#breed,#sex,#and#agePbased#predispositions.# # 5# REFERENCES 6# REFERENCES# # # 1.# Rakich#PM,#D.#LM.#Clinical#signs#and#laboratory#abnormalities#in#23#dogs#with# spontaneous#hypoadrenocorticism.#J#Am#Anim#Hosp#Assoc#1984;20:647P649.# # 2.# 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disease).#The#Compendium#for#Continuing#Education#1998;20:921P934.# # 9.# Melian#C,#Peterson#ME.#Diagnosis#and#treatment#of#naturally#occurring# hypoadrenocorticism#in#42#dogs.#J#Small#Anim#Pract#1996;37:268P275.# # 10.# Feldman#EC.#Adrenal#Gland#Disease.#Textbook#of#Veterinary#Internal#Medicine,#3# ed.#Philadelphia:#W.#B.#Saunders#Co.,#1989;3:1756P1774.# # 11.# Schaer#M.#Autoimmunity#and#Addison's#disease#in#the#dog.#J#Am#Anim#Hosp# Assoc#1985;22:789.# 7# # 12.# Boujon#CE.#Pituitary#gland#changes#in#canine#hypoadrenocorticism:#A#functional# and#immunocytochemical#study.#Journal#of#Comparative#Pathology#1994;111:287.# # 13.# Weller#RE,#Buschbom#RL,#Dagle#GE,#et#al.#Hypoadrenocorticism#in#Beagles# exposed#to#aerosols#of#plutoniumP238#dioxide#by#inhalation.#Radiat#Res#1996;146:688P693.# # 14.# Bondy#PK.#Disorders#of#the#adrenal#cortex.#Williams#Textbook#of#Endocrinology,#7# ed.#Philadelphia:#W.#B.#Saunders#Co.,#1985;7:1756P1774.# # 15.# Nelson#DH.#The#adrenal#cortex:#Physiological#function#and#diseases.#Major# Problems#in#Internal#Medicine,#XVIII#ed.#Philadelphia:#W.#B.#Saunders#Co.,#1980;XVIII:1756P1774.# # 16.# Laureti#S,#De#Bellis#A,#Muccitelli#VI,#et#al.#Levels#of#adrenocortical#autoantibodies# correlate#with#the#degree#of#adrenal#dysfunction#in#subjects#with#preclinical#Addison's#disease.#J# Clin#Endocrinol#Metab#1998;83:3507P3511.# # 17.# Betterle#C,#Pedini#B,#Presotto#F.#Serological#markers#in#Addison's#disease.# Advances#in#Thomas#Addison's#disease.#Bristol:#Journal#of#Endocrinology#Ltd,#1994;2:67P84.# # 18.# Nerup#J.#Addison's#diseasePPserological#studies.#Acta#Endocrinol#(Copenh)# 1974;76:142P158.# # 19.# Nerup#J.#Addison's#disease#P#a#review#of#some#clinical,#pathological#and# immunological#features.#Dan#Med#Bull#1974;21:201P217.# # 20.# Colover#J,#Glynn#LE.#Experimental#isoPimmune#adrenalitis.#Immunology# 1958;1:172P178.# # 21.# Steiner#JW,#Langer#B,#Schatz#DL,#et#al.#Experimental#immunologic#adrenal#injury:# A#response#to#injections#of#autologous#and#homologous#adrenal#antigens#in#adjuvant.#J#Exp#Med# 1960;112:187P202.# # 22.# Witebsky#E,#Milgrom#F.#Immunological#studies#on#adrenal#glands.#II.# Immunization#with#adrenals#of#the#same#species.#Immunology#1962;5:67P78.# 8# # 23.# Boe#AS,#Bredholt#G,#Knappskog#PM,#et#al.#Autoantibodies#against#21Phydroxylase# and#sidePchain#cleavage#enzyme#in#autoimmune#Addison's#disease#are#mainly#immunoglobulin# G1.#Eur#J#Endocrinol#2004;150:49P56.# # 24.# Husebye#ES,#Bratland#E,#Bredholt#G,#et#al.#The#substratePbinding#domain#of#21P hydroxylase,#the#main#autoantigen#in#autoimmune#Addison's#disease,#is#an#immunodominant#T# cell#epitope.#Endocrinology#2006;147:2411P2416.# # 25.# Barker#JM,#Ide#A,#Hostetler#C,#et#al.#Endocrine#and#immunogenetic#testing#in# individuals#with#type#1#diabetes#and#21Phydroxylase#autoantibodies:#Addison's#disease#in#a#highP risk#population.#J#Clin#Endocrinol#Metab#2005;90:128P134.# # 26.# Miguel#RN,#Chen#S,#Nikfarjam#L,#et#al.#Analysis#of#the#interaction#between#human# steroid#21Phydroxylase#and#various#monoclonal#antibodies#using#comparative#structural# modelling.#Eur#J#Endocrinol#2005;153:949P961.# # 27.# Volpe#R.#The#role#of#autoimmunity#in#hypoendocrine#and#hyperendocrine# function:#with#special#emphasis#on#autoimmune#thyroid#disease.#Ann#Intern#Med#1977;87:86P 99.# # 187.# 28.# Volpe#R.#AutoPimmunity#in#the#endocrine#system.#Monogr#Endocrinol#1981;20:1P # 29.# Spinner#MW,#Blizzard#RM,#Childs#B.#Clinical#and#genetic#heterogeneity#in# idiopathic#Addison's#disease#and#hypoparathyroidism.#J#Clin#Endocrinol#Metab#1968;28:795P804.# # 30.# Nigam#R,#Bhatia#E,#Miao#D,#et#al.#Prevalence#of#adrenal#antibodies#in#Addison's# disease#among#north#Indian#Caucasians.#Clin#Endocrinol#(Oxf)#2003;59:593P598.# # 31.# Sekiguchi#Y,#Hara#Y,#Matsuoka#H,#et#al.#Sibling#cases#of#Addison's#disease#caused# by#DAXP1#gene#mutations.#Intern#Med#2007;46:35P39.# # 32.# Wolff#AS,#Erichsen#MM,#Meager#A,#et#al.#Autoimmune#polyendocrine#syndrome# type#1#in#Norway:#phenotypic#variation,#autoantibodies,#and#novel#mutations#in#the# autoimmune#regulator#gene.#J#Clin#Endocrinol#Metab#2007;92:595P603.# 9# # 33.# Rabinowe#SL,#Jackson#RA,#Dluhy#RG,#et#al.#IaPpositive#T#lymphocytes#in#recently# diagnosed#idiopathic#Addison's#disease.#Am#J#Med#1984;77:597P601.# # 34.# Trence#DL,#Morley#JE,#Handwerger#BS.#Polyglandular#autoimmune#syndromes.# Am#J#Med#1984;77:107P116.# # 35.# Kelch#WJ.#Canine#Hypoadrenocorticism#(Canine#Addison's#Disease):#History,# Contemporary#Diagnosis#by#Practicing#Veterinarians,#and#Epidemiology.#Tennessee:#University# of#Tennessee,#1996;1P286.# # 36.# Wagner#R.#Summary#of#the#grant#proposal#to#American#Kennel#Club,#Canine# Health#Foundation.#http://www.beardiehealth.com/Addison'sPCasePandPMayP98PUpdate.htm,# 1998;6.# # 37.# Padgett#G.#Control#of#canine#genetic#diseases:#Howell#Book#House,#1998.# # 38.# Oberbauer#AM,#Bell#JS,#Belanger#JM,#et#al.#Genetic#evaluation#of#Addison's# disease#in#the#Portuguese#Water#Dog.#BMC#Vet#Res#2006;2:15.# # 39.# Famula#TR,#Belanger#JM,#Oberbauer#AM.#Heritability#and#complex#segregation# analysis#of#hypoadrenocorticism#in#the#standard#poodle.#J#Small#Anim#Pract#2003;44:8P12.# # # 10# Chapter"I:"Literature"review" " Physiology"of"the"adrenal"glands"and"their"hormones" “For"a"long"period"I"had"from"time"to"time"met"with"a"very"remarkable"form"of"general" aneamia,"occurring"without"any"discoverable"cause"whatever."The"disease"presented"in"every" instance"the"same"general"character,"pursued"a"similar"course,"and,"with"scarcely"a"single" exception,"was"followed,"after"a"variable"period,"by"the"same"fatal"result."The"appetite"is" impaired"or"entirely"lost;"the"whites"of"the"eyes"become"pearly;"the"pulse"small"and"feeble."The" body"wastes,"slight"pain"or"uneasiness"is"from"time"to"time"referred"to"the"region"of"the" stomach,"and"there"is"occasionally"actual"vomiting,"which"in"one"instance"was"both"urgent"and" distressing;"Neither"the"most"diligent"inquiry,"nor"the"most"careful"physical"examination,"tends" to"throw"the"slightest"gleam"of"light"upon"the"precise"nature"of"the"patient’s"malady;"But"with"a" more"or"less"manifestation"of"the"symptoms"already"enumerated,"we"discover"a"most" 1 " remarkable,"and,"so"far"as"I"know,"characteristic"discoloration"taking"place"in"the"skin .” This" was"the"description"of"10"patients"that"Thomas"Addison,"a"remarkable"physician"and"scientist"at" 2" Guy’s"Hospital"in"London,"recorded"in"1855 . Even"though"this"original"citation"is"156"years"old" and"medicine"has"advanced"tremendously,"Addison’s"description"of"his"namesake"disease"is"still" accurate."Addison’s"disease,"which"is"also"called"adrenal"insufficiency,"hypocorticolism," ! 11" hypocorticism"or"hypoadrenocorticism,"remains"a"diagnostic"challenge"in"humans"and" veterinary"medicine."If"the"diagnosis"is"missed"or"delayed,"it"is"fatal."" Addison’s"disease"is"defined"by"the"following:"“The"bilateral"destruction"or"dysfunction" of"the"adrenal"cortex"gives"rise"to"primary"adrenocortical"insufficiency,"or"Addison’s"disease," and"the"biochemical"pattern"of"this"condition"is"a"deficient"production"of"glucocorticoids," mineralocorticoids"and"androgens,"associated"with"high"levels"of"both"ACTH" (adrenocorticotropic"hormone,"corticotropin,"adrenocorticotrophic"hormone)"and"plasma"renin" 3 activity .”" Adrenal"glands"consist"of"the"outer"adrenal"cortex"and"the"inner"adrenal"medulla"(figure" I.1)."The"medulla"synthesizes"and"secrets"catecholamines."The"cortex"consists"of"three"layers." The"outer"layer"is"the"zona"glomerulosa."This"layer"synthesizes"and"secretes"mineralocorticoids," which"are"steroid"hormones"and"also"provides"precursor"cells"to"the"inner"layers,"the"zona" fasciculata"and"the"zona"reticularis."The"zona"fasciculata"synthesizes"and"secretes" glucocorticoids,"which"are"also"steroid"hormones."The"inner"layer,"the"zona"reticularis," synthesizes"and"secretes"androgens,"another"form"of"steroid"hormones."Cholesterol"is"a" precursor"for"these"hormones"and"can"be"rapidly"converted"into"these"upon"stimulation," therefore"storage"of"steroid"hormones"is"not"necessary."The"biochemical"pathways"of"the" steroidogenesis"are"summarized"in"figure"I.2."The"two"most"important"glucocorticoids"and" mineralocorticoids"are"cortisol"and"aldosterone,"respectively."The"adrenal"cortex"has"a" distinctive"and"very"important"exclusive"role,"much"more"than"the"adrenal"medulla."If"the" cortex"is"absent,"there"is"a"total"lack"of"synthesis"and"secretion"of"steroids"with"fatal" ! 12" consequences"whereas"when"the"medulla"is"absent,"catecholamines"can"still"be"produced"by" 4 the"autonomic"nervous"system ."" Cortisol"synthesis"and"secretion"is"under"the"control"of"ACTH,"which"is"secreted"by"the" anterior"pituitary"gland."The"secretion"of"ACTH"is"controlled"by"CRH"(corticotropin"releasing" hormone)"from"the"hypothalamus."There"is"a"negative"feedback"inhibition"loop"in"place"in" which"cortisol"inhibits"the"secretion"of"CRH"and"ACTH"(figure"I.3)."ACTH"secretion"follows"a" circardian"rhythm"in"humans,"whereas"the"secretion"in"dogs"is"nonYcircardian 5Y8 ."Even"though" only"ACTH"can"directly"stimulate"cortisol"secretion,"stress"(pain,"mental"stress,"illnesses,"etc.)" 6 can"stimulate"CRH"secretion,"and"therefore"can"indirectly"increase"cortisol"secretion ." Aldosterone"on"the"other"hand"is"controlled"by"the"reninYangiotensin"system"with"angiotensin"II" 6 being"the"most"important"direct"stimulator ."The"goal"of"the"reninYangiotensin"system"is"to" 9 maintain"the"extracellular"fluid"volume"secondary"to"sodium"retention ."The"reninYangiotensin" system"is"activated"by"a"reduction"in"the"extracellular"fluid,"by"a"decrease"in"blood"pressure"or" 10 when"the"sodium"concentration"in"the"renal"filtrate"decreases ."Further,"aldosterone"secretion" is"stimulated"by"an"increase"in"extracellular"potassium,"by"hyponatremia"or"by"a"decrease"in" extracellular"pH."Aldosterone"secretion"is"suppressed"by"a"decrease"in"extracellular"potassium," dopamine,"and"atrial"natriuretic"peptide."A"rise"in"blood"pressure"will"also"decrease"aldosterone" 6,8,10 secretion."ACTH"also"has"some"stimulating"effect"on"aldosterone"secretion ! 13" ."In"addition," cortisol"and"aldosterone"both"seem"to"be"affected"by"cytokines,"ion"concentrations"and"many" 4 other"factors ."" Cortisol"affects"almost"all"tissues"and"is"involved"in"a"variety"of"processes,"including" energy"metabolism,"mineral"homeostasis,"immune"functions"and"many"other"cellular"functions" by"gene"regulation,"all"depending"on"its"concentration:"It"helps"to"maintain"blood"pressure," water"balance,"vascular"volume,"vascular"tone,"vascular"permeability,"endothelial"integrity"and" stimulates"erythrocytes."It"increases"the"vascular"response"to"catecholamines,"lipolysis"and" gluconeogenesis"and"decreases"glucose"utilization"during"hunger"in"order"to"maintain" normoglycemia."It"suppresses"inflammation,"counteracts"stress,"and"it"has"a"catabolic"effect"on" 5,6,8,9,11 connective"tissue,"muscle"and"bone ."" 12 Aldosterone’s"function"is"unique"to"the"control"of"mineral"homeostasis ."It"primarily" works"in"the"kidneys,"but"also,"to"a"lesser"extent,"in"the"intestinal"mucosa,"salivary"and"sweat" 6 glands ."It"also"increases"reabsorption"of"sodium"and"chloride"and"excretion"of"potassium"and" hydrogen."In"the"kidneys,"it"inserts"sodium"channels"in"the"luminal"surface"of"the"cortical" collecting"duct"cells"and"stimulates"the"sodiumYpotassium"ATPase"pump,"which"is"located"on" the"basolateral"side"of"these"cells."It"also"raises"the"number"of"open"potassium"channels"in"the" 9,10,13 luminal"membrane ."The"water"homeostasis"is"therefore"maintained"secondary"to" retention"of"sodium."Both"cortisol"and"aldosterone"are"necessary"to"maintain"life."In"animal" models"in"which"the"hypothalamicYpituitaryYadrenal"axis"has"been"experimentally"interrupted," as"well"as"in"human"and"veterinary"patients,"it"has"been"shown"that"an"interruption"of"the" ! 14" hypothalamicYpituitaryYadrenal"axis"most"certainly"ends"in"death"if"appropriate"therapy"is"not" 14,15 initiated"immediately ."" " Addison’s"disease"in"human"medicine" In"this"part"of"the"literature"review,"I"will"cover"human"Addison’s"disease,"including"its" pathophysiology,"symptoms,"diagnosis,"treatment,"and"longYterm"outcome."" " Introduction" Addison’s"disease"is"a"rare,"chronic"endocrine"disorder"in"which"the"adrenal"glands" produce"insufficient"steroid"hormones,"as"described"previously."The"prevalence"of"Addison’s" 16Y19 disease"in"people"in"Western"Europe"is"estimated"to"be"110Y144"cases"per"million"people ." 18 The"incidence"is"estimated"to"be"4.4Y6"new"cases"per"million"population"a"year ,"whereas"in" the"1960s"the"prevalence"was"only"estimated"to"be"between"40"to"70"cases"per"million" 20,21 people ."The"main"cause"of"hypoadrenocorticism"in"surveys"of"human"beings"in"the"first"half" 22 of"the"last"century"was"tuberculosis,"although"a"few"cases"were"classified"as"idiopathic ." Although"tuberculosis"is"now"less"common"in"developed"countries,"the"frequency"of" hypoadrenocorticism"increasing,"thus"it"is"reasonable"to"assume"that"the"proportion"of" 23 autoimmune"adrenal"insufficiency"has"increased ."Currently"approximately"70"to"90%"of"cases" ! 15" of"Addison’s"disease"in"adult"human"beings"are"considered"to"be"the"result"of"immuneY 16,24 mediated"adrenalitis ."However,"two"other"risk"groups"have"been"identified"recently."AIDS" (acquired"immune"deficiency"syndrome)"patients"are"at"high"risk"to"develop"adrenal" insufficiency"(up"to"20%)"and"patients"with"head"trauma"develop"pituitary"insufficiency" 25Y27 frequently ."These"and"causes"such"as"other"infections,"congenital"deficiencies"of" steroidogenic"enzymes,"infiltrative"diseases,"primary"and"secondary"cancers,"drugs"or"surgeryY 16 induced"and"tuberculosis"account"for"the"remaining"10"to"30% ."The"situation"is"different"in" pediatric"patients"in"which"72%"result"from"congenital"adrenal"hyperplasia,"6%"result"from" other"genetic"diseases"and"only"13%"are"the"result"of"an"immuneYmediated"destruction"of"the" 28 adrenal"glands;"the"remainder"is"of"unknown"origin ."" Addison’s"disease"is"divided"into"two"groups,"primary"adrenal"insufficiency"and" secondary"adrenal"insufficiency."In"primary"cases,"the"end"result"is"usually"a"destruction"of"the" adrenal"cortex,"resulting"in"a"total"lack"of"glucocorticoids,"mineralocorticoids,"and"adrenal" androgens."In"some"cases,"the"medulla"gets"destroyed"as"well."Secondary"cases"usually"only" result"in"glucocorticoid"deficiency,"which"means"that"mineralocorticoids"remain"unaffected."It"is" therefore"unlikely"to"see"patients"with"secondary"hypoadrenocorticism"presenting"in"an"acute" Addisonian"crisis."" Primary"causes"include"autoimmune"diseases,"as"well"as"adrenal"infections"and" inflammation"due"to"tuberculosis,"fungal"disease"and"endYstage"AIDS,"bilateral"adrenal" metastasis,"bilateral"adrenalectomy,"adrenal"enzyme"deficiency,"adrenal"hemorrhage"or" ! 16" necrosis"due"to"sepsis"or"coagulation"disorders,"idiopathic,"drugYinduced"and" adrenoleukodystrophy"and"other"congenital"disorders."Secondary"causes"include"pituitary"or" hypothalamic"tumors,"pituitary"irradiation,"pituitary"surgery,"pituitary"(brain)"trauma,"infections" or"inflammatory/"autoimmune"disorders"in"the"pituitary"region,"pituitary"necrosis"or"bleeding" and"acute"interruption"of"prolonged"pharmacologic"glucocorticoid"therapy."Patients"after" adrenal"ectomy,"with"adrenal"hemorrhage"or"necrosis,"after"pituitary"surgery,"with"pituitary" trauma,"necrosis"or"bleeding"and"after"acute"interruption"of"prolonged"glucocorticoid"therapy" 4 show"an"increased"risk"for"developing"signs"of"an"acute"adrenal"crisis ."" Recently,"another"form"of"adrenal"insufficiency"has"been"described."There"is"recognition" of"a"relative"adrenal"insufficiency"in"patients"with"severe"nonYadrenal"or"pituitary"disease."These" people"secrete"an"inappropriate"amount"of"cortisol"to"cope"with"a"stressful"situation"and" supplementation"with"exogenous"glucocorticoids"can"improve"the"outcome"of"the" 12,29,30 disease ." The"autoimmune"form"of"Addison’s"disease,"the"main"focus"of"this"dissertation,"is"a" 16 chronic"disease,"separated"into"five"stages,"called"0"to"4 ."The"only"abnormality"in"stage"0"is" the"presence"of"adrenal"cortex"and/"or"21Yhydroxylase"antibodies;"ACTH,"cortisol,"plasma"renin" activity"and"aldosterone"levels"remain"normal."Patients"with"Addison’s"disease"stage"1"show"an" increased"plasma"renin"activity."Stage"2"includes"the"same"abnormalities,"but"the"cortisol" response"to"ACTH"stimulation"is"decreased."As"the"capacity"for"cortisol"is"further"decreased," stage"3"is"characterized"by"addition"of"a"compensatory"increase"of"ACTH"production."It"appears" ! 17" that"the"zona"glomerulosa,"the"outer"layer"of"the"adrenal"glands"that"produces"aldosterone," might"be"more"susceptible"to"the"autoimmune"attack"and"therefore"may"get"injured"first,"hence" the"increase"in"renin"activity"to"counteract"the"decrease"in"aldosterone"secretion"in"stage"1" alone."The"zona"fasciculata"might"be"protected"longer"due"to"the"local"production"of"cortisone," followed"by"the"progressive"decrease"in"cortisol"in"stages"2"through"4."Very"low"cortisol"levels,"a" very"high"ACTH"concentration"and"clinical"signs"of"the"disease"define"stage"4."Stage"0"is"a" potential"Addison’s"disease"status,"1"to"3"are"considered"to"be"subclinical"Addison’s"disease"and" 31Y35 stage"4"represents"clinical"Addison’s ."The"disease"has"a"very"long"asymptomatic"phase"in" which"the"only"abnormality"is"the"presence"of"circulating"adrenal"cortex"antibodies,"that" recognize"steroidogenic"enzymes."It"was"shown"in"1983"that"the"presence"of"this"autoantibody" has"a"predictive"value"for"disease"development,"when"4"out"of"9"antibody"positive"patients" 36 developed"clinical"Addison’s"disease"within"1"to"31"months"of"followYup ."However,"not"all" autoantibody"positive"patients"develop"Addison’s"disease"and"a"mathematical"risk"stratification" model"for"the"development"of"Addison’s"disease"has"been"developed."The"model"predicts"the" risk"of"developing"Addison’s"disease"as"low,"medium,"and"high,"based"on"five"important" variables:"patient’s"age,"autoantibody"titers,"adrenal"function"status"at"time"of"detection"of" 37 adrenal"autoantibodies,"patient"gender"and"types"of"other"autoimmune"diseases"present ."" Autoimmune"Addison’s"disease"occurs"in"conjunction"with"other"autoimmune"diseases" as"part"of"an"APS"(autoimmune"polyendocrine"syndrome)."Neufeld"and"Blizzard"classified"four" types"of"APSs ! 38,39 ."APS"type"1"consists"of"chronic"candidiasis,"chronic"hypoparathyroidism"and" 18" Addison’s"disease;"APS"type"2"consists"of"Addison’s"disease,"thyroid"autoimmune"diseases"and/" or"type"1"diabetes"mellitus;"APS"type"3,"the"only"one"that"does"not"contain"Addison’s"disease" consists"of"thyroid"autoimmune"diseases"and"other"autoimmune"diseases"without"Addison’s" disease;"and"APS"type"4"consists"of"a"combination"of"two"or"more"autoimmune"diseases"that"do" not"fall"into"any"of"the"other"three"categories"(table"I.1)."There"is"also"the"form"of"Addison’s" disease"as"a"single"immuneYmediated"endocrine"deficiency."" 16 APS"type"1"is"the"early"onset"form"(mean"age"in"the"Betterle"study"was"14"years )."APS" type"1"is"caused"by"a"mutation"in"the"AIRE"(autoimmune"suppressor"or"autoimmune"regulator)" gene,"which"is"located"on"chromosome"21q22.3."Sixty"mutations"within"AIRE"have"been" discovered"so"far"that"can"cause"APS"type"1."Eighty"%"of"people"with"these"mutations"develop" Addison’s"disease"and"are"at"increased"risk"for"mucocutaneous"candidiasis,"hypoparathyroidism" 40Y43 and"type"1"diabetes"mellitus ."" If"Addison’s"disease"develops"in"adults,"it"is"either"related"to"APS"type"2"or"occurs"in" 40 isolation ."APS"type"2"is"a"polygenic"syndrome"that"is"related"to"a"polymorphism"of"the"HLA" (human"leukocyte"antigen)"system,"with"genotypes"HLAYDR3YDQ2"and/or"DR4YDQ8"haplotypes" shown"to"increase"the"risk"to"develop"Addison’s"disease."Further,"a"reduction"of"DR1"and"DR13" 44Y46 might"also"increase"the"risk"for"the"disease ."There"also"seems"to"be"an"association"between" Addison’s"disease"and"a"mutation"in"allele"5.1"of"a"MICA"(MHC"(major"histocompatibility" complex)"class"I"chain"related"A)"gene."This"gene"is"independent"of"the"DR"or"DQ"gene" 47 polymorphism ."Besides"developing"Addison’s"disease,"patients"with"APS"type"2"may"also" ! 19" develop"hypothyroidism,"type"1"diabetes"and"many"other"autoimmune"diseases"like"pernicious" 48 47 anemia,"vitiligo,"alopecia,"celiac"disease "and"gonadal"insufficiencies ."Autoimmune" 16 Addison’s"disease"connected"to"APS"type"4"is"extremely"rare"and"not"a"lot"is"known"about"it ." The"influence"of"environmental"factors"on"disease"development"is"not"well"understood." Stressful"situations"certainly"can"trigger"an"Addisonian"crisis"if"an"autoimmune"process"has" damaged"adrenal"glands."" Betterle,"a"researcher"from"Italy"who’s"main"interest"is"Addison’s"disease,"looked"at"501" human"patients"in"2001"and"published"the"following"epidemiologic"data"(figure"I.4):"the"mean" age"of"the"501"patients"dealing"with"autoimmune"Addison’s"disease"was"31"years,"females"were" almost"twice"as"often"affected"than"males"(1.7"to"1)"and"adults"were"five"times"more"commonly" affected"than"children."APS"type"2"was"most"common"(301"cases,"representing"62"%"of"the" diseased"population)."Patients"with"APS"type"2"were"on"average"35"years"old,"the"female"to" male"ratio"was"2.3"to"1,"and"adults"were"15"times"more"likely"to"be"affected"than"children."The" second"most"common"form"of"autoimmune"hypoadrenocorticism"was"isolated"Addison’s" disease"(90"cases,"18"%)."The"mean"age"of"this"mostly"adult"group"(ratio"4"adults"to"1"child)"was" 32"years"and"males"were"more"likely"affected"than"females"(female"to"male"ratio"0.7"to"1)."APS" type"1"was"represented"by"65"cases"(13"%)."The"mean"age"of"this"group"was"14"years,"with"a" child"to"adult"ratio"of"16"to"1."The"female"to"male"ratio"was"1.9"to"1."APS"type"4"was"the"least" common"(36"cases,"7.2"%)"form."Children"were"8"times"more"likely"to"be"affected"than"adults" 16 were,"and"the"female"to"male"ratio"was"0.9"to"1 ."Of"the"82"%"of"patients"with"autoimmune" Addison’s"disease"that"also"had"one"or"more"subclinical,"clinical"or"latent"autoimmune"disease," ! 20" 62"%"had"thyroid"autoimmune"disease,"40"%"had"gastric"autoimmunity,"39"%"had"premature" ovarian"failure,"24"%"had"type"I"diabetes"mellitus,"11"%"had"chronic"hypoparathyroidism,"10"%" had"chronic"candidiasis,"8"%"vitiligo,"5"%"had"either"celiac"disease"or"alopecia,"4"%"had" autoimmune"hepatitis,"3"%"had"cancer"and"1"%"had"intestinal"malabsorption"or"Sjögren’s" 16 syndrome ."The"findings"of"a"nationwide"Norwegian"study"were"similar."In"this"population," autoimmune"Addison’s"disease"was"accompanied"by"another"clinical"autoimmune"disease"in""" 61"%"of"patients."When"subclinical"cases"were"included,"this"percentage"rose"to"88"%."Of"the""" 61"%"of"patients"with"autoimmune"Addison’s"disease"in"the"Norwegian"population,"47"%"had" concurrent"autoimmune"thyroid"disease,"12"%"had"diabetes"mellitus"type"1,"11"%"had"vitiligo," 49 10"%"had"pernicious"anemia,"and"4"%"had"alopecia ."" " Pathophysiology" Over"time,"it"has"become"more"and"more"clear"that"most"cases"of"human"Addison’s" disease"are"caused"by"autoimmune"destruction"of"the"adrenal"glands."Adrenal"cortex"antibodies" 50 were"first"identified"in"1957 ."For"the"next"30"years,"antibodies"directed"against"a"microsomal" autoantigen"located"in"the"cytoplasm"of"the"zona"glomerulosa,"zona"fasciculata"and"zona" reticularis"were"identified"via"indirect"immunofluorescence"technique"in"51"%"to"61"%"of" patients"with"autoimmune"Addison’s"disease"and"in"close"to"90"%"of"patients"with"newly" diagnosed"(as"opposed"to"patients"on"longYterm"treatment)"autoimmune"Addison’s"disease." 51Y53 Two"%"of"patients"with"Addison’s"disease"due"to"tuberculosis"were"antibody"positive ! 21" ." Then,"in"1992,"three"groups"independently"described"that"the"adrenal"cortex"antibody" 54Y56 recognizes"a"major"enzyme"of"the"steroidYpathway,"21Yhydroxylase ."In"order"to"test"for" 35 antibodies,"an"immunoprecipitation"assay"with" SYmethionine"labeled"recombinant"21Y hydroxylase"was"developed"in"1995,"and"another"using"recombinant"human"21Yhydroxylase" 125 produced"in"yeast"and"labeled"with" 57 I"was"described"in"1997 ."With"these"assays,"it"was" discovered"that"78"%"of"patients"with"autoimmune"Addison’s"disease"tested"positive"for" autoantibody,"including"more"than"90%"of"newly"diagnosed"patients."Positivity"was"also"found" 53 in"1.9"%"of"the"general"population ."In"addition"to"21Yhydroxylase"antibodies,"17Yhydroxylase" antibodies"and"cholesterol"side"chainYcleaving"enzyme"antibodies"can"be"identified"in"serum." 21Yhydroxylase"antibodies"are"by"far"the"most"common,"while"cholesterol"side"chainYcleaving" antibodies"are"less"commonly"found."17Yhydroxylase"antibodies"are"the"least"commonly"found" 58 antibodies ."" Addison’s"disease"has"been"successfully"induced"in"mice"and"rats"by"injecting"adrenal" cortex"extracts."These"animals"showed"clinical"signs"of"adrenal"insufficiency"and"circulating" antibodies"against"adrenal"tissue"were"demonstrated."Upon"autopsy,"the"adrenal"cortices"were" 59,60 infiltrated"with"mononuclear"cells ."NonYobese"diabetic"mice"that"spontaneously"develop" diabetes"mellitus"type"1"may"also"develop"mononuclear"cell"infiltration"in"the"adrenal"glands," 61 but"these"animals"do"not"show"signs"of"adrenal"insufficiency ."Dogs"and"cats"can"develop" autoimmune"adrenal"insufficiency"(experimentally"induced"or"naturally"occurring);"however,"it" ! 22" has"been"proven"difficult"and"unreliable"to"identify"the"circulating"adrenal"antibodies"in"these" species 62Y71 ."" Adrenal"glands"are"small"in"human"patients"with"Addison’s"disease."The"cortex"is" markedly"atrophied"with"various"amounts"of"fibrosis"and"infiltrated"with"lymphocytes,"plasma" cells"and"macrophages,"sometimes"located"in"germinal"centers."Lymphocytes"are"usually"T"cells" and"the"CD4"(cluster"of"differentiation"4)"to"CD8"ratio"is"5"or"6"to"1."Only"5"%"are"B"cells,"of" which"half"are"positive"for"class"II"HLA."If"there"are"adrenocortical"cells"left,"they"are"usually" 72 hyperplastic."However,"the"adrenal"medulla"remains"unchanged ."It"is"not"clear"how"the" adrenal"cortex"gets"destroyed"in"patients"with"autoimmune"Addison’s"disease,"but"it"appears" 73 that"antibodies"do"not"mediate"the"disease "for"the"following"reasons:"The"antigens"involved" are"all"located"intracellularly"and"localized"in"the"endoplasmic"reticulum."Since"plasma" membranes"are"not"permeable"for"immunoglobulins,"destruction"of"the"antigens"can"only"take" 58 place"after"the"destruction"of"the"tissue ."Even"though"antibodies"against"21Yhydroxylase"can" 74 block"the"enzymatic"activity"of"this"enzyme"in"vitro ,"a"passive"transfer"from"mother"to"fetus"of" 75 21Yhydroxylase"antibodies"does"not"cause"disease"in"the"baby "and"the"presence"of"ACTH" 76,77 receptorYblocking"antibodies"could"not"be"verified ."Antibodies"might"activate"the"cytolytic" complement"cascade"though,"which"ultimately"would"lead"to"the"lysis"of"the"cell"or"might" 58 initiate"antibodyYdependent"cellYmediated"immunity,"which"would"lead"to"phagocytosis "and" 31,78,79 complement"fixing"antibodies"have"been"identified"in"a"number"of"patients ! 23" ."On"the" other"hand,"patients"with"autoimmune"Addison’s"disease"have"mononuclear"cells"that"can"be" stimulated"by"21Yhydroxylase"to"secrete"IFNYγ"(interferonYgamma)"and"ILY2"(interleukinY2)."In" vitro,"it"appeared"that"especially"amino"acids"342"to"361"of"the"21Yhydroxylase"enzyme"were" able"to"stimulate"these"mononuclear"cells."This"area"of"the"21Yhydroxylase"may"comprise"a" 80 disease"epitope,"represented"by"cells"carrying"genotype"HLAYDRB1*0404 ."Patients"also"have" 79 81 circulating"IaYpositive"T"cells ,"a"proliferative"T"cell"response"to"an"adrenalYspecific"protein " and"a"defect"in"the"suppressor"of"the"CD4+/CD25+"regulatory"T"cell."The"latter"has"only"been" 82 identified"in"patients"with"APS"type"2 ."These"findings"made"it"plausible"to"believe"that"the" 16 disease"is"caused"by"cellYmediated"immunity,"but"further"studies"are"needed ."" " Symptoms"of"adrenal"insufficiency" Symptoms"of"Addison’s"disease"in"humans"are"nonYspecific"and"likely"to"not"appear"until" after"a"long"subclinical"phase."Since"Addison’s"disease"in"humans"is"mostly"of"immune"mediated" etiology,"most"patients"have"measurable"circulating"adrenal"cortex"and/"or"21Yhydroxylase" 16 antibodies"at"time"of"diagnosis ."Patients"with"autoimmune"adrenal"disease"go"through"a"long" preclinical"phase"of"disease"in"which"antibodies"are"present,"but"clinical"signs"are"not."Most"of" these"patients"also"have"other"concurrent"autoimmune"diseases"as"mentioned"above."Clinical" signs"then"usually"appear"when"at"least"90"%"of"the"glands"are"destroyed,"or"earlier"if"an" 45 extremely"stressful"event"occurs ."Symptoms"and"signs"of"chronic"primary"and"secondary" ! 24" adrenal"insufficiency"include"fatigue,"weakness,"lethargy,"loss"of"energy,"anorexia,"weight"loss," postural"hypotension,"nausea,"vomiting,"myalgia,"general"malaise,"diffuse"abdominal"pain/" gastric"pain,"joint"pain,"dizziness,"salt"craving"and"in"women"loss"of"libido"and"loss"of"axillary"and" 4,16 pubic"hair"(due"to"pronounced"androgen"deficiency) ."Cutaneous"and"mucosal" hyperpigmentation"can"result"from"an"excess"of"opiomelanocortin"and"melanocyte"stimulating" hormone."Also,"ACTH"is"similar"in"structure"to"melanocyte"stimulating"hormone,"causing"it"to" 83 react"with"its"receptor ."Sodium"(in"all"patients),"chloride"and"bicarbonate"concentrations"are" low"at"time"of"diagnosis"and"potassium"concentrations"are"elevated"(in"50"to"70"%)."Ten"to"20"%" 3 of"patients"show"hypercalcemia "and"many"show"fasting"hypoglycemia."The"combination"of"the" 4 two"should"make"the"physician"highly"suspicious"of"adrenal"insufficiency ."Even"though"it"is"not" understood"why,"40"to"50"%"of"patients"are"anemic,"with"10"to"15"%"having"eosinophilia"and" 16 lymphocytosis ."The"acute"patient"shows"the"same"biochemical"abnormalities,"but"presents" with"hypotension"or"hypotensive"shock"due"to"mineralocorticoid"deficiencies"and"the"resulting" hyponatremia"and"plasma"volume"depletion."The"circulation"is"further"decreased"by"an" elevation"of"prostaglandine"E"and"a"decreased"responsiveness"to"norepinephrine"and" angiotensin"II."On"top"of"that,"whatever"caused"the"acute"adrenal"crisis,"for"example"sepsis," 4 surgery"or"trauma,"might"mask"it ."Because"symptoms"are"very"nonspecific,"patients"are" 84,85 commonly"misdiagnosed"with"other"conditions"with"potentially"serious"consequences " ! 25" ."" Diagnosis"of"adrenal"insufficiency" The"most"useful"diagnostic"tests"for"Addison’s"disease"in"humans"are"blood"tests." Cortisol"in"circulation"is"bound"to"cortisol"binding"globulin"(also"called"transcortin),"and"its"level" can"be"decreased"due"to"chronic"liver"or"kidney"disease"or"because"of"an"exposure"to"estrogens." Measured"low"total"cortisol"concentrations"therefore"should"not"be"used"to"confirm"a"diagnosis" of"hypoadrenocorticism."Rather"free"cortisol"levels"should"be"measured"or"calculated"based"on" 86,87 total"cortisol"and"transcortin"measurement ."To"diagnose"adrenal"insufficiency,"it"is" necessary"to"differentiate"between"screening"tests"and"confirmatory"tests."As"a"screening"test," early"morning"basal"cortisol/"free"serum"cortisol"should"be"measured."Levels"greater"than"20" μg/"dL"in"a"patient"not"in"an"acute"stress"situation"rule"out"a"differential"diagnosis"of"adrenal" insufficiency."If"transcortin"concentrations"are"normal,"then"the"normal"morning"cortisol"level" ranges"between"10"to"20"μg/"dL."Levels"less"than"10"μg/"dL"necessitate"more"workup"and"levels" less"than"3"μg/"dL"make"adrenal"insufficiency"very"likely."Furthermore,"plasma"basal"ACTH"in"the" normal"range"or"below"100"pg/"mL"rule"out"a"differential"diagnosis"of"primary" hypoadrenocorticism."Normal"to"low"renin"and"normal"aldosterone"further"make"adrenal" insufficiency"unlikely."However,"elevated"renin"concentrations"are"one"of"the"earliest"changes" seen"with"primary"adrenal"insufficiency."If"the"screening"test"(measurement"of"early"morning" basal"cortisol/"free"serum"cortisol)"result"is"equivocal"and"a"patient"needs"more"workup,"a" stimulation"test"should"be"performed."A"stimulation"test"should"also"be"performed"if"patients" are"positive"for"adrenal"antibodies"but"do"not"show"clinical"signs"yet."The"gold"standard" simulation"test"is"to"take"a"baseline"blood"sample"for"cortisol"and"aldosterone"measurement" ! 26" from"the"patient"followed"by"an"administration"of"1"or"250"μg"of"corticotropin."Cortisol"and" aldosterone"should"be"measured"again"30"and"60"minutes"later."Cortisol"concentrations"of" greater"than"20"μg/"dL"at"any"time"rule"out"a"differential"diagnosis"of"primary"adrenal" insufficiency,"but"do"not"rule"out"the"presence"of"secondary"adrenal"insufficiency."Other" screening"tests"include"stimulation"of"the"pituitaryYadrenal"axis"by"insulinYinduced" hypoglycemia"or"stimulation"with"corticotropinYreleasing"hormone."The"first"test"is"especially" useful"if"there"is"a"suspicion"of"pathology"in"the"hypothalamicYpituitary"axis."A"dose"of"0.1"U"of" regular"insulin"is"administered"intravenously"and"basal,"30,"60"and"90"minute"levels"of"cortisol," corticotropin"and"sometimes"of"growth"hormone"in"cases"of"suspected"multiple"pituitary" hormone"deficiencies"are"measured."A"normal"cortisol"response"would"exceed"20"μg/"dL."The" latter"test"is"helpful"to"differentiate"between"hypothalamic"and"pituitary"origin"of"adrenal" 4 insufficiency ."" Different"tests"should"be"performed"during"acute"illness"in"order"to"initiate"therapy" immediately."The"physician"should"be"suspicious"of"adrenal"insufficiency"in"patients"that"show" unexplained"hypotension"and"that"are"at"high"risk"to"develop"adrenal"insufficiency."This"group" includes"patients"with"AIDS,"patients"that"just"discontinued"glucocorticoid"therapy,"patients" with"autoimmune"diseases,"patients"with"hyperpigmentation,"patients"that"are"chronically"tired" 4 or"patients"that"show"acute"clinical"signs"of"an"adrenal"insufficiency"as"mentioned"above ." Immediate"tests"that"should"be"performed"include"simple"diagnostic"tests"that"give"immediate" results"and"that"are"rapidly"available,"including"measurement"of"sodium,"potassium," bicarbonate"and"cortisol."A"plasma"sample"for"measurement"of"corticotropin,"renin"and" ! 27" aldosterone"should"be"obtained."A"short"corticotropin"stimulation"test,"in"which"a"post" corticotropin"sample"for"the"measurement"of"cortisol"30"min"post"injection"is"taken,"should"also" be"performed."Once"samples"are"obtained"and"tests"are"performed,"therapeutic"intervention" 4 should"be"started"until"results"are"available ."Untreated"cortisol"deficiency"could"also"lead"to"a" modest"elevation"in"thyroid"stimulating"hormone"in"30"%"of"patients,"because"cortisol"usually" inhibits"TSH"(thyroid"stimulating"hormone)"secretion 16,88 ."If"adrenal"insufficiency"can"be" confirmed,"it"is"recommended"to"identify"the"etiology"of"the"disease"in"order"to"address"the" underlying"issue,"screen"for"other"autoimmune"diseases"or"make"sure"that"relatives"are" followed"up"with"and"monitored"appropriately."" Imaging,"including"ultrasound"and"xYrays,"only"appear"to"be"important"if"a"patient"does" 89 not"have"adrenal"autoantibodies ."Enlargement"of"the"adrenal"glands"with"and"without" 3 calcification"can"occur"with"tuberculosis,"cancer,"infections"and"infiltrative"or"vascular"diseases ."" " Treatment"of"adrenal"insufficiency" For"treatment"purposes,"it"is"necessary"to"differentiate"between"the"acute"phase,"and" the"chronic,"maintenance"phase"of"hypoadrenocorticism."The"acute"phase"is"life"threatening" and"therefore"depends"primarily"on"the"patient’s"clinical"condition,"but"treatment"should"be" initiated"immediately"after"diagnostic"samples"have"been"taken."Treatment"in"the"acute"phase" includes"electrolytes,"fluid"and"hydrocortisone"at"a"dose"of"150Y300"mg/"d."The"hydrocortisone" ! 28" 4 is"given"either"intravenously"or"intramuscularly"until"the"patient"is"stable ."At"such"a"high"dose," which"is"also"called"‘physiologic"stress"dosage’,"hydrocortisone"activates"the"mineralocorticoid" receptor"directly"and"additional"mineralocorticoid"supplementation"is"therefore"not"necessary." In"physiologic"concentrations,"cortisol"does"not"activate"the"mineralocorticoid"receptor,"even" though"it"has"the"same"affinity"to"it"as"aldosterone"has."This"is"related"to"the"sudden"intrarenal" inactivation"of"cortisol"to"cortisone"via"11YβYsteroid"dehydrogenase"in"mineralocorticoidY responsive"tissues."Total"inactivation"only"occurs"when"physiologic"concentrations"of"cortisol" 4 are"present ."Once"the"patient"is"stable,"the"dosage"can"be"gradually"decreased."During"acute" phase,"a"lower"hydrocortisone"dosage"could"yield"same"results,"but"no"clinical"studies"have" 4 been"conducted"to"address"this"question ."Further,"the"mineralocorticoid"effect"of"the"high" dosage"of"hydrocortisone"is"desirable."" Maintenance"replacement"therapy"of"glucocorticoids"and"mineralocorticoids"is"given" orally."The"goal"is"to"give"the"lowest"dosage"of"hydrocortisone"that"is"necessary"to"avoid" symptoms"of"adrenal"insufficiency"and"to"mimic"physiologic"cortisol"production."Good"followYup" hormone"tests,"like"they"exist"with"thyroxine"replacement"therapy"for"hypothyroidism,"are"not" defined"for"treatment"of"hypoadrenocorticism"with"glucocorticoids."The"evaluation"of"the" 90,91 clinical"response"therefore"is"extremely"important ."However,"some"authors"believe"that" measurement"of"24Yhour"free"cortisone"in"urine"(urinary"free"cortisol)"is"a"way"to"determine" 92,93 adequacy"of"cortisone"replacement"therapy ."Daily"cortisol"production"is"estimated"to"be"at" 2 " 4 8"mg/"m / d,"measured"via"isotope"dilution"methodology ."Cortisol"secretion"follows"a"diurnal" ! 29" rhythm"in"humans."Over"time,"cortisol"replacement"therapy"has"been"decreased"from"30"mg/"d" of"hydrocortisone"or"even"more"to"15"to"25"mg/"d"of"which"15"to"10"mg"are"given"after"waking" up"and"5"to"10"mg"are"given"in"the"afternoon"or"early"evening"to"mimic"the"diurnal"rhythm." Twenty"five"to"50"mg"of"cortisone"acetate"per"day"divided"in"two"or"three"doses"can"also"be" used."This"dosage"is"still"slightly"above"the"physiologic"concentration"but"is"probably"needed"to" offset"the"inactivation"of"cortisol"into"cortisone"after"oral"intake."Patient"and"family"education"is" extremely"important,"as"is"that"the"patient"carries"a"medical"emergency"alert"card"with"him"at" all"times."Even"though"replacement"therapy"has"been"gradually"decreased"over"time"from"30" mg"or"more"of"hydrocortisone"plus"fludrocortisone"per"day,"patients"seldom"suffer"from"an" acute"adrenal"crisis,"partly"because"they"are"taught"to"increase"the"dosage"by"50"to"300"%" during"acute"disease"or"extreme"stressful"situations,"including"but"not"limited"to"illness,"injury," vomiting,"surgical"procedures,"pregnancies,"etc. 4,90 ."To"maintain"electrolyte"homeostasis,"50"to" 47 16 200"μg"fludrocortisone"per"day"is"given ."A"diet"rich"in"sodium"might"be"beneficial"as"well ."In" theory,"excess"salt"is"especially"needed"during"summer,"because"the"sweat"of"a"person"suffering" from"adrenal"insufficiency"contains"more"salt"as"compared"to"a"healthy"individual." Mineralocorticoid"dosage"can"be"easily"monitored"by"measurement"of"blood"pressure,"plasma" 94 renin"activity,"and"electrolyte"levels ."Women"with"adrenal"insufficiency"might"show"additional" benefit"from"replacement"with"dehydroepiandrosterone"to"compensate"for"the"inability"to" 4 produce"androgens ."" " ! 30" LongYterm"outcome"of"adrenal"insufficiency" One"might"think"that"people"with"Addison’s"disease,"once"diagnosed,"have"a"normal"life" expectancy."However,"even"when"patients"are"diagnosed"and"treated"correctly,"retrospective" studies"have"shown"that"the"standardized"mortality"ratios"of"both"women"and"men"with" primary"adrenal"failure"are"more"than"2Yfold"increased,"compared"to"the"general"population."A" recent"study"estimates"these"standardized"mortality"rates"at"2.9,"95"%"CI"(confidence"interval)" 2.7Y3.0"and"2.5,"95"%"CI"2.3Y2.7,"respectively."The"risk"seems"to"be"especially"high"when"patients" are"diagnosed"during"childhood."Also,"patients"with"APS"1"have"a"higher"standardized"mortality" 95,96 rate"compared"to"patients"with"APS"2,"4.6,"95%"CI"3.5Y6.0"and"2.1,"95%"CI"1.9Y2.4 exact"reasons"for"that"are"not"fully"understood.""" " " ! 31" ."The" Addison’s"disease"in"dogs" In"this"part"of"the"literature"review,"I"will"cover"Addison’s"disease"in"dogs,"including"its" pathophysiology,"symptoms,"diagnosis,"treatment,"and"longYterm"outcome."" " Introduction" " Addison’s"disease"is"a"chronic"endocrine"disorder"in"which"the"adrenal"glands"produce" insufficient"steroid"hormones,"as"described"previously."Hypoadrenocorticism"is"not"uncommon" in"the"dog."The"average"small"animal"veterinarian"sees"1400"to"1600"dogs/"year"and"combined" with"the"estimated"incidence"rate,"a"fullYtime"veterinarian"would"be"expected"to"diagnose"0.17" to"0.53"dogs"with"hypoadrenocorticism"per"year."The"average"two"veterinarian"practice"in" which"each"veterinarian"sees"around"1500"dogs/"year"should"therefore"expect"to"diagnose"one" 97 case"per"year ."" Several"breeds"have"been"reported"to"have"a"10Y50"%"higherYthanYaverage"risk"for" hypoadrenocorticism."These"breeds"include"the"Bearded"Collie,"West"Highland"White"Terrier," Standard"Poodle"(and"other"Poodles),"Portuguese"Water"Dog,"Leonberger,"Great"Dane,"Airedale" Terrier,"Basset"Hound,"Wheaten"Terrier,"Rottweiler,"Springer"Spaniel"and"Nova"Scotia"Duck" Tolling"Retriever."Breeds"at"lower"risk"include"Boston"Terrier,"Lhasa"Apso,"Yorkshire"Terrier"and" mixedYbreed"dogs 5,97Y102 ."Excluding"the"studies"of"Kelch"and"Peterson,"no"one"takes"into" account"the"breed"distribution"in"the"overall"population"and"studies"might"therefore"be" ! 32" misleading."Kelch"points"out"however"that"“results"are"remarkably"consistent"in"that"in"no"case" 103 was"a"breed"found"to"be"at"increased"risk"in"one"dataset"and"at"decreased"risk"in"another .”" These"two"studies"find"that"Great"Danes,"Poodles"(see"table"I.2"for"more"details"on"Poodles)"and" 99,103 West"Highland"White"Terriers"are"at"increased"risk ."Portuguese"Water"Dogs,"Rottweilers," and"Soft"Coated"Wheaten"Terriers"have"been"reported"by"Peterson"to"be"at"increased"risk," 99 which"has"not"been"confirmed"by"Kelch ."A"decreased"risk"among"Golden"Retrievers,"Lhasa" 99 Apsos"and"Yorkshire"Terriers"has"been"reported"by"Peterson ."Kelch"confirms"the"decreased" 103 risk"in"the"latter"two,"but"cannot"confirm"it"for"the"Golden"Retriever ."Kelch’s"and"Peterson’s" findings"are"summarized"in"tables"I.2"and"I.3."In"certain"breeds"that"are"at"higher"risk"to"develop" primary"hypoadrenocorticism,"the"condition"is"considered"to"be"heritable"with"a"possible" familial"predisposition"in"the"Portuguese"Water"Dog,"Leonberger,"Standard"Poodle,"Bearded" 5,102,104 Collie"and"Nova"Scotia"Duck"Tolling"Retriever ."An"autosomal"recessive"mode"of" inheritance"has"been"suggested"in"the"Nova"Scotia"Duck"Tolling"Retriever,"the"Standard"Poodle" and,"most"likely,"in"the"Portuguese"Water"Dog."This"mode"of"inheritance"has"not"been" 100Y102,105 confirmed"for"the"Bearded"Collie ."" The"duration"of"the"disease,"from"time"of"diagnosis"to"death,"related"or"unrelated"to" Addison’s"disease,"is"approximately"4.9"years"with"adequate"medical"replacement"therapy."In"a" steadyYstate,"the"prevalence"equals"the"incidence"times"duration"of"disease"(P=I*D)."In"his" dissertation"from"1996,"Kelch"analyzed"three"different"groups"of"dogs"with" hypoadrenocorticism"and"reported"the"following:"the"first"group"consists"of"244"cases"that"were" ! 33" found"in"the"veterinary"literature;"the"second"group"(n=266)"of"dogs"enrolled"in"a"clinical"study" to"evaluate"the"microcrystalline"drug"desoxycorticosterone"pivalate;"and"the"third"group" consists"of"376"cases"obtained"from"colleges"of"veterinary"medicine"and"other"veterinary" referral"hospitals."The"prevalence"estimates"in"these"three"groups"are"0.6/"1000"dogs,"1.7/"1000" 103 dogs"and"0.32/"1000"dogs,"respectively,"with"a"point"estimate"of"1.8/"1000"dogs ."" Different"studies"in"veterinary"medicine"throughout"the"world"reveal"similar"results" 97,103,106Y110 concerning"incidence,"prevalence,"age"of"onset,"breed"and"gender"distribution ." However,"the"most"current"study"was"done"in"France"between"1997"and"2001"and"was" presented"during"the"meeting"of"the"European"Society"of"Veterinary"Internal"Medicine"in"Spain" 109 in"2004 ."It"revealed"a"lower"prevalence"of"0.003/"per"1000"dogs"than"previously"reported 109 but"the"diagnosis"was"based"on"low"cortisol"and"aldosterone"concentrations"post" administration"of"ACTH,"whereas"other"studies"only"required"low"plasma"cortisol" concentrations"post"ACTH"administration 109 ."The"study"also"excluded"dogs"of"unknown"status" and"dogs"that"weren’t"followedYup"three"years"post"diagnosis,"leading"to"a"decrease"in"total" number"of"cases"and"therefore"a"smaller"prevalence."I"therefore"believe"that"this"study"is" misleading"rather"than"helpful."" The"most"useful"epidemiologic"data"in"dogs"is"extractable"from"the"Kelch"and"Peterson" 99,103 study ."As"mentioned"above,"most"studies"do"not"take"into"account"the"prevalence"of"a" breed"distribution"in"the"overall"population"and"are"therefore"misleading."Kelch"and"Peterson" however"calculated"odds"ratios"from"the"number"of"cases"in"a"particular"breed"compared"with" ! 34" ," the"number"of"that"breed"seen"overall 97,99,103 ."Peterson’s"reference"population"consists"of" n=3512"dogs"that"were"examined"at"the"Animal"Medical"Center"in"New"York"City"between" January"1979"and"January"1993"that"did"not"have"hypoadrenocorticism."Included"dogs"were" 99 randomly"selected"from"all"examined"dogs,"and"each"individual"dog"was"only"included"once ." Kelch’s"control"group"consisted"of"patients"from"1000"randomly"selected"veterinarians"from"the" American"Veterinary"Medical"Association’s"list"of"small"animal"practitioners."A"questionnaire" was"sent"to"these"veterinarians"asking"for"data"on"dogs"diagnosed"with"hypoadrenocorticism" and"for"controls"which"were"defined"as"the"next"and"the"fifth"next"dog"in"the"veterinarian’s" database"that"were"owned"by"a"different"owner."The"response"rate"was"56"%;"9"%"were" excluded."The"number"of"active"dog"patients"per"veterinarian"was"also"reported."Data"(age"of" onset,"female"to"male"ratio,"mean"body"weight"at"onset)"from"different"studies"are"summarized" in"table"I.4."The"average"age"of"diagnosis"of"the"summarized"studies"was"4.8"years,"with"the" average"in"these"studies"ranging"from"4.0"to"6.0"years."The"average"body"weight"at"onset"was" 20.5"kg"(range:"10.2"to"28.5"kg)"with"a"female"to"male"ratio"of"65"to"35."" The"point"estimate"for"age"in"the"three"different"groups"defined"by"Kelch"is"4.9"years" (range"5"weeks"to"15"years) 103 ."Other"studies"obtained"similar"results"(range"4"weeks"to"16" years"with"an"average"age"of"onset"of"4"to"5"years),"reinforcing"that"the"disease"can"be" diagnosed"in"very"old"dogs"or"in"very"young"dogs."The"average"reported"age"of"onset"for"the" 8,97Y102 Nova"Scotia"Duck"Tolling"Retriever"is"only"2.6"years ."Kelch"calculated"odds"ratios"that" show"that"age"is"associated"with"the"occurrence"of"the"disease."The"probability"increases"with" age"and"peaks"in"the"age"group"4"to"7"years"(1.9,"p<.0001) ! 35" 103 "(table"I.5)." All"studies"approximate"the"risk"for"females"developing"hypoadrenocorticism"compared" to"males"as"close"to"2"to"1"(except"Bearded"Collies,"Portuguese"Water"Dogs"and"Standard" Poodles"in"which"males"are"as"much"affected"as"females"are)"and"results"are"remarkably"similar," even"though"most"of"the"studies"do"not"take"into"account"the"sex"distribution"in"the"entire"dog" population 5,97,99Y103,105Y107,110Y112 ."Peterson"does"consider"the"sex"distribution"and" 99 concludes"that"females"are"more"likely"to"develop"disease"than"males ."SexYspecific"incidence" estimates"from"Kelch’s"data"sets"(veterinary"referral"hospitals,"mail"survey"from"Ciba"Animal" Health"veterinarians,"and"from"randomly"selected"veterinarians)"conclude"that" ovariohysterectomized"females"are"more"likely"to"be"affected"than"intact"females,"that" castrated"males"are"more"likely"to"be"affected"than"intact"males,"and"that"neutered"dogs"in" general"are"more"likely"to"develop"hypoadrenocorticism"than"intact"ones"(table"I.6) 97,103 ."" This"data"contradicts"Peterson’s"study,"which"reports"that"intact"females"had"an" increased"probability"of"disease"compared"to"ovariohysterectomized"females."Peterson" calculated"odds"ratios"of"developing"hypoadrenocorticism"for"gender."Relative"to"sexually"intact" males"which"had"the"lowest"risk,"intact"females"had"the"highest"risk"(5.55,"p<0.001)"(table" 99 I.7) ."The"association"between"spaying"and"hypoadrenocorticism"could"be"influenced"by"the" age"at"which"dogs"are"ovariohysterectomized,"which"can"explain"the"different"outcome."Dogs" are"usually"spayed"at"a"very"young"age." " " ! 36" Pathophysiology" " It"has"been"reported"that"up"to"10"%"of"dogs"with"Addison’s"disease"might"present"with" ‘atypical’"primary"Addison’s"disease,"in"which"serum"electrolytes"(and"sometimes"aldosterone" 5,111 levels)"are"normal"at"diagnosis ."However,"it"is"suspected"that"these"dogs"develop" electrolyte"abnormalities"later"on"during"progression"of"disease"and"a"discrepancy"of"specialists" opinions"remains"on"whether"this"condition"should"be"named"or"treated"differently"than"the" classic"form"of"Addison’s"disease."" Naturally"occurring"primary"Addison’s"disease,"with"a"lack"of"both"glucocorticoids"and" mineralocorticoids"results"from"destruction"or"atrophy"of"all"layers"of"the"adrenal"cortex."The" primary"cause"for"this"destruction"is"hypothesized"to"be"immuneYmediated,"and"bilateral" adrenocortical"atrophy"with"mononuclear"infiltrates"has"been"described 71,113 71,114 assessed,"a"small"proportion"of"dogs"showed"antiYadrenal"antibodies ."Also,"when" ."Studies"however" were"difficult"to"replicate."Other,"mostly"rare"causes"in"the"dog"for"primary"Addison’s"disease" are"infiltrative"diseases"including"fungal"infections"(Histoplasma,"Blastomyces,"Coccidioides," Cryptococcus),"neoplastic"diseases,"amyloidosis,"trauma,"coagulopathy,"and"iatrogenic"due"to" mitotane"(Lysodren,"oYp’YDDD;"BristolYMyersYSquibb,"Princeton,"New"Jersey,"USA)"and" 5,7,111 trilostane"(Vetoryl,"Dechra"Veterinary"Products,"Overland"Park,"Kansas,"USA)"use ." Iatrogenic"cases"of"primary"hypoadrenocorticism"are"relatively"common"and"result"from" oversuppression"of"the"adrenal"glands"during"treatment"of"hyperadrenocorticism"(Cushing’s" syndrome)."Whereas"most"humans"with"Addison’s"disease"have"other"autoimmune"diseases," ! 37" autoimmune"polyglandular"syndrome"in"the"dog"is"uncommon."Out"of"187"dogs"that"were" diagnosed"with"primary"hypoadrenocorticism,"only"28"(15%)"had"at"least"one"other"confirmed" 5 autoimmune"disease ."Other"autoimmune"diseases"present"were"hypothyroidism"in"16"dogs," insulinYdependent"diabetes"mellitus"in"14"dogs,"hypoparathyroidism"in"three"dogs"and"two"had" 5 azoospermia .""" Naturally"occurring"secondary"hypoadrenocorticism"is"related"to"insufficient"ACTH" secretion"because"of"a"pituitary"lesion,"such"as"neoplasia,"trauma"or"inflammation 5,111 ," resulting"in"atrophy"of"the"zonae"fasciculata"and"reticularis."Aldosterone"secretion"and"serum" electrolytes"are"maintained 5,6 ;"clinical"signs"therefore"result"from"a"lack"of"cortisol."This" condition"is"sometimes"very"difficult"to"diagnose"since"blood"work"and"other"tests"can"remain" normal."The"exact"incidence"of"this"condition"is"not"known,"but"it"is"rareY"estimates"range"from" 5,111 4"%"to"24"% "of"all"dogs"with"hypoadrenocorticism."" Iatrogenic"secondary"hypoadrenocorticism"is"the"most"common"form,"and"is"caused"by"a" sudden"withdrawal"of"chronic"use"of"injectable,"oral,"ophthalmic,"otic"or"topical"exogenous" 5 steroids ."The"use"of"exogenous"steroids"suppresses"ACTH"secretion,"which"in"turn"causes"the" zona"reticularis"and"zona"fasciculata"to"atrophy."Hypoadrenocorticism"then"occurs"when" exogenous"steroids"are"withdrawn"too"quickly."" ! 38" Tertiary"hypoadrenocorticism"is"caused"by"a"lack"or"decrease"of"CRH"secretion,"which"in" turn"causes"a"decrease"in"ACTH"secretion."The"clinical"manifestation"is"then"the"same"as"in" 5 secondary"hypoadrenocorticism,"which"results"from"a"lack"of"ACTH ."" " Symptoms"of"adrenal"insufficiency" " For"the"purpose"of"this"research"and"review,"I"will"from"now"on"focus"on"dogs"with"the" classic"form"of"primary"Addison’s"disease."" The"clinical"characteristics"of"primary"hypoadrenocorticism"in"the"dog"have"been"well" 5,99,104,108,110,115Y120 documented"in"the"veterinary"literature ."Clinical"signs"that"are"observed" and"reported"by"the"owner"are"caused"by"deficiencies"of"glucocorticoids"and"mineralocorticoids" and"are"very"similar"in"dogs"and"humans."The"lack"of"glucocorticoids"causes"gastrointestinal" signs"including"anorexia"(88"%"to"95"%),"vomiting"and"regurgitation"(68"%"to"75"%),"diarrhea""" (35"%),"and"abdominal"pain"(8"%);"renal"signs"including"polyuria"and"polydipsia"(17"%"to"25"%);" neurologic"signs"including"shaking,"shivering"and"tremors"(17"%"to"27"%),"and"collapse"(10"%);" and"nonYspecific"systemic"signs"including"lethargy"and"depression"(85"%"to"95"%),"weakness""" (51"%"to"75"%),"and"weight"loss"(40"%"to"50"%)."Also"noted"can"be"fasting"hypoglycemia"from" impaired"energy"metabolism,"hematemesis,"hematochezia,"melena,"ataxia,"seizures,"difficulty" breathing,"hair"loss,"muscle"cramps"in"front"and"back"limbs,"prior"response"to"nonspecific"fluidY" and"glucocorticoid"therapy"and"statements"that"a"dog"has"a"hard"time"handling"stressful" situations."Clinical"signs"are"nonYspecific,"and"mimic"these"of"gastrointestinal"disease,"renal" ! 39" failure"or"neurological"disease,"which"makes"finding"a"diagnosis"difficult."Clinical"signs"may"also" only"appear"episodically"or"so"weakly"that"they"remain"unnoticed"in"25"%"to"43"%"of"cases 5,99,111 might"therefore"take"up"to"52"weeks"before"a"dog"is"diagnosed"correctly 5,99 ."It" ."Some"dogs" 5 become"sick"acutely"and"very"severely ."Such"an"acute"phase"can"be"triggered"by"stress," 5,11 including"boarding,"traveling,"grooming"or"changes"in"routine"or"habitat ." Mineralocorticoid"insufficiency"might"not"cause"clinical"signs"if"the"electrolyte"balance"is" not"disturbed."However,"if"hyperkalemia"and"hyponatremia"occur,"above"mentioned"clinical" signs"worsen"and"cardiac"abnormalities"occur"due"to"hyperkalemia."Hyperkalemia"causes" 5,8,9,13 decreased"myocardial"excitability,"slowed"conduction"and"finally"cardiac"arrest ." Hyponatremia"causes"hypovolemia"and"hypotension"as"well"as"reduces"cardiac"output." Hypoperfusion"exacerbates"hyperkalemia"and"leads"to"many"other"clinical"features"including" elevation"of"liver"and"kidney"enzymes,"azotemia,"low"urine"specific"gravity,"shaking,"trembling" and"muscle"weakness." " Diagnosis"of"adrenal"insufficiency" " Diagnosing"hypoadrenocorticism"in"dogs"can"be"difficult,"because"its"clinical"signs"are" 99 nonspecific"and"can"mimic"those"of"other"conditions ."Upon"physical"examination"by"a"trained" veterinarian,"dogs"present"with"lethargy"and"depression"(87"%),"a"thin"physique"(82"%)," weakness"(66"%"to"69"%),"dehydration"(42"%),"shock"and"collapse"(24"%"to"29"%),"hypothermia" ! 40" (15"%"to"34"%),"bradycardia"(22"%"to"25"%),"weak"pulse"(22"%),"melena"and"hematochezia""""""" (17"%),"and"abdominal"pain"(7"%) 5,11,99,104,119,120 ."More"than"oneYthird"of"affected"dogs"are" presented"in"a"lifeYthreatening"condition"because"the"disease"goes"unrecognized"until"there"is" 97,107 an"acute"adrenal"crisis ."Emergency"treatment"in"these"situations"is"intensive,"expensive" 97 and"timeYconsuming ."Other"diagnostic"procedures"that"should"be"performed"include" 5 laboratory"tests,"diagnostic"imaging"and"an"electrocardiogram ."" Up"to"95"%"of"dogs"with"primary"hypoadrenocorticism"are"hyperkalemic,"up"to"86"%"are" hyponatremic,"40"%"are"hypochloremic"and"up"to"95"%"show"a"decreased"sodium"to"potassium" 5,111,119 ratio"of"less"than"27"to"1 ."These"changes"can"happen"isolated"or"together,"are"not" pathognomonic"for"the"disease,"but"the"clinician"should"be"suspicious"of"primary"Addison’s" disease"if"present."If"changes"are"absent"however,"primary"hypoadrenocorticism"still"remains"a" possibility."Sometimes"the"sodium"to"potassium"ratio"drops"below"15"to"1,"which"should" increase"the"suspicion"of"hypoadrenocorticism."Hyperkalemia"might"lead"to"cardiac"arrest,"first" decreasing"the"myocardial"excitability"and"slowing"conduction"and"then,"if"more"severe,"finally" causing"the"heart"to"stop 5,8,9,13 .""Differential"diagnoses"for"these"electrolyte"changes"include" gastrointestinal"disease"(vomiting,"diarrhea,"duodenal"perforation,"gastric"torsion),"urinary"tract" disease"(renal"failure,"postYobstructive"diuresis),"liver"failure,"parasitic"infection" (pseudohypoadrenocorticism"due"to"whipworms),"congestive"heart"failure,"chylothorax," metabolic"acidosis"(diabetic"ketoacidosis,"respiratory"acidosis)"or"pregnancy."Electrolyte" ! 41" changes"could"also"be"a"laboratory"artifact"related"to"suboptimal"sample"quality"(lipemia," hemolysis,"thrombocytosis"and/or"leukocytosis) 5,10,121 ."" SixtyYsix"%"to"95"%"of"dogs"with"primary"hypoadrenocorticism"have"preYrenal"azotemia," 5,8,9,104,111,119,120 caused"by"a"decrease"in"renal"perfusion"and"glomerular"filtration"rate ."Even" though"preYrenal"azotemia"usually"causes"an"increase"in"urinary"specific"gravity,"60"%"to"88"%"of" dogs"with"primary"Addison’s"disease"show"a"decrease"in"urinary"specific"gravity,"most"likely" caused"by"chronic"urinary"sodium"loss,"which"consequently"leads"to"renal"medullary"washout," 5,8,9,104,111,119 loss"of"the"normal"medullary"concentration"gradient"and"hence"a"dilute"urine ." This"decrease"and"the"most"likely"presence"of"elevation"of"BUN"and"creatinine"due"to" gastrointestinal"bleeding,"renal"hypoperfusion"or"hypovolemia,"usually"respond"well"to"fluid" therapy"(if"adequate),"whereas"dogs"with"renal"failure"do"not."SixtyYsix"%"to"95"%"are" hyperphosphatemic,"most"likely"a"result"from"the"decreased"glomerular"filtration"rate"and" 119,120 decreased"renal"excretion"of"phosphorus ."" Approximately"50"%"of"dogs"develop"metabolic"acidosis,"because"of"the"inability"of"the" kidneys"to"excrete"hydrogen"ions"without"the"presence"of"aldosterone."Hypovolemia," hypotension"and"hypoperfusion"further"exacerbate"the"metabolic"acidosis,"which"ranges"from" mild"to"severe"(in"less"than"10"%) 5,6,8,9,111,120 ."The"metabolic"acidosis"causes"potassium"to" move"from"the"intracellular"space"into"the"extracellular"space"in"exchange"for"hydrogen"ions." This"then"causes"the"hyperkalemia"to"worsen ! 5,8,9,13 42" ."" Elevation"of"total"calcium"occurs"in"up"to"30"%"of"dogs"with"primary" hypoadrenocorticism,"even"though"ionized"calcium"might"remain"within"the"normal"range." Most"authors"hypothesize"that"many"factors"contribute"to"the"development"of"hypercalcemia," including"hemoconcentration,"decreased"glomerular"filtration"rate,"decreased"calcium" secretion,"development"of"hyperkalemia"and"dehydration."Hypercalcemia"in"an"Addisonian"dog" usually"does"not"cause"clinical"signs"and"resolves"quickly"with"adequate"rehydration"and" glucocorticoid"replacement"therapy."Special"therapy"to"treat"hypercalcemia"is"not" 5,104,119,120 required ."Differential"diagnoses"for"hypercalcemia"include"neoplasia,"primary" hyperparathyroidism,"vitamin"D"toxicity"and"granulomatous"disease"processes 5,104,119 ." Hypoglycemia"develops"in"22"%"of"dogs"with"primary"hypoadrenocorticism"and"is"related" to"the"lack"of"glucocorticoidYmediated"gluconeogenesis"and"mobilization"of"glycogen"stores." Also,"cortisol"antagonizes"the"action"of"insulin,"and"the"decreased"cortisol"concentration"further" intensifies"hypoglycemia."Clinical"signs"caused"by"hypoglycemia"however"remain"very" uncommon."Differential"diagnoses"causing"hypoglycemia"include"sepsis,"liver"disease,"starvation" or"maldigestion,"hunting"dog"hypoglycemia,"insulinomas,"or"excessive"use"of"exogenous"insulin." Improper"samples"or"sample"handling"can"also"cause"an"artificially"decreased"glucose" concentration 5,104,111,120 ." Seventeen"%"to"39"%"of"affected"dogs"develop"moderate"to"severe"hypoalbuminemia." Hypoalbuminemia"in"dogs"with"hypoadrenocorticism"is"caused"by"anorexia,"gastrointestinal"loss" ! 43" and"decreased"synthesis."Differential"diagnoses"include"liver"disease,"gastrointestinal"disease"or" 5,104,119 kidney"disease ."" Alanine"aminotransferase"and"aspartate"aminotransferase"are"mildly"to"moderately" elevated"in"30"%"to"50"%"of"patients."It"is"not"yet"understood"what"causes"this"elevation,"but" contributing"factors"include"poor"cardiac"output,"hypotension,"poor"perfusion"and"concurrent" 5,104,111,119,120 immuneYmediated"hepatopathy ."Eighteen"%"of"dogs"with"primary" 5 hypoadrenocorticism"present"with"hypocholesterolemia ."" The"lack"of"glucocorticoids"causes"boneYmarrow"suppression,"which"may"result"in"mild" anemia."Gastrointestinal"bleeding"can"exacerbate"the"anemia,"and"mild"to"moderate" normocytic,"normochromic,"nonregenerative"anemia"is"present"in"21"%"to"25"%"of"patients."The" anemia"might"be"masked"by"hemoconcentration 5,111,119,122 ."" The"white"blood"cell"count"might"range"from"lowYnormal"to"mildly"increased,"related"to" concurrent"infection."Ten"%"to"20"%"of"dogs"with"primary"hypoadrenocorticism"are"eosinophilic," 5,104,111,119,120 and"absolute"lymphocytosis"exists"in"10"%"to"13"%"of"patients ."" The"use"of"diagnostic"imaging"or"measurement"of"blood"pressure"in"dogs"with"primary" hypoadrenocorticism"is"of"questionable"merit"for"routine"diagnosis."Hypotension"most"likely"is" 5 present"in"the"dog"with"hypoadrenocorticism,"but"there"is"insufficient"data"in"the"literature ." Diagnostic"imaging"is"usually"included"in"the"workYup"of"a"critically"ill"patient"and"might"reveal" changes"that"are"related"to"hypovolemia,"including"microcardia,"reduced"vena"cava"size," ! 44" pulmonary"hypoperfusion,"and"microhepatica."Megaesophagus"has"been"reported"as"a" 5,99,104,111,119 concurrent"disease"in"dogs"with"hypoadrenocorticism ."A"recent"study"compared" adrenal"gland"size"measured"via"ultrasound."Enrolled"were"14"healthy"dogs"and"28"dogs"with" hypoadrenocorticism."Adrenal"glands"are"significantly"smaller"in"dogs"with"Addison’s"disease" that"also"show"mineralocorticoid"insufficiency,"compared"to"healthy"dogs."The"difference"in"the" comparison"of"healthy"dogs"with"dogs"with"hypoadrenocorticism"without"mineralocorticoid" 123 insufficiency"approaches"significance ."" Changes"seen"on"electrocardiogram"(ECG)"are"related"to"hyperkalemia,"and,"even" though"not"completely"correlated"to"rising"potassium"levels,"get"more"significant"when"levels" increase."Performing"an"ECG"enables"the"clinician"to"initiate"immediate"and"potentially" lifesaving"therapy."Continuous"monitoring"helps"assessing"the"response"to"therapy."Changes" seen"on"an"ECG"result"in"changes"in"cell"membrane"excitability"and"depress"the"heart’s" conduction"system"due"to"hyperkalemia,"and"might"also"be"affected"by"hyponatremia"and" 124,125 metabolic"acidosis ."Bradyarrhythmias,"tachyarrhythmias,"conduction"disturbances," sinoventricular"rhythm,"ventricular"fibrillation,"and"asystoles"can"be"identified"in"46"%"of"dogs" 124 with"primary"Addison’s"disease ."In"a"different"study,"47"%"of"dogs"showed"atrial"standstill," nd rd 29%"bradycardia,"6%"atrial"or"ventricular"extrasystoles,"and"5"%"a"2 "or"3 "degree"heart" 99 block ." ! 45" It"is"not"sufficient"to"measure"baseline"cortisol"levels"alone"to"diagnose"Addison’s" disease,"as"dogs"with"nonYadrenal"illness"sometimes"have"very"low"baseline"cortisol" concentrations,"which"could"result"in"a"misdiagnosis."The"negative"predictive"value"is"high" though,"in"cases"where"the"baseline"cortisol"concentration"is"≥"55.2"nmol/"L"in"dogs"that"have" not"received"exogenous"steroids,"mitotane,"ketoconazole"or"trilostane,"and"the"dog"is"unlikely" 126 to"have"hypoadrenocorticism ."Baseline"cortisol"measurement"alone"has"100"%"sensitivity" and"98"%"specificity"for"hypoadrenocorticism"if"the"level"is"≤"27.6"nmol/"L"and"100"%"sensitivity" but"only"78"%"specificity"if"the"level"is"≤"55.2"nmol/"L 126 ."An"ACTH"stimulation"test,"should" always"be"performed"when"baseline"cortisol"concentrations"are"between"27.6"nmol/"L"and"55.2" nmol/"L"or"in"case"the"baseline"cortisol"concentration"is"≥"55.2nmol/"L,"but"the"primary" 126 differential"diagnosis"is"hypoadrenocorticism ."" Since"all"of"the"aboveYmentioned"laboratory"abnormalities,"and"most"of"the"clinical" signs,"are"nonYspecific"or"not"precise"enough,"the"gold"standard"test,"the"ACTH"stimulation"test," has"to"be"performed"whenever"primary"hypoadrenocorticism"is"suspected"in"order"to"confirm" 5 or"to"rule"out"the"diagnosis ."The"ACTH"stimulation"test"is"easy"to"perform,"safe,"rapid,"reliable," and"is"usually"easy"to"interpret"as"long"as"prior"administration"of"chronic"exogenous"steroids" can"be"excluded"(suppression"of"pituitaryYadrenal"axis)"as"well"as"prior"administration"of" prednisone,"prednisolone"or"hydrocortisone,"which"crossYreact"in"immunoassays 5,104,127 ."The" principle"of"the"ACTH"stimulation"test"is"as"follows:"synthetic"ACTH,"which"contains"the"active" portion"of"the"hormone"(amino"acids"1"to"24),"or"a"gel"preparation"from"porcine"pituitary" ! 46" extract"which"contains"the"39Y"amino"acid"fullYlength"hormone,"are"administered"after"a" baseline"blood"sample"has"been"taken 128 ."A"oneYhour"post"stimulation"sample"(synthetic"ACTH)" or"a"twoYhour"post"stimulation"sample"(ACTH"gel)"is"then"obtained."The"administered"ACTH,"like" the"endogenous"ACTH"from"the"pituitary"gland,"stimulates"the"zona"fasciculata"and"zona" reticularis"to"produce"cortisol,"and"the"zona"glomerulosa"to"produce"aldosterone."The" stimulation"test"therefore"assesses"the"ability"of"the"adrenal"cortices"to"respond"to"maximum" stimulation"and"therefore"gives"information"on"how"much"responsive"adrenal"cortex"is"present." Cortisol"is"measured"in"both"samples."The"additional"measurement"of"aldosterone"might"help"to" differentiate"between"primary,"secondary"and"tertiary"hypoadrenocorticism,"but"might"not" yield"additional"information"in"a"dog"with"primary"hypoadrenocorticism"and"the"assay"is"not" readily"available."If"a"dog"with"primary"Addison’s"disease"does"not"have"any"functional"adrenal" cortex,"no"stimulation"of"either"cortisol"or"aldosterone"postYACTH"administration"can"be"seen."A" typical"dog"with"primary"hypoadrenocorticism"would"have"pre"and"post"cortisol"results"of"≤55.2" nmol/"L"(normal"range"preYACTH"13.8"to"137.9"nmol/"L,"post"ACTH"151.8"to"469""""nmol/" L) 5,111,126,128,129 ."" To"perform"an"ACTH"stimulation"test,"either"synthetic"ACTH"or"ACTH"gel"are" recommended"and"yield"very"similar"results."It"is"recommend"to"inject"2.2U/"kg"ACTH"gel" 5,128 intramuscularly,"with"the"post"sample"taken"2"hours"post"injection ."ACTH"gel"needs"to"be" kept"refrigerated"at"all"times."The"use"of"compounded"ACTH"gel"is"not"recommended"because" 128,130 of"concerns"with"consistent"bioavailability ! ."There"are"two"protocols"available"on"how" 47" much"synthetic"ACTH"is"necessary"to"cause"maximum"stimulation."The"classic"test"uses"one"vial" of"synthetic"ACTH"(cosyntropin,"Cortrosyn;"Amphastar"Pharmaceuticals,"Rancho"Cucamonga," California,"USA)"(250"μg),"given"intravenously"or"intramuscularly"with"the"post"stimulation" sample"taken"one"hour"later."If"an"animal"is"severely"dehydrated,"absorption"of"intramuscularly" given"ACTH"might"be"prolonged,"and"rather"the"intravenous"route"should"be"used" instead 99,119,131 ."The"alternative"protocol"recommends"the"use"of"5"μg/"kg"given" 5,131Y134 intravenously,"with"a"maximum"dose"of"250"μg ."A"prospective"crossover"study" comparing"the"two"dosages"in"dogs"with"clinical"signs"of"Addison’s"disease"did"not"reveal" 133 statistically"significant"differences"between"the"two"dosages ."The"clinician"should"therefore" be"encouraged"to"use"the"lower"dosage"since"synthetic"ACTH"is"extremely"expensive"and" sometimes"hard"to"come"by."An"open"vial"of"synthetic"ACTH"remains"effective"when" refrigerated"for"21"days"or"up"to"six"months"when"kept"at"Y20°C"in"a"non"selfYdefrosting" 128,135,136 freezer " ."" " Treatment"of"adrenal"insufficiency" " As"in"human"medicine,"treating"a"dog"with"acute"primary"Addison’s"disease"is"different" from"treating"a"dog"with"chronic"adrenal"insufficiency."" " The"acute"patient"is"usually"presented"in"an"emergency,"with"severe"dehydration," hypovolemia,"hypotension,"electrolyte"abnormalities"including"hyperkalemia"and"hypoglycemia," ! 48" and"metabolic"acidosis."Counteracting"these"changes"immediately"on"top"of"sufficient" 5,111,137 corticosteroid"replacement"therapy"is"important " ."" An"animal"in"an"Addisonian"crisis"requires"aggressive"intravenous"fluid"replacement" therapy"to"correct"the"hypovolemia,"which,"if"uncorrected,"is"the"main"cause"of"death"in"an" Addisonian"crisis."It"has"been"shown"that"intraperitoneal"or"subcutaneous"fluid"therapy"is"not" adequate"in"such"a"situation."A"largerYgauge,"short"catheter"in"a"peripheral"vein"can"be"placed" quickly"and"allows"for"infusion"of"larger"volumes"than"a"jugular"catheter."After"placing"the" catheter,"if"necessary"via"venous"cutYdown,"a"baseline"blood"sample"should"be"collected"for" diagnostic"purposes"to"rule"in"or"out"the"diagnosis,"before"starting"fluid"therapy"at"a"shockYrate" of"60"to"90"mL/"kg/"h"for"one"to"two"hours"with"0.9"%"sodium"chloride"solution."Sodium"chloride" solution"has"the"benefit"of"replacing"the"sodium"loss,"reducing"the"hyperkalemia"and"bettering" the"metabolic"acidosis"without"providing"more"potassium."If"necessary,"lactated"Ringer’s"or" NormosolYR"can"be"used,"as"using"these"fluids"is"still"far"better"than"not"giving"fluids"at" all 5,111,137 ."By"giving"fluids,"hyperkalemia"usually"improves"because"of"hemodilution,"increased" renal"perfusion,"and"in"turn"increased"urine"output,"and"by"shifting"potassium"from"the" 5,137 extracellular"space"to"the"intracellular"space"as"metabolic"acidosis"gets"better ."If"lifeY threatening"bradyarrhythmia"is"present,"additional"therapy"is"indicated"to"counteract" hyperkalemia"immediately."0.2"Units/"kg"of"regular"insulin,"given"intravenously,"rapidly"lowers" serum"potassium"levels"by"driving"potassium"into"the"cells."The"effect"however"only"lasts"for"up" to"30"minutes."Dextrose"should"be"given"at"the"same"time"and"blood"glucose"levels"have"to"be" monitored"carefullyY"ideally"every"30"to"60"minutesY"to"avoid"hypoglycemia."Another"option"to" ! 49" decrease"hyperkalemia"is"to"use"bicarbonate."Bicarbonate"does"not"take"effect"for"an"hour"after" administration,"but"lasts"for"several"hours."1"to"2"mEq/kg"is"given"slowly"intravenously." 111,137 Hydrogen"leaves"the"cells"and"potassium"is"thereby"driven"into"the"cells ."Bicarbonate"can" also"be"used"to"correct"metabolic"acidosis,"especially"if"the"acidosis"does"not"resolve"with"fluid" therapy"alone."The"goal"when"using"bicarbonate"to"counteract"and"correct"acidosis"is"not"to" completely"correct"the"acidosis,"but"rather"to"increase"pH"to"7.2"and"the"HCO3"to"12"""""""""" mmol/"L 137 ."The"formula"0.3"*"body"weight"(kg)"*"(24Ypatient’s"HCO3)"can"be"used"to"estimate" 137 the"bicarbonate"deficit ."Calcium"gluconate"also"decreases"hyperkalemia:"0.5"to"1"mL/"kg"or"2" to"10"mL/"dog"of"a"10"%"calcium"gluconate"solution"given"as"a"slow"infusion"over"15"minutes" antagonizes"the"effects"of"hyperkalemia"on"cell"membranes,"therefore"allowing"normal"action" potentials."The"effect"is"very"sudden,"but"also"does"not"last"for"more"than"30"minutes."Further," it"is"recommended"to"monitor"the"ECG"while"giving"the"calcium"gluconate"infusion."If" bradycardia"worsens,"SYT"segment"elevates,"or"QYT"shortens,"the"infusion"should"be"stopped" immediately."Clinicians"usually"prefer"the"use"of"insulin"or"calcium"gluconate"if"fluid"therapy"is" not"enough"to"lower"potassium"concentrations."While"giving"these,"fluid"therapy"has"to"be" continued 136,137 ."" Dextrose"should"be"used"to"treat"hypoglycemia."Either"a"bolus"of"0.5"mL"to"1"mL"of"50%" dextrose"(given"diluted"to"prevent"phlebitis)"if"clinical"signs"of"hypoglycemia"exist"or"2.5"%"to""""" 5"%"added"to"the"fluids"if"no"clinical"signs"of"hypoglycemia"exist,"should"be"given ! 50" 137 ."" The"second"most"important"treatment"besides"fluid"therapy"includes"glucocorticoid" supplementation."Recommended"products"are"0.5"to"4"mg/"kg"of"rapid"acting"dexamethasone" sodium"phosphate"given"intravenously,"15"to"20"mg/"kg"of"prednisolone"sodium"succinate"(over" 3"minutes)"given"intravenously,"or"5"mg/"kg"bolus"(over"5"minutes,"followed"by"1"mg/"kg"every"6" hours)"or"0.3mg/"kg/"h"intravenously"given"hydrocortisone"sodium"succinate."Glucocorticoids" should"be"given"repeatedly"every"2"to"6"hours,"which"approximates"3"to"10"times"the" physiological"requirements."Prednisolone"and"hydrocortisone"interfere"with"cortisolYimmunoY assays"via"crossYreaction"with"the"assay"antibodies."ACTH"stimulation"tests"should"therefore"be" completed"prior"to"administration"or"dexamethasone"has"to"be"used,"which"does"not"crossY react"in"the"cortisol"assays."Hydrocortisone"also"has"some"mineralocorticoid"activity."The"goal"is" to"improve"vascular"and"GI"(gastroYintestinal)"integrity"and"to"help"improve"blood"pressure"and" 137 circulation"volume " ." Supplementation"with"mineralocorticoids"during"an"acute"crisis"is"of"lesser" benefit 5,104,111,119,120 ."The"two"mineralocorticoid"products"currently"on"the"market"are"the" long"acting,"injectable"desoxycorticosterone"pivalate"and"the"short"acting"oral"fludrocortisone." In"the"acutely"vomiting"patient,"oral"medication"might"not"get"absorbed"properly."Once"DOCP"is" injected,"it"lasts"approximately"25"days."However,"DOCP"is"a"very"safe"drug"and"appears"to"not" have"any"side"effects,"even"if"administered"to"a"completely"healthy"animal."Each"individual" clinician"should"therefore"decide"on"his"or"her"own"on"whether"to"administer"it"during"acute" 5,119,136,138,139 crisis"or"not ! ."" 51" " Fluid"therapy"and"glucocorticoid"therapy"are"most"important"in"the"treatment"of"an" acute"Addisonian"crisis."Other"symptoms"that"should"be"addressed"(treatment"suggestions"in" parentheses),"depending"on"severity,"include"anemia"due"to"ulceration"(transfusion),"GI" ulceration"(GI"protectants"including"sucralfate,"HY2"blockers,"protonYpump"inhibitors,"broad" spectrum"antibiotics"in"case"of"sepsis),"and"vomiting"(antiemeticsY"prochlorperazine"is" 5 contraindicated"though) ."These"treatment"suggestions"are"guidelines"and"need"to"be"adjusted" depending"on"treatment"response"and"results"during"hourly"monitoring"and"they"should"be" continued"as"long"as"necessary."Once"hyperkalemia"reaches"the"nonYlifeYthreatening"level"of"<" 6.5"mmol/L,"electrolyte"and"venous"gas"do"not"have"to"be"rechecked"hourly,"but"rather"every"6" to"8"hours."Care"should"also"be"taken"that"sodium"concentrations"do"not"rise"more"than"12" mmol/L"per"day,"as"a"faster"rise"could"lead"to"pontine"myelinolysis,"which"is"a"nonYinflammatory" neurological"disorder"with"clinical"signs"including"lethargy,"weakness,"dysphagia,"trismus,"and" ataxia"progressing"to"hypermetria"and"spastic"quadriparesis."This"syndrome"and"its"signs"are" 137,138,140Y142 rare"in"veterinary"medicine,"but"if"present,"they"could"take"months"to"resolve ." Most"patients"are"completely"stable"within"24"to"48"hours"and"should"be"weaned"off" emergency"treatment,"switched"over"to"chronic"treatment"as"described"in"the"following" 129 paragraph"and"monitored"for"reoccurrence"of"clinical"signs"prior"to"discharge " ."" A"dog"with"primary"Addison’s"disease"needs"to"be"supplemented"with"glucocorticoids" and"mineralocorticoids"for"the"rest"of"its"life."Oral"prednisone"should"be"initially"given"at"a" physiologic"dosage"of"0.22"mg/"kg"twice"daily"and"then"tapered"down"to"the"lowest"dose" necessary"to"maintain"wellYbeing"of"the"patient"without"seeing"unwanted"side"effects"such"as" ! 52" polyuria,"polydipsia,"panting,"and"polyphagia."Just"as"a"human"patient"would"be"instructed"to" do,"the"owner"of"a"dog"with"hypoadrenocorticism"should"be"instructed"to"increase"the"dose"by" 2"to"10"times"the"physiologic"dose"whenever"a"stressful"situation"occurs"or"is"foreseen."The" dose"should"only"be"adjusted"every"6"weeks"to"see"how"a"patient"is"doing"on"a"lower"dose" 5 before"attempting"to"decrease"it"further ."" " For"mineralocorticoid"supplementation,"there"are"currently"two"choices"available,"DOCP" and"fludrocortisone"acetate,"as"mentioned"above."DOCP"is"a"longYacting"preparation."The"short" acting"DOCA"(desoxycorticosterone"acetate)"was"developed"in"the"1940’s,"but"is"no"longer" available."DOCP"is"the"long"acting"ester"of"DOCA"and"DOCP"has"no"glucocorticoid"activity."For" veterinary"use,"it"is"formulated"in"a"microcrystalline"suspension,"which"has"to"be"given" intramuscularly"and"lasts"for"25"days."The"initial"dose"is"2.2"mg/"kg."It"starts"working"within"1"h" post"injection."It"is"a"very"safe"drug"and"very"well"tolerated."However,"DOCP"is"not"inexpensive," therefore"there"are"many"prospective"studies"on"what"is"the"most"economical"dosage"to"use" and"what"might"be"more"costYeffective,"for"example"giving"a"higher"than"recommended"dose" and"then"going"for"a"longer"interval"or"the"other"way"around."Clinicians"at"this"point"remain" uncertain."The"manufacturer"recommended"protocol"is"as"follows:"injecting"2.2"mg/"kg" intramuscularly"on"day"0,"then"recheck"(electrolytes"and"physical"examination)"at"day"12"and" day"25."The"peak"effect"of"DOCP"is"at"day"12."If"hyperkalemia"and/"or"hyponatremia"exist"at" recheck"day"12,"the"DOCP"dose"should"be"increased"by"5"to"10"%,"or"decreased"by"5"to"10"%"in" case"of"hypokalemia"and/"or"hypernatremia."The"frequency"of"injection"should"be"shortened"by" 5,119,136 one"day"or"prolonged"by"one"day"if"these"changes"occur"during"recheck"at"day"25 ! 53" ."The" second"mineralocorticoid"choice"for"managing"a"dog"with"primary"hypoadrenocorticism"is"oral" fludrocortisone"acetate,"which"is"a"synthetic"glucocorticoid"with"predominantly" mineralocorticoid"activity."The"starting"dose"is"0.01"mg/"kg"given"twice"daily;"however,"this"dose" usually"needs"to"be"increased"within"the"first"year"of"treatment."Treatment"goal"is"to"maintain" normal"electrolyte"concentrations."Since"fludrocortisone"acetate"does"exert"glucocorticoid" activity,"up"to"50"%"of"dogs"do"not"require"additional"prednisone"supplementation."However," the"fludrocortisone"dose"that"is"necessary"to"maintain"normal"electrolyte"concentrations"is" often"times"so"high"that"many"dogs"start"showing"signs"of"hypercortisolism."Finding"the"right" dosage"with"this"drug"is"therefore"very"difficult,"and"it"should"be"recommended"to"switch"these" patients"to"DOCP."Even"though"a"diet"rich"in"salt"might"be"beneficial"for"humans,"the"feeding"of" table"salt"to"these"dogs"remains"controversial " 5,119 ."" In"my"opinion,"it"is"necessary"to"teach"the"owner"of"a"dog"diagnosed"with"primary" hypoadrenocorticism"about"the"disease,"and"that"certain"clinical"signs"have"a"huge"impact"on" how"to"adjust"the"glucocorticoid"and"mineralocorticoid"dose."In"brief,"vomiting,"diarrhea," lethargy,"or"inappetence"suggest"that"the"glucocorticoid"dose"needs"to"be"increased."Polyuria" and"polydipsia"suggests"that"the"glucocorticoid"dose"needs"to"be"decreased."However,"the" dosage"of"mineralocorticoids"should"not"be"changed"without"the"evaluation"of"serum" electrolytes."" " " ! 54" LongYterm"outcome"of"adrenal"insufficiency" " Kintzer"reports"that"more"than"80"%"of"dogs"have"a"good"to"excellent"response"to" 106 therapy"and"a"fair"response"to"treatment"is"seen"in"13"% was"4.7"years 106 ."Median"survival"time"of"these"dogs" ."Kelch"estimates"a"comparable"4.9"years"as"average"disease"duration 103 ."There" appears"to"be"no"difference"in"survival"time"between"dogs"treated"with"fludrocortisone"versus" DOCP."Of"the"dogs"that"died"during"the"study,"97%"died"of"reasons"other"than" 103 hypoadrenocorticism ."" " Addison’s"disease"in"human"and"veterinary"medicine" Overall,"it"is"apparent"that"there"are"many"similarities"between"hypoadrenocorticism"in" dogs"and"humans."The"major"clinical"signs"and"laboratory"changes"of"hypoadrenocorticism"in" dogs"and"humans"are"similar."Females"are"more"affected"than"males"(average"of"all"cited" studies:"dogs:"64"%"to"36"%;"humans"53"%"to"47"%),"and"in"both"species"it"is"a"disease"of"the" younger"to"middle"aged"individual"(dogs:"4.7"years"(range"0.1"to"15"years);"humans:"36"years" (range"5"to"79"years))."" Incidences"and"prevalences"are"higher"in"dogs,"which"might"be"related"to"the"smaller" genetic"pool"in"dogs"and"degree"of"inbreeding"(dogs:"13"to"60/"100,000"dogs/"year"incidence," ! 55" 600"to"3200/"1,000,000"dogs"prevalence;"humans:"0.5"to"0.83/"100,000"people/"year"incidence," 5"to"140/"1,000,000"people"prevalence)."" The"main"difference,"the"unknown"etiology"of"Addison’s"disease"in"dogs,"versus" immuneYmediated"in"humans,"is"the"basis"for"the"work"in"this"dissertation."The"autoimmune" etiology"of"primary"Addison’s"disease"in"dogs"has"not"been"well"defined,"and"up"to"date,"most" cases"are"still"classified"as"idiopathic."" " " ! 56" APPENDIX ! 57" Appendix" " Figure"I.1:""Schematic"showing"the"cellular"zonation"of"the"adrenal"cortex"and"blood"flow" through"the"cortex"to"the"collecting"veins"in"the"medulla"(retrieved"on"07/02/2011"from" http://www.hakeemYsy.com/main/files/adrenal.jpg)."(For"interpretation"of"the"references"to" color"in"this"and"all"other"figures,"the"reader"is"referred"to"the"electronic"version"of"this" dissertation)."" " " ! " 58" Figure"I.2:"Steroidogenesis"(from:"Annual"Rev"Physiology"(2001)"63:193);"Cholesterol"is" either"obtained"from"the"diet"or"synthesized"from"acetate"by"a"CoA"reductase"enzyme."The" adrenal"cortex"(also"ovaries"and"testes)"use"cholesterol"to"produce"a"range"of"steroid" hormones,"including"aldosterone,"cortisol"and"testosterone/estradiol." " ! 59" Figure"I.3:"The"hypothalamicYpituitaryYadrenal"axis"(from:"Addison’s"Disease"in"the"Dog," Catherine"ScottYMoncrieff,"05/15/2011,"retrieved"from:" http://www.vetgrad.co.uk/show10MinuteTopUp.php?type=&Entity=10MinuteTopUps&Entity= 10MinuteTopUps&ID=51"on"09/09/2011)." " " ! " 60" Table" I.1:" Classification" of" autoimmune" polyendocrine" syndrome" (APS)" in" humans" (adapted"from"Betterle"Endocr"Dev"2011;20:161Y72)." APS"type"1" Chronic"candidiasis"+"chronic"hypoparathyroidism"+"Addison’s"disease" APS"type"2" Addison’s"disease"+"thyroid"autoimmune"diseases"and/"or"type"1"diabetes" mellitus" APS"type"3" Thyroid"autoimmune"diseases"+"other"autoimmune"diseases"(excluding" Addison’s"disease)" APS"type"4" Combinations"of"two"or"more"autoimmune"diseases"not"falling"into"the" above"categories"(i.e."Addison’s"disease"and"autoimmune"gastritis,"or" vitiligo,"or"alopecia"or"celiac"disease)" " " ! " 61" Figure" I.4:" Main" features" of" autoimmune" Addison’s" disease" and" their" subtypes" in" humans"(adapted"from"Betterle"Endocr"Dev"2011;20:161Y72)." Autoimmune!Addison's!disease!(AD)! !501!cases!! Mean!age!31!years!! F/M!=!1.7/1!! Adults/children!=!5/1! ! APSC2! Isolated!AD! APSC1! APSC4! 310!cases!(62%)! 90!cases!(18%)! 65!cases!(13%)! 36!cases!(7.2%)! Mean!age!35!years! Mean!age!32!years! Mean!age!14!years! Mean!age!33!years! F/M!=!2.3/1! F/M!=!0.7/1! F/M!=1.9/1! F/M!=!0.9/1! Adults/children!=! Adults/children!=! Children/adults!=! Adults/children!=! 15/1! 4/1! 16/1! 8/1! ! " ! 62" Table"I.2:"Breeds"that"are"found"to"be"at"increased"risk"of"developing" hypoadrenocorticism"(CI=confidence"interval,"N/A=data"not"available)" Kelch Breed"of"dog" 103 Odds" ratio" 99 " Peterson " P"value" 95%"CI" Odds" ratio"" P"value" 95%"CI" Airedale"Terrier" 2.61" <.05" 1.06Y6.56" N/A" N/A" N/A" Basset"Hound" 3.38" <.001" 1.50Y7.79" 3.90" .060" 0.94Y12.22" Bearded"Collie" 4.19" <.01" 1.20Y18.36" N/A" N/A" N/A" 2.52" <.05" 1.06Y6.06" 3.90" .060" 0.94Y12.22" 7.63" <.0001" 2.50Y31.09" 11.98" <.001" 4.65Y29.94" 3.55" <.0001" 2.55Y4.93" N/A" N/A" N/A" N/A" N/A" N/A" 8.90" <.001" 4.43Y17.32" N/A" N/A" N/A" 46.66" .002" 3.73Y245.9" 1.25" >.05" 0.71Y2.21" 2.6" .014" 1.22Y5.05" German"Shorthaired" Pointer" Great"Dane" Poodle"(Standard," Miniature,"Toy)" Poodle"(Standard)" Portuguese"Water" Dog" Rottweiler" " " ! " 63" Table"I.2"(cont’d):" Springer"Spaniel" West"Highland" White"Terrier" Soft"Coated" Wheaten"Terrier" 2.54" <.01" 1.29Y5.04" 5.85" .051" 0.99Y24.56" 5.93" <.0001" 2.69Y13.46" 11.42" <.001" 4.69Y26.97" N/A" N/A" N/A" 6.68" .039" 1.11Y29.49" " " ! " 64" Table"I.3:"Breeds"that"are"found"to"be"at"decreased"risk"to"develop"hypoadrenocorticism" (CI=confidence"interval;"N/A=data"not"available)" Kelch Breed"of"dog" 103 Odds" ratio" 99 " Peterson " P"value" 95%"CI" Odds" ratio" P"value" 95%"CI" Boxer" .12" <.05" .00Y.77" N/A" N/A" N/A" Chihuahua" .83" >.05" .22Y2.67" .18" .058" .00Y1.04" Cocker"Spaniel" .51" <.01" .30Y.85" .31" .097" .04Y1.15" Dalmatian" .00" <.01" .00Y.50" N/A" N/A" N/A" Golden"Retriever" .77" >.05" .49Y1.21" .00" .001" .00Y.49" Labrador"Retriever" 1.00" >.05" .72Y1.38" N/A" N/A" N/A" Lhasa"Apso" .22" <.01" .04Y.75" .00" .036" .00Y.92" Mixed"Breed" .76" .072" .56Y1.04" N/A" N/A" N/A" Pit"Bull"Terrier" .00" <.05" .00Y.84" .35" .103" .00Y1.26" Pomeranian" .12" <.05" .00Y.77" N/A" N/A" N/A" .73" >.05" .33Y1.55" .00" .068" .00Y1.07" Schnauzer"(Giant," Standard,"Miniature)" " " ! " " 65" Table"I.3"(cont’d):" Shetland"Sheepdog" .15" <.001" .03Y.47" N/A" N/A" N/A" Shih"Tzu" .25" <.05" .05Y.83" N/A" N/A" N/A" Yorkshire"Terrier" .14" <.01" .02Y.58" .00" .003" .00Y.56" "" " " ! " 66" Table"I.4:"Mean"age"and"body"weight,"female"to"male"ratio"in"dogs"with" hypoadrenocorticism"(female"intact"(FI),"male"intact"(MI),"female"spayed"(FS),"male"neutered" (MN),"SD=standard"deviation,"SEM=standard"error"of"the"mean,"N/A=data"not"available)" " Mean"age"of" Kelch"(262) 103 (Fi:"mi:"fs:"mn)"" (SD,"SEM,"Range)" (SD,"SEM,"range)" 103 Mean"bodyweight"" onset"in"years" Kelch"(244) Female:"Male" [%]" [kg]" " 4.3"(N/A,"N/A,"0.2Y12)" 65:35"(17:22:48:13)" N/A" " 4.9"(3.0,"0.2,"0.3Y13)" 68:32"(6:13:62:19)" 23.4"(13.6,"0.8,"2.7Y 72.7)" Kelch"(376) 99 103 Peterson " Melián 107 Willard Lifton " 110 143 " " " 5.4"(3.2,"0.2,"0.1Y15)" 68:32"(14:13:54:19)" N/A" 4.0"(N/A,"N/A,"0.3Y14)" 71:29"(16:16:55:13)"" 20"(N/A,"N/A,"2.3Y64.5)" 4.5"(N/A,"N/A,"1Y12)" 67:33"(12:10:55:24)"" 10.2"(2.5Y46.1)" 4.2" 68:32" N/A" 6.0"(N/A,"N/A,"2.8Y11)" 56:44" 28.5"(3.2Y43)" 66:34"" 20.5"(10.2Y28.5)" Average"(Range)" 4.8"(4.0Y6.0)" " " " ! 67" Table"I.5:"Hypoadrenocorticism"and"age"(CI=confidence"interval)" 103 Odds"of"a"dog"developing"hypoadrenocorticism"based"on"age"(Kelch"study )" Age" Odds"ratio" P"value" 95%"CI" 0Y1" 0.12" <.0001" 0.06Y0.22" 1Y4" 1.01" >.05" 0.78Y1.29" 4Y7" 1.90" <.0001" 1.49Y2.42" 7Y10" 1.51" <.01" 1.16Y1.98" >10" 1.06" >.05" 0.78Y1.43" " " ! " 68" Table"I.6:"Hypoadrenocorticism"incidence"and"gender" 103 Estimated"incidences"by"gender"(Kelch"Study )" Gender" Estimated"cases/1000/year" All"dogs"of"all"genders" 0.6" Females" 0.7" Ovariohysterectomized"females" 1.0" Intact"females" 0.3" Males" 0.4" Castrated"males" 0.6" Intact"males" 0.2" " " ! " 69" Table"I.7:"Odds"ratios"for"hypoadrenocorticism"and"gender" Odds"of"a"dog"developing"hypoadrenocorticism"based"on"gender"" (Relative"to"intact"males)" 99 (Peterson"study )" Sex" Odds"ratio" P"value" 95%"CI" Ovariohysterectomized"females"" 1.44" 0.158" 0.88Y2.36" Castrated"males" 1.83" 0.026" 1.07Y3.11" Intact"females" 5.55" <0.001" 3.75Y8.37" " " ! " 70" REFERENCES ! 71" References" " 1." Addison"T."On"the"Constitutional"and"Local"Effects"of"Diseases"of"The"SupraYRenal" Capsules."London:&Samuel&Highley"1855." " 2." Jeffcoate"W."Thomas"Addison:"one"of"the"three""Giants""of"Guy's"Hospital."Lancet" 2005;365:1989Y1990." " 3." 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" " ! 84" Chapter#II:#Pathogenesis#of#autoimmune#diseases# # Introduction# Autoimmune#diseases#occur#when#an#organism#fails#to#recognize#its#own#cellular# components#as#self,#and#therefore#mounts#an#immune#response#against#its#own#cells#and# tissues.#Another,#more#precise#definition#defines#an#autoimmune#disease#as#a#“clinical# syndrome#caused#by#the#activation#of#T#cells#or#B#cells,#or#both,#in#the#absence#of#an#ongoing# 1 infection#or#other#discernible#cause” .#Autoimmune#diseases#affect#five#percent#of#the# population#in#Western#countries;#the#most#common#autoimmune#diseases#include#Celiac# disease,#diabetes#mellitus#type#1,#systemic#lupus#erythematosus,#Sjögren’s#syndrome,# Hashimoto’s#thyroiditis,#Graves’#disease,#idiopathic#thrombocytopenic#purpura,#rheumatoid# arthritis,#and#hypersensitivity.# # For#many#years,#it#was#believed#that#autoreactive#T#cells#and#B#cells#were#eliminated# during#clonal#deletion,#leaving#only#immune#cells#specific#for#foreign#antigen#recognition.#It#is# now#understood#and#accepted#that#a#low#level#of#autoreactivity#is#normal#and#physiologic#and# vital#for#normal#immune#function#and#to#enhance#an#immune#response#in#its#early#stage#when# 2 the#availability#of#foreign#antigen#is#limited .## Stefanova#et#al.#demonstrated#that#selfPMHC#identification#maintains#the# 3 responsiveness#of#CD4+#(cluster#of#differentiation#4)#T#cells#in#the#absence#of#antigens .#To# ! 85# prove#this,#an#antiPMHC#(major#histocompatibility#complex)#Class#II#antibody#was#injected#into# mice.#This#antibody#temporarily#prevented#CD4+#T#cellPMHC#interaction.#Harvested#naïve#CD4+# T#cells#from#these#mice#showed#decreased#responsiveness#to#the#presentation#of#an#antigen,# thus#they#were#able#to#show#that#selfPMHC#recognition#maintains#the#responsiveness#of#CD4+#T# 3 cells#when#foreign#antigen#is#not#present .#Further,#a#low#level#of#autoimmunity#might#help# CD8+#T#cells#in#the#recognition#of#cancerous#cells,#therefore#reducing#the#incidence#of#certain# cancers.## 1 Autoantigens#help#to#form#the#repertoire#of#mature#lymphocytes ,#and#B#and#T#cells#in# 4,5 the#periphery#need#constant#exposure#to#autoantigens#in#order#to#survive .#Some#have# speculated#that#lymphocytes#are#able#to#distinguish#self#from#nonPself.#However,#there#is#no# central#difference#in#foreign#antigens#and#self#antigens,#and#it#appears#that#the# microenvironment#is#important#for#development#of#an#immune#response,#and#that#this#usually# 6 only#occurs#in#the#presence#of#inflammatory#cytokines .#For#this#physiologic#autoreactivity# transition#to#a#pathologic#reaction,#additional#stimuli#are#necessary.#The#most#common#insults# include:#1)#loss#of#immunologic#tolerance,#2)#genetic#susceptibility,#3)#environmental#and# internal#triggers#and#changes#in#pathologic#processes#during#autoimmunePdisease#progression,# 1 and/#or#4)#different#mechanisms#of#tissue#injury .#The#fact#that#certain#insults#are#necessary,# and#a#genetic#predisposition#by#itself#does#not#automatically#result#in#the#development#of#an# autoimmune#disease,#is#clearly#illustrated#by#the#at#best#50#%#penetrance#of#autoimmune# diseases#in#identical#twins#raised#in#the#same#environment.#Certain#factors#are#necessary#that# ! 86# create#a#perfect#storm#of#events#leading#to#autoimmune#disease.#This#is#also#demonstrated#in# the#Venn#diagram#of#autoimmune#diseases,#as#further#discussed#at#the#end#of#this#review.#The# four#abovePmentioned#potential#mechanisms#that#contribute#to#the#development#of# autoimmune#diseases#are#discussed#in#the#following#paragraphs.#At#the#end,#I#will#also#briefly# discuss#the#function#of#the#relatively#recently#discovered#Th17#(T#helper#17)#cells.## # Immunologic#tolerance# # Most#autoimmune#diseases#are#related#to#the#loss#of#immunological#tolerance,#resulting# in#T#cells#and#antibodyPproducing#B#cells#that#recognize#selfPantigens#as#nonPself#or#foreign 7,8 .# Although#the#exact#mechanisms#of#immunologic#tolerance#remain#unclear,#different#but#not# mutually#exclusive#theories#have#been#proposed,#and#if#any#cells#escape#any#of#these#processes,# an#autoimmune#disease#may#be#triggered.#Below,#several#mechanisms#that#promote#or# facilitate#immunologic#tolerance#are#briefly#described.## # Clonal&deletion:#In#1960,#Frank#M.#Burnet#and#Peter#B.#Medaware#received#the#Nobel# Prize#in#Physiology#for#their#“discovery#of#acquired#immunological#tolerance”.#They#discovered# 9 that#selfPreactive#lymphoid#cells#are#destroyed#during#development#of#the#immunePsystem .# During#development,#T#and#B#lymphocytes#interact#with#self#antigens.#If#their#unique#antigen# receptor#recognizes#self#antigens,#these#cells#are#then#deleted#through#apoptosis#or# programmed#cell#death.#For#example,#T#cells#developed#in#the#thymus#originally#express#neither# CD4#nor#CD8.#Later,#they#acquire#both#CD4#and#CD8#and#express#low#levels#of#αβ#T!cell!receptor# ! 87# (TCR).#These#cells#are#positively#selected#after#interacting#with#class#I#or#class#II#MHC#molecules.# Cells#with#low#affinity#for#MHC#are#positively#selectedP#unselected#cells#die#by#apoptosis,#a# process#called#"death#by#neglect".#The#elected#T#cells#are#presented#with#selfPpeptides# presented#in#the#context#of#MHC#molecules#by#dendritic#cells.#Any#T#cells#with#high#affinity# receptors#for#MHC#+#selfPpeptide#undergo#clonal#deletion.#Any#TPcells#that#escape#this#process# may#cause#an#autoimmune#disease,#and#genetic#mutations#that#disrupt#T#cell#deletion#in#the# thymus#can#result#in#catastrophic#autoimmunity.#For#example,#a#genetic#mutation#in#the# autoimmune#regulator#(AIRE)#is#seen#in#patients#with#autoimmune#polyendocrinopathy# syndrome#type#1#(APSP1),#with#clinical#manifestation#of#hypoparathyroidism,#primary# adrenocortical#failure#and#chronic#mucocutaneous#candidiasis.#A#transgenic#mouse#knockPout# 10 has#been#created#to#study#the#exact#mechanisms .#AIRE,#a#human#transcription#factor#that#is# expressed#in#the#medulla#of#the#thymus,#is#important#in#the#inhibition#of#autoimmune#diseases.# AIRE#initiates#the#transcription#of#many#organ#specific#genes#in#the#thymus.#The#epitopes#of# proteins,#which#are#usually#only#expressed#in#the#periphery,#then#bind#to#T#cells.#The#‘autoP reactive’#T#cells#that#bind#these#proteins#are#then#eliminated#via#the#process#of#negative# selectionP#thereby#reducing#the#occurrence#of#autoimmunity#later#on.#By#this#mechanism,# maturing#thymocytes#become#tolerant#towards#peripheral#organs#or#selfPproteins.#One#of#these# proteins#for#example#is#insulin.#Its’#transcription#is#initiated#by#AIRE#in#the#thymus.#AIRE#is#also# 11,12 expressed#in#many#other#tissues .#During#BPcell#development,#a#similar#strategy#as# described#for#T#cells#is#used#to#eliminate#selfPreactive#B#cells.## Clonal&anergy:#Anergy#is#another#process#that#induces#tolerance,#modifying#the#immune# ! 88# system#to#prevent#selfPdestruction.#Clonal#anergy#was#first#described#by#Gustav#Nossal#for#B# lymphocytes#and#later#on,#in#very#similar#fashion,#by#Ronald#Schwartz#and#Marc#Jenkins#for#T# lymphocytes 13P19 .#Self#reactive#TP#or#BPcells#are#inactivated#and#therefore#cannot#increase#an# 16 immune#reponse .#If#an#antigenPpresenting#cell#does#not#have#coPstimulatory#molecules#CD80# or#CD86#when#presenting#an#antigen#to#a#T#cell,#the#T#cell#becomes#anergic#(nonresponsive).# Further,#when#TPcells#are#activated#through#CTLA4,#Interleukin#(IL)P2#production#is#inhibited#and# they#become#anergic.#When#B#cells#are#exposed#to#large#amounts#of#soluble#antigen,#surface# IgM#is#downPregulated,#surface#Fas#molecules#are#upPregulated#and#they#become#anergic.#FasP ligand#bearing#T#cells#interact#with#these,#resulting#in#their#death#via#apoptosis.#The#clinical# significance#of#clonal#anergy,#beyond#reducing#autoimmune#diseases,#is#seen#in#some#infectious# diseases,#including#HIV#(human#immunodeficiency#virus).#HIV#seems#to#utilize#the#immune# system’s#use#of#tolerance#induction#and#is#therefore#able#to#evade#the#immune#system.#Anergy# might#however#also#be#significant#for#therapeutic#uses,#for#example#after#organ#transplants#or# to#induce#activated#lymphocytes#to#become#unresponsive#with#autoimmune#diseases#like# diabetes#mellitus,#multiple#sclerosis,#and#rheumatoid#arthritis.## Idiotype&network&and&anti3idiotype&antibody:#During#a#process#called#tolerization,# antibodies#are#produced#against#specific#antibodyPidiotypes.#Such#an#antibody#might#prevent# the#B#cell#receptor#from#interacting#with#its#specific#antigen.#Naturally#existing#antibodies# 20,21 further#are#able#to#build#a#network#and#are#able#to#neutralize#selfPreactive#antibodies ! 89# .## Clonal&ignorance:#Host#immune#responses#ignore#selfPantigens.#T#cells#reactive#to#selfP antigen#mature#and#migrate#to#the#periphery,#where#they#might#never#meet#the#appropriate# antigen.#These#cells#might#then#die,#because#a#stimulus#is#missing.#BPcells#that#escape#deletion# might#never#meet#their#specific#antigen#or#the#specific#antigen#and#might#also#die#because#lack# 22 of#stimulus .## Peripheral&tolerance:#If#T#and#B#cells#do#not#undergo#clonal#deletion,#they#could#cause# an#autoimmune#response#once#they#reach#lymphoid#organs#in#the#periphery.#Therefore,#the# immune#system#has#additional#peripheral#checkpoints#to#delete#the#cells#that#did#not#undergo# 23 deletion .## Activation3induced&cell&death:#Besides#producing#cytokines#when#activated#with#an# antigen,#T#cells#also#express#FasL,#which#binds#to#Fas,#which#in#turn#triggers#apoptosis#via# activation#of#caspaseP8.#Mice#with#mutations#in#Fas#or#FasL#develop#severe#autoimmune# diseases#and#usually#die#within#the#next#6#months.#In#humans,#similar#mutations#cause#a# 24 lymphoproliferative#disease#called#autoimmune#lymphoproliferative#syndrome .# Regulatory&T&cells:#Normal#CD4+#T#cells#express#CD25,#but#regulatory#T#cells,#which#have# recently#been#discovered,#also#express#the#forkhead#family#transcription#factor#Foxp3#(forkhead# box#P3),#by#which#they#can#be#differentiated#from#normal#CD4+#T#cells.#Foxp3#is#required#for# regulatory#T#cell#development#and#function,#although#the#mechanism#by#which#regulatory#T# cells#suppress#other#T#cells#is#not#fully#understood.#It#has#been#suggested#that#one#mechanism# includes#the#production#of#cytokines#such#as#TGFPβ#(Transforming#growth#factor#beta)#and#ILP10,# ! 90# which#have#an#immunosuppressive#action.#The#fact#that#a#mutation#in#humans#in#Foxp3#causes# a#deadly#autoimmune#syndrome#(IPEX:#immunodysregulation#polyendocrinopathy#enteropathy# XPlinked#syndrome)#proves#that#regulatory#T#cells#play#an#important#role#in#preventing# 25,26 autoimmune#disease .## # Genetic#susceptibility# # The#deletion#or#overexpression#of#genes#predisposes#mice#to#autoimmune#diseases#and# more#than#25#genes#have#been#identified#so#far.#This#clearly#shows#the#effect#of#gene#mutations# on#development#of#autoimmune#diseases.#The#genes#that#were#modified#encode#for#cytokines,# antigenPcoreceptors,#elements#of#the#cytokineP#or#antigenPsignaling#cascade,#costimulatory# molecules#or#molecules#that#promote#apoptosis#or#inhibits#it,#and#molecules#that#clear#antigens# 1 or#antigenPantibody#complexes .#However,#in#these#experiments,#genetic#engineering#of#one# single#gene#did#not#automatically#cause#autoimmune#diseases,#but#it#rather#predisposed#the# animal#to#develop#one.#This#means#that#disease#development#overall#depends#on#the#total# genetic#background#of#the#host.#Further,#some#genetic#mutations#predisposed#the#mice#to# develop#more#than#one#autoimmune#disease,#showing#that#the#development#of#certain# 1 autoimmune#diseases#may#have#the#same#pathway .#Findings#in#human#medicine#confirmed# the#results#of#these#studies;#however,#it#was#shown#that#a#genetic#locus#is#more#likely#to#cause# an#increased#susceptibility#to#an#autoimmune#disease#rather#than#a#single#gene#mutation#alone,# ! 91# as#opposed#to#the#case#with#autoimmune#proliferative#syndrome#and#polyglandular# endocrinopathy.#Certain#loci#also#seem#to#be#responsible#for#causing#more#than#just#one# autoimmune#disease.#A#single#mutation#can#increase#susceptibility,#but#the#development#of# disease#phenotype#depends#on#other#genes.#Oftentimes#autoimmune#diseases#are#multigenic,# with#many#susceptibility#genes#working#together#to#cause#disease#phenotype.#The#genetic# background#also#influences#the#severity#of#disease#as#some#people#do#not#show#disease#and# have#a#normal#immune#function,#besides#carrying#the#polymorphism.#Some#genes#also#posses#a# much#higher#risk#than#others#as#it#is#the#case#with#especially#the#ones#that#are#related#to#the# major#histocompatibility#complex,#T#cell#receptors#or#immunoglobulins.#And#then#there#are# 27,28 certain#HLA#alleles#that#protect#against#disease,#even#if#the#susceptibility#allele#is#present .# Further,#it#has#been#observed#that#more#than#one#autoimmune#disease#appears#within#a#family,# 27P30 suggesting#that#some#genes#at#certain#loci#predispose#patients#to#more#than#one#disease .# For#some#time#it#was#hard#to#understand#why#some#people#with#the#same#serologic# abnormalities#did#not#necessarily#have#the#same#tissue#changes.#However,#an#explanation#has# been#found#in#an#animal#model,#demonstrating#that#the#vulnerability#of#an#organ#to#an#immuneP mediated#destruction#is#genetically#determined.#The#animal#model#demonstrated#the#presence# 31,32 of#a#variable#threshold#to#cardiac#and#renal#damage # .## Further#underlying#the#importance#of#genetic#factors#on#the#development#of# autoimmune#diseases#are#the#results#of#many#epidemiologic#studies,#which#have#demonstrated# familial#clustering,#the#higher#rate#of#similar#autoimmune#diseases#in#monozygous#versus# dizygous#twins,#and#the#fact#that#almost#75#%#of#the#more#than#23.5#million#Americans#that#are# ! 92# affected#with#autoimmune#diseases#are#women 33,34 .#The#exact#reason#why#women#are# overrepresented#is#not#well#understood#and#could#be#related#to#genetics,#but#reasons#could#also# include#that#they#mount#a#larger#inflammatory#response#when#stimulated#than#men,#the# involvement#of#sex#hormones,#history#of#pregnancy#and#therefore#exposure#to#antigens#of#the# baby,#imbalanced#X#chromosome#inactivation#or#other#complex#XPlinked#susceptibility# 35 mechanisms .## # Environmental#triggers,#internal#triggers#and#changes#in#pathologic# processes#during#autoimmunePdisease#progression# Epidemiologic#studies#of#twins#(lowerPthanPexpected#rate#of#same#autoimmune#diseases# 36 among#monozygotic#twins )#and#genetically#similar#populations#living#in#different#locations# show#that#an#environmental#exposure#is#necessary#to#trigger#autoreactivity,#even#if#a#genetic# 37,38 predisposition#is#present ,#as#shown#by#the#change#of#incidence#of#certain#autoimmune# disease#when#people#move#away#(for#example#with#type#1#diabetes,#multiple#sclerosis,#and# pemphigus#foliaceus).#Besides#an#environmental#exposure,#a#change#in#internal#environment#or# infections#could#also#trigger#disease.#Unfortunately,#the#trigger#for#most#autoimmune#diseases# is#unknown.#For#example,#certain#chemicals#(for#example#Procainamide)#can#cause#drugP induced#lupus#erythematosus,#estrogens#may#exacerbate#it,#cigarette#smoking#increases#risk#for# incidence#and#severity#of#rheumatoid#arthritis,#most#likely#due#to#increased#citrullination#of# ! 93# proteins,#penicillin#and#cephalosporin#can#bind#to#the#redPcell#membrane,#causing#production#of# an#antibody#which#causes#hemolytic#anemia,#and#the#blockade#of#TNFPα#(Tumor#necrosis#factor# alpha),#used#in#patients#with#Crohn’s#disease#or#rheumatoid#arthritis,#may#induce#antinuclear# 39 antibodies,#systemic#lupus#erythematosus#and#multiple#sclerosis .## Microbial#agents#can#trigger#an#autoimmune#disease,#related#to#polyclonal#activation,# release#of#previously#sequestered#antigens,#or#molecular#imitation#(or#mimicry).#Polyclonal# activation#is#believed#to#be#the#reason#for#the#increased#incidence#of#autoimmune#diseases#in# 40 rodents#that#are#exposed#to#microbes .#The#microbes#can#cause#inflammation,#and#a#release#of# 40,41 sequestered#antigens,#which#then#can#result#in#autoimmunity .#Similarities#exist#in#humans.# In#humans,#it#has#been#shown#that#inflammation#(in#the#absence#or#presence#of#infection)#can# 42 cause#polyclonal#activation#and#autoreactivity .#The#activation#of#B#cells#by#T#cells#can#be# bypassed#and#B#cells#can#secrete#large#amounts#of#antibodies#without#activation.#Polyclonal# activation#of#B#cells#can#happen#by#directly#binding#to#the#βPsubunit#of#the#T#cell#receptor#in#a# nonPspecific#fashion.#Interesting#on#the#other#hand#is#that#some#studies#have#found#that# parasitic#infections#in#humans#are#actually#associated#with#reduced#activity#of#autoimmune# diseases 43,44 .#The#parasite#might#influence#the#hosts#immune#response#in#order#to#protect#itself,# probably#by#secreting#antiPinflammatories#or#by#interfering#with#the#hosts#signaling#mechanism.## With#molecular#imitation,#it#has#been#speculated#that#an#infection#causes#the#initial# activation#of#lymphocytes#that#mediate#the#disease.#Antibodies#that#are#produced#during#initial# ! 94# infection#crossPreact#with#autoantigens#(antigenicPcross#reactivity),#which#sustain#the#activation# 1 even#after#the#foreign#antigen#is#eliminated .#This#mechanism#has#been#speculated#to#cause# rheumatic#fever#(due#to#streptococcal#infection) 48 45P47 40,49 jejuni) ,#autoimmune#diabetes#(coxsackievirus) ,#GuillainPBarré#syndrome#(Campylobacter# ,#and#multiple#sclerosis#(EpsteinPBarr,# 50 influenzavirus#type#A,#human#papillomavirus) .## Further,#regulatory#cells#carry#an#importance#in#the#development#of#autoimmune# diseases.#Some#regulatory#cells#mature#in#the#thymus,#and#others#must#be#activated#by# autoantigens#in#the#periphery.#Important#autoreactivityPcontrolling#regulatory#cells#include#CD1# restricted#T#cells,#T#cells#with#γ/δ#receptors,#CD4+CD25+#T#cells,#and#T#cells#that#produce# 51 cytokines,#which#in#turn#suppress#autoreactive#cells .#The#importance#of#these#regulatory#cells# in#the#development#of#autoimmunity#was#demonstrated#in#epidemiologic#studies.#It#was#shown# that#monozygotic#twins#that#are#discordant#for#diabetes#have#different#levels#of#CD1#restricted#T# 52 cells,#where#the#affected#twin#has#much#lower#levels#than#the#unaffected#twin .#The#exact# 1 mechanisms#in#which#antigens#activate#regulatory#T#cells#are#not#clearly#understood .## Epitope#spreading#occurs#during#disease#progression,#that#is#when#the#disease# progresses#from#activation#to#chronic#and#more#autoantigen#attacked#by#T#cells#and#antibodies# becomes#available.#Both#autoreactive#B#and#T#cells#add#to#epitope#spreading.#Autoreactive#B# cells#not#only#present#antigen#to#T#cells,#but#they#also#make#novel#peptides#of#proteins#and# protein#complexes#and#present#these#to#naïve#T#cells.#This#causes#a#cascade#to#start,#with#T#cells# ! 95# activating#more#autoreactive#B#cells,#and#B#cells#then#presenting#even#more,#additional#selfP epitopes.#The#result#is#increased#autoreactivity#to#many#more#autoantigens.#Further,#different# cells#contribute#to#chronic#disease#compared#to#initial#activation,#including#cytokines#and#other# 53P57 inflammatory#mediators,#which#also#contributes#to#epitope#spreading .## A#T#cellP#B#cell#discordance#has#been#recognized#in#for#example#Celiac#disease.#B#cells,# which#recognize#transglutaminase,#are#receiving#assistance#from#T#cells#that#recognize#gliadin.#It# is#hypothesized#that#a#B#cell#that#is#specific#for#IgGFc#(immunoglobulin#gamma#Fc#region)#can# recruit#and#get#help#from#a#T#cell#that#responds#to#an#antigen#coPendocytosed#with#IgG#by#B# cells.## Autoimmune#diseases#in#humans#are#mostly#restricted#to#a#few#antigens,#including#some# that#are#known#to#have#signaling#roles#in#the#immune#reaction.#Spontaneous#autoimmunity#may# occur#when#abnormal#signals#are#sent#to#parent#B#cells#through#membrane#bound#ligands# (including#B#cell#receptor#for#antigen,#IgG#Fc#receptors,#CD21,#TollPlike#receptors#9#and#7),#after# binding#of#an#antibody#to#certain#antigens.#This#idea,#together#with#the#idea#of#T#cell#B#cell# 58 discordance,#shaped#the#basis#for#the#hypothesis#of#selfPperpetuating#autoreactive#B#cells .#It#is# hypothesized#that#these#cells#survive#due#to#subversion#of#both#the#feedback#signal#via#the#B# cell#receptor#and#of#the#T#cell#help#pathway.#Negating#the#negative#signals#causing#B#cell#self# tolerance,#without#automatically#losing#T#cell#self#tolerance.#This#is#called#aberrant#B#cell# receptorPmediated#feedback.# ! 96# Antigenic#epitopes#that#are#found#in#the#antigenPbinding#portion#of#an#immunoglobulin# molecule#are#called#idiotypes.#If#an#idiotype#on#an#antiviral#antibody#and#a#host#cell#receptor#for# the#virus#in#question#crossPreact,#autoimmunity#can#occur.#The#host#cells#are#then#attacked#by# 59 the#antiPidiotype#antibodies,#because#the#hostPcell#receptor#is#seen#as#the#virus .# Cytokines#either#promote#function#of#helper#T#cells#type#1#or#helper#T#cells#type#2.#The# cytokines#that#promote#function#of#helper#T#cells#type#2#appear#to#have#a#role#in#prevention#of# exaggeration#of#proPinflammatory#immune#responses#of#type#1#T#cells.#A#few#cytokines#that#are# in#this#group#are#ILP4,#ILP10,#and#TGFPβ.#This#phenomenon#is#called#‘cytokine#dysregulation’.## Dendritic#cells#present#antigens#to#active#lymphocytes.#If#the#dendritic#cells#are#defective# in#apoptosis,#it#could#result#in#an#inappropriate#systemic#activation#of#lymphocyte#apoptosis#and# 60 as#a#consequence#a#decline#in#selfPtolerance .# Over#time,#the#effector#cells#and#inflammatory#mediators#of#an#autoimmune#disease# might#change,#which#makes#it#very#difficult#for#treatment.#A#treatment#that#is#beneficial#early#on# could#lose#its#effectiveness,#or#might#even#be#harmful#later#on.#These#changes#have#been# demonstrated#in#studies#in#animals,#but#can#also#be#experienced#during#treatment#of#many# autoimmune#diseases#in#humans,#of#which#the#most#common#are#Crohn’s#disease,#and# rheumatoid#arthritis.#Blockade#of#TNFPα#in#patients#with#either#disease#is#of#huge#benefit,#but# induces#production#of#antinuclear#antibodies#in#10%#of#treated#patients#and#systemic#lupus# erythematosus#in#a#few.#The#question#remains#whether#naïve#cells#or#memory#cells#cause# 1,39 progression#of#disease ! .## 97# Further#under#investigation#in#the#pathogenesis#of#autoimmune#diseases#is#the#role#of# regulatory#T#cells,#natural#killer#cells,#and#γδ#T#cells.## # Autoimmune#mechanisms#of#tissue#injury# The#effector#mechanisms#of#the#immune#system#may#be#directed#against#selfPantigens,# as#described#previously.#Since#the#adaptive#immune#system#is#not#able#to#remove#an# autoantigen,#the#immune#response#persists#and#might#get#stronger,#because#of#the#constant# supply#of#new#autoantigen.#T#cells#and#autoantibodies#cause#tissue#damage#during#an# autoimmune#disease.#T#cells#induce#cytolysis#through#perforinPinduced#necrosis#or#through# granzyme#BPinduced#apoptosis.#Type#1#and#type#2#helper#T#cells#cause#tissue#damage#through# 61P64 the#production#of#cytokines#and#through#recruitment#of#inflammatory#cells#and#mediators IgE,#IgM,#and#IgG#appear#to#be#of#importance#during#autoimmune#diseases,#even#though#IgE# does#not#seem#to#have#a#major#role.#They#have#been#identified#in#some#autoimmune#diseases,# for#example#in#autoimmune#vasculitis,#an#inflammatory#disease#of#blood#vessels#that#is#also# known#as#ChurgPStrauss#vasculitis.#However,#it#has#not#been#proven#that#the#IgE#antibodies# mediate#the#disease.#Autoimmune#diseases#that#cause#tissue#damage#by#means#similar#to#a# type#II#hypersensitivity#response#are#very#common#though.#The#IgG#or#IgM#response#is#against# autoantigens#that#are#located#on#cell#surfaces#or#the#extracellular#matrix,#which#then#causes# injury.#Injury#related#to#autoimmunity#similar#to#a#type#II#response#involves#the#formation#of# ! 98# .# immune#complexes,#containing#antibodies#to#soluble#autoantigens.#These#autoimmune#diseases# are#usually#systemic#and#are#characterized#by#autoimmune#vasculitis.## # Th17#cells# # Th17#cells,#a#subset#of#T#helper#cells,#were#first#described#in#the#literature#between#2005# 65P67 and#2007 .#The#T#helper#cell#differentiation#process#and#their#protective#and#harmful#role# are#shown#in#figure#II.1.#Cytokines#transforming#growth#factor#beta#(questionable#in#humans),# 68,69 ILP1β,#ILP6,#ILP21,#and#ILP23#contribute#to#their#formation#in#humans#and#mice ,#as#well#as# other#proteins#including#signal#transducer#and#activator#of#transcription#3,#retinoicPacidP 68 receptorPrelated#orphan#receptors#alpha#and#gamma .#Interferon#gamma,#ILP4,#and#ILP12#have# been#shown#to#negatively#regulate#Th17#differentiation#in#man,#as#well#as#ILP2,#ILP25,#ILP27,#and# ILP35#in#mice#(see#figure#II.2).## # Th17#cells#are#highly#inflammatory#in#nature,#and#excessive#amounts#of#Th17#have#a#key# role#in#the#development#of#autoimmune#diseases#and#have#been#linked#to#multiple#sclerosis,# psoriasis,#autoimmune#uveitis,#juvenile#diabetes,#rheumatoid#arthritis,#inflammatory#bowl# 70P75 disease,#arthritis#and#Crohn’s#disease ,#most#likely#by#causing#inflammation#and#tissue# 76 injury.#Their#physiologic#role#is#to#defend #against#extracellular#pathogens#at#epithelial#and# 77 mucosal#barriers,#including#Klebsiella#and#Citrobacter#bacterial#species #by#producing#cytokines# ! 99# 78 (effector#cytokines#are#ILP17#(especially#ILP17A#and#F),#ILP21,#and#ILP22 ),#which#stimulate# epithelial#cells#to#produce#antiPmicrobial#proteins.#Especially#ILP17A#and#ILP17F#recruit,#activate,# and#are#involved#in#the#recruitment#of#neutrophils.#A#lack#of#Th17#would#leave#a#host#vulnerable# to#opportunistic#infections.#The#role#of#Th17#cells#in#carcinogenesis#remains#under# 79 investigation .# # Conclusions# In#order#to#develop#superior#treatments#for#autoimmune#diseases,#it#will#be#necessary#to# better#understand#the#exact#mechanisms#that#control#development#of#autoimmune#disease# phenotype.#A#model#for#the#development#of#autoimmune#diseases#needs#to#include#genetic# predispositions,#environmental#factors#and#immune#regulations#or#dysreglations,#as# schematized#in#figure#II.3,#and#as#described#previously.#Besides#other#markers#of#predisposition# to#autoimmune#diseases,#and#other#genes#that#are#important#in#the#regulation#of#an#immune# response,#MHC#molecules#regulate#and#form#the#specificity#of#the#adaptive#immune#response# and#have#been#associated#with#the#development#of#autoimmune#diseases.#The#genetic#makeup# of#humans#determines#how#the#immune#system#reacts#to#antigenic#confronts#from#the# environment.#Further,#it#is#responsible#for#staying#tolerant#towards#selfPantigens.#Under#certain# conditions,#an#infection,#an#inappropriate#immune#response#to#crossPreactive#selfPantigens,# and/#or#the#failure#of#regulatory#mechanisms,#may#cause#tissue#damage.#Severe#tissue#damage# ! 100# can#further#strengthen#the#immune#response,#and#a#clinical#manifest#autoimmune#disease# results.# ! 101# APPENDIX ! 102# Appendix# Figure#II.1:#Heterogeneity#in#helper#T#cell#fates.#The#helper#T#cell#differentiation#process# is#initiated#by#signaling#from#dendritic#cell#to#T#cell#in#the#lymph#node,#resulting#in#division#and# differentiation.#The#mature#helper#T#cells#and#their#signature#transcription#factors#are# illustrated.#Cytokines#play#a#critical#role#in#the#induction#or#repression#of#the#lineages.#The# different#helper#T#cell#subsets#have#distinct#protective#and#pathological#roles.#Host#defense#is# orchestrated#by#the#three#major#fates,#Th1,#Th2,#and#Th17.#Adaptive#regulatory#T#(aTreg)#cells# can#downregulate#immune#responses,#although#a#physiological#role#in#vivo#is#yet#uncertain.#The# mature#helper#T#cell#progeny#must#eventually#exit#the#lymph#node#and#migrate#to#infected# tissue#to#exert#their#function#in#host#defense.#Some#of#the#mature#progeny#may,#instead,# migrate#to#B#cell#follicles#to#promote#antibody#subclasses#that#will#suit#the#particular#immune# response#(from:#Reiner,#S.#L.,#Development#in#Motion:#Helper#T#Cells#at#Work,#Cell#129,#April#6,# 2007,#pages#33#–#36).# # ! 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112# # 67.# Harrington#LE,#Hatton#RD,#Mangan#PR,#et#al.#Interleukin#17Pproducing#CD4+# effector#T#cells#develop#via#a#lineage#distinct#from#the#T#helper#type#1#and#2#lineages.#Nat$ Immunol#2005;6:1123P1132.# # 68.# Dong#C.#TH17#cells#in#development:#an#updated#view#of#their#molecular#identity# and#genetic#programming.#Nat$Rev$Immunol#2008;8:337P348.# # 69.# Manel#N,#Unutmaz#D,#Littman#DR.#The#differentiation#of#human#T(H)P17#cells# requires#transforming#growth#factorPbeta#and#induction#of#the#nuclear#receptor#RORgammat.# Nat$Immunol#2008;9:641P649.# # 70.# Cua#DJ,#Sherlock#J,#Chen#Y,#et#al.#InterleukinP23#rather#than#interleukinP12#is#the# critical#cytokine#for#autoimmune#inflammation#of#the#brain.#Nature#2003;421:744P748.# # 71.# Duerr#RH,#Taylor#KD,#Brant#SR,#et#al.#A#genomePwide#association#study#identifies# IL23R#as#an#inflammatory#bowel#disease#gene.#Science#2006;314:1461P1463.# # 72.# Langrish#CL,#Chen#Y,#Blumenschein#WM,#et#al.#ILP23#drives#a#pathogenic#T#cell# population#that#induces#autoimmune#inflammation.#J$Exp$Med#2005;201:233P240.# # 73.# Murphy#CA,#Langrish#CL,#Chen#Y,#et#al.#Divergent#proP#and#antiinflammatory#roles# for#ILP23#and#ILP12#in#joint#autoimmune#inflammation.#J$Exp$Med#2003;198:1951P1957.# # 74.# Nakae#S,#Nambu#A,#Sudo#K,#et#al.#Suppression#of#immune#induction#of#collagenP induced#arthritis#in#ILP17Pdeficient#mice.#J$Immunol#2003;171:6173P6177.# # 75.# Zheng#Y,#Danilenko#DM,#Valdez#P,#et#al.#InterleukinP22,#a#T(H)17#cytokine,# mediates#ILP23Pinduced#dermal#inflammation#and#acanthosis.#Nature#2007;445:648P651.# # 76.# Steinman#L.#A#brief#history#of#T(H)17,#the#first#major#revision#in#the#T(H)1/T(H)2# hypothesis#of#T#cellPmediated#tissue#damage.#Nat$Med#2007;13:139P145.# # 77.# Weaver#CT,#Harrington#LE,#Mangan#PR,#et#al.#Th17:#an#effector#CD4#T#cell#lineage# with#regulatory#T#cell#ties.#Immunity#2006;24:677P688.# # 78.# Ouyang#W,#Kolls#JK,#Zheng#Y.#The#biological#functions#of#T#helper#17#cell#effector# cytokines#in#inflammation.#Immunity#2008;28:454P467.# ! 113# # 79.# Wu#S,#Rhee#KJ,#Albesiano#E,#et#al.#A#human#colonic#commensal#promotes#colon# tumorigenesis#via#activation#of#T#helper#type#17#T#cell#responses.#Nat$Med#2009;15:1016P1022.# # # ! 114# Chapter#III:#Preliminary#research#leading#to#my#hypothesis# # Retrospective#study:#histology#and#histopathology#of#normal#canine# adrenal#glands#and#adrenal#glands#from#Addison’s#disease#dogs# Twenty@four#adrenal#gland#tissue#slides#from#dogs#diagnosed#with#adrenal#insufficiency# were#stained#with#hematoxylin#and#eosin#stain#(H&E)#and#examined.#Normal#adrenal#glands# were#obtained#during#senior#student#surgery#labs#and#were#used#as#negative#controls.#In#the# Addison’s#disease#adrenal#glands,#the#severity#of#inflammation#was#subjectively#graded#as#mild# (1),#moderate#(2),#or#severe#(3)#by#two#independent#observers#depending#on#the#amount#of# inflammation#observed#(Rick#and#Williams).#Ten#cases#were#classified#as#grade#1,#eight#as#grade# 2,#and#five#as#grade#3#(figures#III.1#and#III.2).#One#adrenal#gland#did#not#show#inflammation;# however,#in#this#case#the#adrenal#cortex#was#collapsed#and#atrophied.#As#would#be#expected#in# immune@mediated#disease,#the#inflammation#was#primarily#lymphocytic#early#in#the#course#of# the#disease,#and#became#mostly#macrophagic#coincident#with#the#destruction#and#loss#of#gland# parenchyma.#None#of#the#12#control#adrenal#glands#had#any#evident#inflammatory#cells#present# within.#Immunohistochemistry#was#utilized#to#identify#the#lymphocytes#as#either#T#cells#(CD3+)# (cluster#of#differentiation#3+)#or#B#cells#(CD79a+).#Again,#as#would#be#expected#for#an#immune@ mediated#disease,#the#majority#of#cells#are#T#cells#(figure#III.3).## # ! 115# Detection#of#anti@adrenal#autoantibodies#using#Western#blot#analysis# # # I#followed#the#protocol#of#the#Diagnostic#Center#for#Population#and#Animal#Health# (DCPAH),#Michigan#State#University#(MSU),#East#Lansing,#MI,#USA,#Western#blot#(WB)# PARASOP.0026.01#(Standard#operating#procedure#for#detection#of#Sarcoystic#neurona#by#WB).# Freshly#collected#adrenal#glands#were#homogenized#in#50#mM#Tris,#containing#0.4#mM#KI,#and#1# mM#EDTA,#and#centrifuged#at#4°C#and#1,000#x#g#for#10#minutes.#The#antigen#preparations#were# used#immediately#or#stored#at#@80°C.#Following#SDS@PAGE#(sodium#dodecyl#sulfate# polyacrylamide#gel#electrophoresis),#antigens#were#transferred#to#nitrocellulose#sheets.# Subsequently,#the#sheets#were#incubated#with#negative#and#suspect#positive#sera#or#plasma# (normal#or#slightly#lipemic#samples#only),#and#antigen@antibody#complexes#were#visualized#by# incubating#with#a#peroxidase@conjugated#goat#anti@canine#IgG#(immunoglobulin#gamma),#heavy# and#light#chain,#and#a#substrate@chromogen#mixture.# # # I#analyzed#a#total#of#17#samples#(14#affected#dogs,#three#healthy#control#dogs).#Out#of# the#14#affected#dogs,#five#showed#a#distinct#band#around#20#kDa#(figure#III.4).#None#of#the#three# negative#control#samples#showed#this#band.#However,#human#microsomal#autoantigen#21@ hydroxylase#weighs#55#kDa.#In#humans#that#have#been#recently#diagnosed#with# hypoadrenocorticism,#there#is#a#proliferative#T#cell#response#to#an#18#kDa#to#24#kDa#molecular# weight#protein#fraction 1,2 .#This#protein#fraction#is#adrenal#specific#and#corresponds#in#weight#to# the#band#that#I#observed#in#this#preliminary#experiment.#WB#is#a#reliable#and#sensitive# diagnostic#test#for#the#detection#of#antibody#in#serum.#However,#it#is#also#very#sensitive#to#slight# ! 116# changes#in#conditions.#Further,#antigen#preparations#should#be#purified#to#decrease#nonspecific# binding.## # Detection#of#21@hydroxylase#antibodies#with#a#commercially#available# radioimmunoassay#for#the#detection#of#human#21@hydroxylase# antibodies# A#commercially#available#radioimmunoassay#for#the#identification#of#21@hydroxylase# # autoantibodies#in#human#serum#is#distributed#by#Kronus® (Boise,#Idaho,#USA).#This#assay#was# used#according#to#the#manufacturer#recommendations#and#three#different#dog#populations# were#analyzed.#First,#canine#serum#or#plasma#samples#from#dogs#with#a#confirmed#diagnosis#of# hypoadrenocorticism,#based#on#ACTH#(adrenocorticotropic#hormone)#stimulation#test,#and#no# history#of#chronic#exogenous#steroid,#Lysodren,#trilostane#or#ketoconazole#use#were#used,# representing#the#sick#dog#group#(SD).#Second,#serum#or#plasma#samples#from#breeds#that#are# reported#to#have#a#higher#incidence#of#hypoadrenocorticism#compared#to#the#general#dog# population,#or#from#affected#lines#of#dogs#were#used,#and#represented#a#group#of#dogs#at#higher# risk#to#develop#the#disease#compared#to#the#general#dog#population#(HR).#Serum#or#plasma# samples#from#healthy#dogs#sent#to#us#for#thyroid#evaluation#for#registration#by#the#Orthopedic# Foundation#for#Animals#were#used#as#negative#(healthy)#control#samples#(LR).#The#samples#were# stored#at#@18°C#prior#to#analysis.#By#definition,#positive#samples#contained#>1#U/mL#antibodies# which#is#the#same#cut@off#established#for#human#samples.#Results#are#summarized#in#table#III.1.## ! 117# Non@parametric#statistical#analyses#showed#a#difference#among#groups#(Kruskal@Wallis,# p=0.001)#and#Mann@Whitney#with#Bonferroni#correction#showed#differences#between#individual# groups:## • Significant#difference#between#SD#to#LR#(p=0.0003)#and#HR#to#LR#(p=0.0013)# • No#significant#difference#between#SD#to#HR#(p=0.8338)# The#21@hydroxylase#antibody#activity#was#reduced#in#two#out#of#two#positive#samples# after#incubation#with#an#excess#amount#of#unlabeled#21@hydroxylase#indicating#that#they# contained#specific#21@hydroxylase#antibodies.## Unfortunately,#this#assay#gave#very#poor#replication#and#the#differences#in#raw#counts# per#minutes#(cpm)#between#supposedly#positive#and#supposedly#negative#samples#were#less# then#200#cpm.#Although#preliminary#data#with#this#strategy#were#promising,#I#concluded#that# this#method#did#not#provide#adequate#specificity#for#detecting#canine#21@hydroxylase# antibodies.## # Detection#of#21@hydroxylase#antibodies#with#radioactively#labeled# amino#acid#capture#probe# The#protein#sequence#for#canine#21@hydroxylase#is#published.#There#is#78#%#homology#of# amino#acid#sequence#between#canine#and#human#21@hydroxylase.#The#autoantibody#epitope#is# well#described#in#the#human#literature#and#is#reported#to#be#located#at#amino#acids#298@356.#N@ terminal#deletions#up#to#280#had#no#effect#on#binding.#C@terminal#deletions#281@494#on#the# other#hand#showed#a#marked#effect#on#antibody#binding.#However,#a#conformational#epitope# ! 118# might#exist.#All#antibodies#reacted#to#full#length#21@hydroxylase#that#was#expressed#in#vitro 3@5 .# According#to#this#information,#a#21#amino#acid#peptide#(YKDRARLPLLNATIAEVLRLR)#was# designed.#The#iodination#was#performed#according#to#standard#protocol.#We#iodinated#2#µg#of# protein,#over#50#%#of#the#iodine#was#bound#to#the#protein#and#we#saved#a#total#of#0.3#mCi#of# iodinated#protein.## # # Analysis#of#54#samples#from#dogs#with#hypoadrenocorticism#and#10#control#samples#was# performed.#The#assay#procedure#was#the#same#as#recommended#for#the#radioimmunoassay#for# the#identification#of#human#21@hydroxylase#antibodies#in#human#serum#commercially#available# through#Kronus®#(see#above).#Results#are#summarized#in#Table#III.2.## Mann@Whitney#statistical#analysis#with#Bonferroni#correction#showed#a#significant# difference#in#counts#between#the#two#individual#groups:## • Significant#difference#between#SD#to#LR#(p=0.0343)## The#biggest#problem#in#this#experiment#was#the#presence#of#a#nonspecific#binding#raw# count#of#greater#than#2000/#min.#I#tried#different#incubation#times,#different#sample#and#tracer# volumes,#different#dilutions,#and#different#capturing#mechanisms,#utilizing#protein#A,#protein#G,# and#N6.#I#also#added#various#amounts#of#protein#from#different#species#to#the#reaction#buffers.# Results#remained#confusing#and#not#explainable,#and#I#concluded#that#there#must#be#some#kind# of#matrix#interferences#between#molecules#in#the#serum#or#plasma#samples#with#the#tracer.## # # ! 119# Detection#of#adrenal#autoantibodies#with#indirect#immunofluorescence# technique# Normal#to#slightly#hemolyzed,#previously#assayed#canine#serum#or#plasma#samples,#with# confirmed#diagnosis#of#hypoadrenocorticism#based#on#ACTH#stimulation#test#and#no#history#of# chronic#exogenous#steroid,#Lysodren,#trilostane#or#ketoconazole#use,#were#used#as#positive# control#samples.#Normal#to#slightly#hemolyzed#serum#or#plasma#samples#from#healthy#dogs#sent# to#us#for#thyroid#evaluation#for#registration#by#the#Orthopedic#Foundation#for#Animals#were# used#as#negative#control#samples.#The#samples#were#stored#at#@18°C#prior#to#analysis.#Adrenal# tissue#from#healthy#beagle#dogs#were#collected#and#immediately#frozen#and#stored#at#@80°C# until#used#for#preparing#cryostat#sections.#None#of#the#adrenal#tissues#had#been#stored#longer# than#six#months#at#the#time#of#this#experiment.#Working#temperature#for#cryo@sectioning#was# approximately#@20°C.#The#frozen#tissues#were#transferred#onto#slides#and#air@dried#for#30# minutes.#Then,#standard#indirect#immunofluorescence#procedures#were#performed#with#both# unfixed#and#acetone#fixed#(10#minutes)#tissues.#I#was#not#able#to#differentiate#supposedly# positive#from#supposedly#negative#serum#samples.#After#consultation#with#Dr.#Ludek#Vajner,# who#has#considerable#experience#with#indirect#immunofluorescence#technique#(iIFA),#especially# with#thyroid#tissues,#we#used#his#protocol#to#detect#thyroglobulin#autoantibodies#(TgAA)#in#sera# on#frozen#canine#thyroid#cryostat#sections.#Thyroid#glands#were#harvested#from#healthy#beagle# dogs#and#immediately#frozen#in#liquid#nitrogen#and#stored#at#@80°C#until#they#were#used#for#this# experiment.#Serum#plasma#samples#positive#or#negative#for#TgAA#were#identified#using#a# ! 120# commercially#available#ELISA#(Enzyme@linked#immunosorbent#assay) test#(Oxford#Biomedical# Research,#Rochester,#MI,#USA)#in#our#laboratory.#Using#iIFA,#I#was#able#to#differentiate#TgAA# positive#serum#samples#from#TgAA#negative#serum#samples#(figure#III.5).## Using#the#same#technique#for#adrenal#antibody#analysis,#I#was#not#able#to#differentiate# supposedly#positive#from#supposedly#negative#samples#because#of#severe#background.#The#use# of#different#blocking#agents#including#5%#non@fat#dried#milk#and#serum#from#different#species# did#not#decrease#background.# It#became#apparent,#that#it#is#difficult#to#establish#and#find#a#proper#working#technique# without#having#a#confirmed#positive#serum#or#plasma#sample#available.#I#decided#not#to#repeat# this#experiment#until#the#creation#or#finding#of#a#known#positive#control#sample.## ## # ! # 121# APPENDIX ! 122# #Appendix# Figure#III.1:#Adrenal#gland#histology,#2.5x#magnification#hematoxylin#and#eosin#stain.## Normal'control'adrenal'gland' Grade'3'adrenal'gland' # ! Grade'1'adrenal'gland' No'inflamma3on,'cortex'collapsed' # 123# Figure#III.2:#Adrenal#gland#histology,#20x#magnification,#hematoxylin#and#eosin#stain.# Grade&3&adrenal&gland& Grade&1&adrenal&gland& # # # ! # 124# Figure#III.3:#Adrenal#gland#histology,#20x#magnification,#CD3#and#CD79a# immunohistochemistry.# CD3,&20x& CD79a,&20x& # # ! # 125# Figure#III.4:#Detection#of#anti#adrenal#antibodies#with#Western#blotting.# # # # ! # 126# Table#III.1:#Results#with#a#commercially#available#radioimmunoassay#for#the#detection#of# 21@hydroxylase#autoantibodies#in#human#serum.# # Median# 25%#percentile# 75%#percentile# [U/mL]# [U/mL]# [u/mL]# 18#(34%)# 0.8# 0.7# 1.1# 21# 9#(43%)# 0.8# 0.6# 1.2# 13# 0#(0%)# 0.5# 0.5# 0.7# Population# Sick#dogs# (SD)# High#risk# Positive# 53# dogs#(HR)# Low#risk#dogs# (LR)# # Table#III.2:#Results#with#radioactively#labeled#amino#acid#capture#probe.# # Sick#dogs# Median#raw# Median# 25%#percentile# 75%#percentile# counts# [U/mL]# [U/mL]# [U/mL]# 54# 6166.5# 0.8# 0.7# 1.1# 10# 4587.5# 0.5# 0.5# 0.7# Population# (SD)# Low#risk# dogs#(LR)# # # ! # 127# Figure#III.5:#Detection#of#adrenal#autoantibodies#with#indirect#immunofluorescence# technique.# # # # ! # 128# REFERENCES ! 129# References# ! 172.# 1.! Betterle#C,#Morlin#L.#Autoimmune#Addison's#disease.#Endocr'Dev#2011;20:161@ # 2.# Freeman#M,#Weetman#AP.#T#and#B#cell#reactivity#to#adrenal#antigens#in# autoimmune#Addison's#disease.#Clin'Exp'Immunol#1992;88:275@279.# # 3.# Song#YH,#Connor#EL,#Muir#A,#et#al.#Autoantibody#epitope#mapping#of#the#21@ hydroxylase#antigen#in#autoimmune#Addison's#disease.#J'Clin'Endocrinol'Metab#1994;78:1108@ 1112.# # 4.# Betterle#C,#Greggio#NA,#Volpato#M.#Clinical#review#93:#Autoimmune# polyglandular#syndrome#type#1.#J'Clin'Endocrinol'Metab#1998;83:1049@1055.# # 5.# Wedlock#N,#Asawa#T,#Baumann@Antczak#A,#et#al.#Autoimmune#Addison's#disease.# Analysis#of#autoantibody#binding#sites#on#human#steroid#21@hydroxylase.#FEBS'Lett# 1993;332:123@126.# # # ! 130# Chapter#IV:#Hypothesis#and#specific#aims# # # Based#on#what#is#known#about#Addison’s#disease#in#humans#and#the#results#of#the# preliminary#experiments,#I#formulated#the#following#hypothesis#and#specific#aims:## # Hypothesis# # As#in#human#hypoadrenocorticism,#antiC21Chydroxylase#antibody#production#occurs#in# naturally#occurring#primary#canine#hypoadrenocorticism.#The#antibody#production#precedes# clinical#disease,#and#is#more#prevalent#in#dog#breeds#susceptible#to#developing# hypoadrenocorticism.## # Specific#aims# 1)#To#establish#a#diagnostic#test#to#detect#canine#antiCadrenal#autoantibodies;## 2)#To#determine#whether#antiCadrenal#autoantibodies#are#present#in#dogs#with# hypoadrenocorticism;## 3)#To#determine#whether#development#of#antiCadrenal#autoantibodies#has#breed,#sex,# and#ageCbased#predispositions.# ! 131# Chapter$V:$The$expression$of$canine$216hydroxylase$ $ Introduction$ The$overall$amino$acid$homology$between$canine$and$human$216hydroxylase$is$78$%.$As$ described$in$preliminary$research,$a$radioimmunoassay$for$the$detection$of$216hydroxylase$ antibodies$in$humans$was$tried$for$use$in$dogs.$Results$were$unsatisfactory.$Therefore,!our$ strategy$was$to$express$recombinant$canine$216hydroxylase$for$use$as$an$antigen$to$detect$ autoantibodies$by$either$Western$blot$(WB)$or$by$enzyme6linked$immunosorbent$assay$(ELISA).$$ To$get$a$feeling$and$get$used$to$molecular$techniques,$I$decided$to$do$the$following$ preliminary$approach,$which$will$not$be$covered$in$detail$in$this$dissertation:$I$obtained$canine$ adrenal$glands$within$30$sec$of$euthanasia$and$quick6froze$them$in$liquid$nitrogen.$Total$RNA$ was$extracted$using$TRIZOL$according$to$the$instructions$provided$by$the$manufacturer$and$ then$reverse6transcribed$via$Superscript$III®$enzyme,$using$random$hexamers.$Primers$were$ designed$in$the$5’$and$3’$untranslated$region$of$the$canine$CYP21A2$gene$and$the$whole$ transcript$was$amplified$using$TaKaRa$LA$PCR$Kit$version$2.1.$NotI$and$BamHI$restriction$sites$ were$added$by$PCR$mutagenesis$5’$of$the$translational$start$codon$and$3’$of$the$termination$ codon,$respectively.$The$sequence$was$then$compared$to$the$published$sequence$and$no$ mutations$were$detected.$A$GST$(glutathione$S6transferase)$fusion$protein$expression$vector$ was$digested$with$the$same$restriction$enzymes,$and$the$cDNA$fragment$subcloned.$This$fused$ the$entire$canine$216hydroxylase$cDNA$in$frame$3'$of$GST.$This$expression$construct$was$ ! 132$ transformed$into$BL21$(DE3)$cells$and$expression$of$the$216hydroxylase6GST$fusion$protein$was$ induced$with$IPTG$(Isopropyl$β6D616thiogalactopyranoside).$Expression$of$the$recombinant$ fusion$protein$was$confirmed$by$SDS6PAGE$(sodium$dodecyl$sulfate$polyacrylamide$gel$ electrophoresis),$analyzing$both$soluble$and$insoluble$fractions$of$the$bacterial$pellet.$Only$a$ minimal$amount$of$216hydroxylase$was$expressed$and$found$primarily$in$the$insoluble$fraction$ (figure$V.1).$Since$the$yield$was$unsatisfactory,$I$changed$my$approach.$$ The$new$approach,$namely$the$generation$of$a$panel$of$different$fusion$proteins$(GST$ and$His$(hexa$histidine)$tags)$generated$according$to$the$instructions$provided$by$Arase$and$ 1 Waterman $will$be$discussed$here.$In$short,$the$membrane$anchor$and$basic$region$of$P450c21$ 2 was$deleted$and$replaced$with$MAKKTSSKGK$from$CYP2C3 $and$chaperone$proteins$were$co6 expressed$to$increase$solubility$and$yield.$$ $ Materials$and$methods$ Constructs:*Cloning$and$construction$of$the$modified$canine$216hydroxylase$fusion$ 3 proteins$was$done$according$to$published$methods .$In$order$to$facilitate$expression$of$the$216 hydroxylase,$the$membrane$anchor$and$basic$region$of$P450c21$was$replaced*with$ MAKKTSSKGK$from$CYP2C3$as$indicated$in$the$primers$by$small$letters.$A$nested$PCR$ (polymerase$chain$reaction)$was$done$using$forward$primer$5’6$ acatcatctaaaggtaagCTCCCACCTCTTGTCCCT$63’$and$reverse$primer$5’6$ GCGGCCGCTCATGGGTGCTGGCCACGT$63’$in$the$first$reaction$and$forward$primer$5’6$ ! 133$ GGGAGGGATCCCCatggctaaaaagacatcatctaaaggtaag$63’$(the$underline$indicating$the$BamHI$site)$ and$reverse$primer$5’6$GCGGCCGCTCATGGGTGCTGGCCACGT$63’$(the$underline$indicating$the$ NotI$site)$in$the$second$reaction,$using$the$previously$obtained$template$and$TaKaRa$LA$PCR$Kit$ version$2.1$(TaKaRa$Bio$Inc.,$Otsu,$Shiga,$Japan).$The$reverse$primer$contained$a$stop$codon$ immediately$following$the$NotI$site.$The$PCR$product$was$isolated$(Qiagen$PCR$Purificarion$Kit,$ Qiagen,$Valencia,$CA,$USA)$and$ligated$into$the$TOPO$vector$(Invitrogen,$Carlsbad,$CA,$USA)$for$ sequencing$using$M13$forward6$and$M13$reverse$primers,$as$well$as$internal$forward$primer$5’6$ gccatcatctgtcacctcac$63’$and$internal$reverse$primer$5’6$cgaatcccgtacagagaccc$63’.$The$sequence$ was$compared$with$the$modified$canine$216hydroxylase$derived$from$the$unmodified$canine$ 216hydroxylase$(GenBank$accession$number$BAB79541.1).$Plasmid$DNA$was$then$extracted$ with$standard$miniprep$and$digested$with$BamHI$and$NotI.$The$resulting$digest$was$gel$purified$ using$a$gel$purification$kit$(Qiagen)$and$ligated$into$vectors$pET28c$and$pGEX65X63,$respectively,$ digested$with$the$same$restriction$enzymes,$thus$creating$the$modified$canine$216hydroxylase/$ pET28c$and$the$modified$canine$216hydroxylase/$pGEX65X63$construct.$Both$ligation$products$ were$then$transformed$into$E.!coli$DH5α$maximum$efficiency$cells.$In$order$to$replace$the$ BamH1$site$with$NdeI$and$to$remove$the$stop$codon$at$the$3’$end,$the$modified$canine$216 hydroxylase/$pGEX65X63$DNA$was$used$as$a$template$for$PCR$with$forward$primer$5’6$ GGTCGTGGGATCCATATGGCTAAAAAGACA$63’$(the$underline$indicating$the$NdeI$site)$and$ reverse$primer$5’6$CGATGCGGCCGCTAATGGGTGCTGGCC$63’$(the$underline$indicating$the$NotI$ site).$The$PCR$product$was$isolated$(Qiagen$PCR$Purificarion$Kit)$and$ligated$into$the$TOPO$ vector$(Invitrogen)$for$sequencing$as$described$above.$After$sequence$analysis,$the$NdeI/NotI$ ! 134$ fragment$was$cut$out$and$subcloned$into$pET42b,$yielding$the$expression$plasmid$modified$ canine$216hydroxylase/$pET42b.$$ Chaperone$vector$pG6KJE8$(TaKaRa)$was$transformed$into$BL21$(DE3)$Gold$cells$(Agilent$ Technologies$Inc.,$Santa$Clara,$CA,$USA)$and$C41(DE3)$cells$(Lucigen$Corporation,$Middletown,$ 4 WI,$USA),$which$were$made$competent$using$a$standard$CaCl2$protocol .$The$three$previously$ obtained$constructs$were$then$transformed$into$these$BL21$(DE3)$Gold,$and$C41$(DE3)$cells,$ respectively,$both$containing$the$chaperone$expression$plasmid$pG6KJE8,$and$screened$on$LB$ plates$supplemented$with$the$appropriate$antibiotic.$For$an$overview$of$the$final$expression$ systems$see$table$V.1.$$ Small/scale*induction:$From$the$three$expression$systems$described$above$in$the$two$ different$hosts,$a$2.5$mL$Lysogeny$broth$(LB)$culture$with$the$appropriate$antibiotics$was$grown$ overnight$at$37°C$under$constant$shaking.$The$next$day,$this$culture$was$transferred$into$250$ mL$of$LB,$again$containing$the$appropriate$antibiotics.$The$culture$was$shaken$constantly$at$ 37°C.$Once$an$OD$between$0.4$and$0.6$was$reached$(with$some$selected$conditions,$expression$ was$not$initiated$until$the$OD$reached$1.0),$a$negative$control,$of$1.5$mL$culture$was$taken$and$ spun$down$at$4°C$at$12,000$rpm$for$1$min$and$the$pellet$was$kept$at$680°C$until$further$analysis.$ The$culture$was$allowed$to$cool$down$to$the$temperature$at$which$the$expression$was$done.$To$ each$culture,$1$mM$δ6aminolevulinic$acid$(precursor$of$heme$biosynthesis)$was$added.$216 hydroxylase$fusion$protein$expression$was$then$induced$with$1$mM$IPTG.$In$addition$to$the$1$ mM$IPTG$and$1$mM$δ6aminolevulinic$acid,$the$following$additions$were$tested$in$separate$ ! 135$ cultures:$5$ng/$mL$Tetracycline$(for$the$induction$of$chaperone$proteins$GroES,$and$GroEL);$4$ mg/$mL$of$Arabinose$(for$the$induction$of$chaperone$proteins$DnaK,$DnaJ,$and$GrpE),$5$ng/$mL$ Tetracycline,$and$4$mg/$mL$of$Arabinose;$5$ng/$mL$Tetracycline,$and$2$%$ethanol$(to$increase$ solubility).$To$evaluate$optimal$temperature$expression$conditions,$each$condition$was$also$ grown$at$three$different$temperatures$(16°C,$27°C,$and$37°C).$All$these$conditions$resulted$in$ 90$different$set6ups$(indeed,$I$was$that$crazy!).$All$these$cultures$were$shaken$for$another$72$ hours,$taking$1.5$mL$samples$at$0.5,$1,$2,$4,$8,$12,$16,$20,$24,$28,$32,$36,$40,$48,$and$72$h$post$ induction.$These$samples$were$spun$down$as$described$previously$and$kept$at$680°C$until$ analysis.$At$the$end$of$the$experiment,$the$remainder$was$spun$down$at$4,400$x$g$for$30$min$at$ 4°C$and$the$pellets$were$kept$at$680°C$until$further$analysis.$Most$of$the$1350$different$ samples$were$analyzed$for$expression$levels,$and$solubility$of$fusion$protein.$$ Determination*of*cellular*location*of*the*21/hydroxylase*fusion*proteins*by*cell* fractionation:*First,$the$cell$pellets$that$were$obtained$from$the$1.5$mL$mini6cultures$were$ suspended$in$150$μL$of$freshly$prepared$solution$of$lysozyme$(1$mg/$mL),$20$%$sucrose,$30$mM$ Tris$HCl$(pH$8.0),$1$mM$EDTA$(Ethylenediaminetetraacetic$acid)$(pH$8.0),$and$placed$on$ice$for$ 10$min.$Cells$were$then$recovered$by$centrifugation$in$a$tabletop$microcentrifuge$at$12,000$ rpm$for$1$min$at$4°C.$Second,$the$resulting$pellet$was$resuspended$in$400$μL$of$0.1$M$Tris$HCl$ (pH$8.0)$and$cells$were$broken$open$by$freezing$and$thawing$for$three$times$in$liquid$nitrogen.$ The$suspension$was$spun$down$for$5$min$at$12,000$rpm$at$4°C.$Third,$membrane$proteins$were$ solubilized$by$incubating$the$pellet$in$500$μL$1$%$Triton$X6100$for$10$min$at$4°C.$The$solution$ ! 136$ was$spun$again$for$5$min$at$12,000$rpm$at$4°C$and$all$obtained$fractions,$including$the$ remaining$pellet,$were$loaded$onto$SDS6PAGE$for$analysis.$$ Large/scale*expression*of*fusion*proteins*21/hydroxylase:*For$electro6elution,$the* modified$canine$216hydroxylase$in$pET42b/$pGKJ6E8$and$the$modified$canine$216hydroxylase$in$ pGEX65X63/$pGKJ6E8$were$chosen.$From$each$construct,$a$10$mL$LB$culture$with$the$ appropriate$antibiotics$was$grown$overnight$at$37°C$under$constant$shaking.$The$next$day,$this$ culture$was$transferred$into$1000$mL$of$LB,$again$containing$the$appropriate$antibiotics.$The$ culture$was$shaken$constantly$at$37°C.$Once$the$OD$reached$between$0.4$and$0.6,$the$culture$ was$allowed$to$cool$down$to$27°C.$To$each$culture,$1$mM$δ6aminolevulinic$acid$was$added.$216 hydroxylase$fusion$protein$expression$was$induced$with$1$mM$IPTG$and$the$culture$was$shaken$ for$another$38$hours.$For$electro6elution,$the$cultures$were$split$into$100$mL$aliquots$and$spun$ down$at$4°C$for$30$min$at$4,400$x$g$and$the$pellets$were$kept$at$680°C$until$further$analysis.$ Fractionation$was$done$as$described$previously,$increasing$the$buffer$volumes$of$the$first$and$ second$step$to$10$mL,$and$of$the$third$to$15$mL.$* $ For$ELISA$development,$pET42b/$pGKJ6E8$was$used$and$the$whole$1$L$bacteria$culture$ was$centrifuged$at$4,400$x$g$(F10S$rotor)$for$30$min$at$4°C$and$pellets$were$kept$at$680°C$until$ needed.$$ $ $ ! 137$ Results$ * Constructs:*The$translated$sequence$was$compared$with$the$modified$canine$216 hydroxylase$and$7$silent$mutations$were$detected$(T659C,$G1011A,$A1143G,$A1278C,$C1386T,$ C1539T,$A1581G).$The$constructs$were$therefore$used$during$the$remainder$of$this$study.$* Small/scale*induction*and*determination*of*cellular*location*of*the*21/hydroxylase* fusion*proteins*by*cell*fractionation:*The$layout$of$the$experiment$enabled$me$to$confidently$ identify$the$expressed$216hydroxylase$fusion$proteins$(216hydroxylase6GST:$75$kDA,$216 hydroxylase6His:$50$kDa)$as$well$as$chaperone$proteins$DnaK$(70$kDa),$DnaJ$(40$kDa),$GrpE$(22$ kDa),$GroES$(10$kDa),$and$GroEL$(60$kDa),$by$comparing$the$uninduced$cultures$and$the$ different$conditions$with$one$another,$respectively.$It$also$became$clear$that$induction$of$DnaK$ made$identification$of$216hydroxylase6GST$difficult.$Protein$fractionation$enabled$me$to$ determine$the$exact$location$of$the$expressed$modified$canine$216hydroxylase.$The$first$ fraction$represents$the$periplasmic,$the$second$the$cytoplasmic,$and$the$third$fraction$the$ membrane$bound$protein.$The$Laemmli$buffer$resuspended$left6over$pellet$represents$the$ inclusion$body$protein.$In$short,$analysis$by$SDS6PAGE$showed$that$most$of$the$expressed$216 hydroxylase$remained$in$the$insoluble$fraction,$independent$of$construct,$expression$ temperature,$addition$of$ethanol,$OD$at$induction,$and$the$co6expression$of$chaperone$ proteins.$The$host$cell$did$not$have$an$effect$on$expression$yield,$either.$Optimal$conditions$for$ expression$were$established$to$be$38$hours$at$27°C$without$the$induction$of$chaperone$ proteins.$The$expression$system$containing$the$modified$canine$216hydroxylase$in$pET42b$and$ pGKJ6E8$resulted$in$more$expression$of$the$216hydroxylase6His$than$the$expression$system$ ! 138$ with$the$modified$canine$216hydroxylase$in$pET28c$and$pGKJ6E8.$In$general,$protein$expression$ was$higher$in$BL21$(DE3)$Gold$cells$than$C41$(DE3)$cells.$$ Large/scale*expression*of*fusion*proteins*21/hydroxylase:*With$the$previously$ established$conditions,$I$was$able$to$repeatedly$express$robust$levels$of$216hydroxylase6His$and$ 216hydroxylase6GST.*** $ Discussion* $ Protein$production$in$E.!coli$enables$the$researcher$to$produce$a$large$amount$of$the$ protein$of$interest$for$further$studies.$The$most$extensively$used$expression$systems$for$the$ expression$of$recombinant$proteins$are$the$GST6$and$His6tag$fusion$protein$systems.$$ $ Unfortunately,$it$is$very$difficult$to$produce$recombinant$P450$enzymes$in$E.!coli.$It$has$ been$shown$that$modifications$and/$or$the$co6expression$of$chaperone$proteins$are$necessary.$ For$example,$successful$expression$of$216hydroxylase$in$E.!coli!depends$on$N6terminal$ 1 modification$and$co6expression$with$chaperone$proteins$GroEL$and$GroES .$The$use$of$ molecular$chaperones$GroES$and$GroEL$has$also$been$shown$to$be$essential$for$the$expression$ of$this$class$of$enzymes$such$as$mitochondrial$vitamin$D36$hydroxylase$CYP27B1 5,6 .$In$the$ CYP2B$subfamily,$it$has$been$shown$that$expression$levels$are$increased$by$truncation$of$the$N6 7 1 terminal$anchor .$The$same$has$been$confirmed$for$several$other$P450$enzymes .$Other$groups$ replaced$the$basic$region$of$difficult$to$express$P450s$with$ones$that$can$be$efficiently$ ! 139$ expressed.$The$basic$regions$of$CYP2C11$and$CYP17$have$been$used$to$increase$the$expression$ 8,9 of$the$aromatase$CYP19 $and$that$of$CYP2E1$has$been$used$for$the$overproduction$of$CYP2C8$ 10 and$CYP2A6 .$Arase$and$Waterman$used$the$modified$sequence$of$the$basic$region$ MAKKTSSKGK$from$CYP2C3$for$the$successful$expression$of$bovine$P450c21$upon$co6expression$ 1 with$chaperones$GroES$and$GroEL .$This$modified$sequence$had$previously$been$used$for$the$ 11 expression$of$soluble$and$monomeric$forms$of$P450s .$$$ $ Besides$these$difficulties,$key$points$in$the$production$of$soluble,$intact$fusion$proteins$ are$to$optimize$several$factors$including$selection$of$host$strain,$growth$temperature,$cell$ density$at$time$of$induction,$length$of$expression,$addition$of$ethanol$to$growth$media,$and$co6 12 expression$of$chaperone$proteins .$In$both$above6mentioned$systems,$the$expression$of$ inserts$is$under$the$control$of$the$IPTG6inducible$tac$promoter.$Both$the$pGEX$vector,$as$well$as$ the$used$pET$vectors,$have$an$internal$lacI$gene,$whose$product$binds$to$the$operator$region$of$ the$tac$promoter.$This$binding$prevents$expression$until$induction$by$IPTG.$Even$though$this$ represents$a$tight$control$over$expression$of$insert,$basal$levels$of$expression$(leaky$expression)$ might$take$place,$making$it$more$difficult$to$identify$the$protein$of$interest$in$the$expression$ sample$compared$to$the$uninduced$control.$However,$my$controls$allowed$me$to$clearly$ identify$the$protein$of$interest.$$ A$wide$variety$of$E.!coli$host$strains$can$be$used$for$cloning$and$expression$with$pGEX$ and$pET$vectors.$Specially$engineered$strains,$which$are$more$suitable$for$expression$of$fusion$ proteins,$are$preferred.$An$example$of$such$a$host$is$E.!coli$strain$BL21$(DE3),$a$strain$defective$ ! 140$ in$OmpT$and$Lon$protease$production 13616 .$In$this$work,$strains$BL21$(DE3)$and$C41$(DE3)$were$ compared.$The$C41$(DE3)$is$a$mutant$host$strain$of$the$BL21$(DE3),$which$was$selected$for$ growing$to$high$saturation$density$and$for$the$continuous$production$of$proteins$without$toxic$ 17 effects$when$compared$to$the$regular$BL21$(DE3)$cells .$C41$(DE3)$host$strain$is$frequently$ used$to$overcome$the$toxicity$associated$with$overexpressing$recombinant$proteins$that$use$ 17 the$bacteriophage$T7$RNA$polymerase$expression$system .$Often$times$sufficient$protein$ expression$can$not$be$achieved$in$BL21$(DE3)$cells$because$of$bacterial$cell$death$caused$by$the$ foreign$protein.$My$studies$have$shown$that$the$regular$host$strain$BL21$(DE3)$was$superior.$ High6level$expression$of$fusion$proteins$might$result$in$the$formation$of$an$insoluble$ product,$called$inclusion$body.$An$inclusion$body$is$the$formation$of$a$dense$precipitated$ aggregate$in$which$the$expressed$fusion$protein$is$complexed$with$RNA,$making$it$very$difficult$ to$get$into$solution.$To$avoid$formation$of$inclusion$bodies,$a$variety$of$growth$parameters$ should$be$investigated:$lowering$the$temperature$to$between$16°C$and$30°C,$inducing$for$a$ shorter$time,$inducing$at$a$higher$cell$density,$adding$ethanol$to$the$growth$media,$the$co6 expression$of$chaperone$proteins,$and$increasing$aeration.$All$possibilities$were$evaluated,$but$ the$216hydroxylase$fusion$proteins$primarily$remained$in$the$insoluble$fraction.$Chaperone$ proteins$have$been$shown$to$increase$solubility$of$expressed$fusion$proteins.$The$formation$of$ inclusion$bodies$can$be$a$result$of$improper$folding$of$expressed$proteins$and$chaperone$ proteins$are$involved$in$the$proper$protein$folding$process.$$ Since$none$of$the$suggested$techniques$worked,$I$decided$to$use$the$formation$of$ inclusion$bodies$as$a$means$to$purify$the$protein$of$interest,$even$though$the$solubilization$ ! 141$ later$on$proved$to$be$difficult,$too.$$ $ ! $ 142$ APPENDIX ! 143$ Appendix$ Figure$V.1:$The$216hydroxylase6GST$fusion$protein$is$primarily$found$in$the$insoluble$ fraction.$(6):$uninduced;$(+):$induced$with$IPTG.$$ Supernatant) Pellet) 21.OH.GST) $ $ ! $ 144$ Table$V.1:$Overview$of$the$final$expression$systems.$ Plasmids$ • Modified$canine$216 Tag$ Protein$(inducer)$ Resistant$marker$ • N6terminal$glutathione$ • Modified$canine$216 • Ampicillin$ hydroxylase$in$pGEX6 S*transferase$(GST)! hydroxylase$(IPTG)$ $ 5X63$ $ $ $ • pGKJ6E8$ • None$ • DnaK6DnaJ6GrpE$(L6 • Chloramphenicol$ Arabinose)$ • GroES6GroEL$(Tetra)$ • Modified$canine$216 hydroxylase$in$pET28c$ • pGKJ6E8$ • N6terminal$6x$histidine$ • Modified$canine$216 • Kanamycin$ $ hydroxylase$(IPTG)$ • None$$ $ • DnaK6DnaJ6GrpE$(L6 • Chloramphenicol$ Arabinose)$ GroES6GroEL$(Tetra)$ • Modified$canine$216 hydroxylase$in$pET42b$ • pGKJ6E8$ • C6terminal$6$x$histidine$ • Modified$canine$216 • Kanamycin$ $ hydroxylase$(IPTG)$ • None$ • DnaK6DnaJ6GrpE$(L6 • Chloramphenicol$ Arabinose)$ GroES6GroEL$(Tetra)$ $ $ ! $ $ 145$ $ Figure$V.2:$The$soluble$proteins$expressed$in$pET42b/$pGKJ6E8,$clearly$showing$that$ hardly$any$216hydroxylase6His$is$in$this$fraction.$Different$conditions$were$tested$(IPTG:$1$mM$ IPTG;$AA:$1$mM$δ6aminolevulinic$acid;$Neg:$no$IPTG;$Tetra:$5$ng/$mL$Tetracycline;$Arab:$4$mg/$ mL$of$Arabinose),$which$enabled$me$to$identify$the$216hydroxylase$and$the$chaperone$proteins,$ and$determine$their$location.$$ $ $ $ $ ! 146$ Figure$V.3:$Inclusion$body$proteins$expressed$in$pET42b/$pGKJ6E8,$showing$that$the$216 hydroxylase6His$is$primarily$in$this$fraction.$Different$conditions$were$tested$(IPTG:$1$mM$IPTG;$ AA:$1$mM$δ6aminolevulinic$acid;$Neg:$no$IPTG;$Tetra:$5$ng/$mL$Tetracycline;$Arab:$4$mg/$mL$of$ Arabinose),$which$enabled$me$to$identify$the$216hydroxylase$and$the$chaperone$proteins,$and$ determine$their$location.$$ $ $ $ $ $ ! $ 147$ Figure$V.4:$Fractionation$of$pET42b/$pGKJ6E8,$demonstrating$that$the$216hydroxylase6 His$fusion$protein$is$expressed$into$inclusion$bodies$(induced$with$IPTG$to$the$left,$without$IPTG$ to$the$right).$$$ $ $ $ $ $ $ $ ! 148$ Figure$V.5:$Fractionation$of$pGEX65X63/$pGKJ6E8,$demonstrating$that$the$216 hydroxylase6GST$fusion$protein$is$expressed$into$inclusion$bodies$(induced$with$IPTG$to$the$left,$ without$IPTG$to$the$right).$$ $ $ $ $ ! 149$ REFERENCES ! 150$ References$ $ 1.$ Arase$M,$Waterman$MR,$Kagawa$N.$Purification$and$characterization$of$bovine$ steroid$216hydroxylase$(P450c21)$efficiently$expressed$in$Escherichia!coli.$Biochem!Biophys!Res! 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Endocrinol$1990;73:73680.$ $ 3.$ Jochum$C,$Beste$M,$Stone$D,$et$al.$Development$and$in$vitro$characterization$of$ canine$CD406Ig.$Vet!Immunol!Immunopathol$2008;123:2606265.$ $ 4.$ Sambrook$J,$Russell$DW.$Molecular!cloning:!A!laboratory!manual.$Molecular$ Cloning:$A$laboratory$manual,$3rd$ed.$Cold$Spring$Harbor:$Laboratory$Press,$2001:1326150.$ $ 5.$ Uchida$E,$Kagawa$N,$Sakaki$T,$et$al.$Purification$and$characterization$of$mouse$ CYP27B1$overproduced$by$an$Escherichia!coli!system$coexpressing$molecular$chaperonins$ GroEL/ES.$Biochem!Biophys!Res!Commun$2004;323:5056511.$ $ 6.$ Sakaki$T,$Kagawa$N,$Yamamoto$K,$et$al.$Metabolism$of$vitamin$D3$by$ cytochromes$P450.$Front!Biosci$2005;10:1196134.$ $ 7.$ Scott$EE,$Spatzenegger$M,$Halpert$JR.$A$truncation$of$2B$subfamily$cytochromes$ P450$yields$increased$expression$levels,$increased$solubility,$and$decreased$aggregation$while$ retaining$function.$Arch!Biochem!Biophys$2001;395:57668.$ $ 8.$ Kagawa$N,$Cao$Q,$Kusano$K.$Expression$of$human$aromatase$(CYP19)$in$ Escherichia!coli$by$N6terminal$replacement$and$induction$of$cold$stress$response.$Steroids$ 2003;68:2056209.$ $ 9.$ Kagawa$N,$Hori$H,$Waterman$MR,$et$al.$Characterization$of$stable$human$ aromatase$expressed$in$Escherichia!coli.$Steroids$2004;69:2356243.$ ! 151$ $ 10.$ Iwata$H,$Fujita$K,$Kushida$H,$et$al.$High$catalytic$activity$of$human$cytochrome$ P450$co6expressed$with$human$NADPH6cytochrome$P450$reductase$in$Escherichia!coli.$Biochem! Pharmacol$1998;55:131561325.$ $ 11.$ Cosme$J,$Johnson$EF.$Engineering$microsomal$cytochrome$P450$2C5$to$be$a$ soluble,$monomeric$enzyme.$Mutations$that$alter$aggregation,$phospholipid$dependence$of$ catalysis,$and$membrane$binding.$J!Biol!Chem$2000;275:254562553.$ $ 12.$ Smith$DB,$Johnson$KS.$Gene.$Gene,$1988:31640.$ $ 13.$ Baker$TA,$Grossman$AD,$Gross$CA.$A$gene$regulating$the$heat$shock$response$in$ Escherichia$coli$also$affects$proteolysis.$Proc!Natl!Acad!Sci!U!S!A$1984;81:677966783.$ $ 14.$ Strauch$KL,$Beckwith$J.$An$Escherichia!coli!mutation$preventing$degradation$of$ abnormal$periplasmic$proteins.$Proc!Natl!Acad!Sci!U!S!A$1988;85:157661580.$ $ 15.$ Grodberg$J,$Dunn$JJ.$ompT$encodes$the!Escherichia!coli$outer$membrane$ protease$that$cleaves$T7$RNA$polymerase$during$purification.$J!Bacteriol$1988;170:124561253.$ $ 16.$ Sugimura$K,$Higashi$N.$A$novel$outer6membrane6associated$protease$in$ Escherichia!coli.$J!Bacteriol$1988;170:365063654.$ $ 17.$ Miroux$B,$Walker$JE.$Over6production$of$proteins$in$Escherichia!coli:$mutant$ hosts$that$allow$synthesis$of$some$membrane$proteins$and$globular$proteins$at$high$levels.$J! Mol!Biol$1996;260:2896298.$ $ $ ! 152$ Chapter$VI:$Purification$of$fusion$proteins$218hydroxylase8glutathione$S8 transferase$(218hydroxylase8GST)$and$218hydroxylase86xhistidine$(218 hydroxylase8His)$ $ Introduction$ $ The$purification$of$the$218hydroxylase$fusion$proteins$remained$very$difficult,$because$ most$of$the$expressed$protein$was$in$the$insoluble$inclusion$bodies,$even$after$optimization$of$ expression$conditions$as$discussed$previously.$Pure$fusion$proteins$were$required,$however,$for$ immunization$of$dogs$and$rabbits$for$antibody$production$(218hydroxylase8GST)$(218 hydroxylase8glutathione$S8transferase),$for$running$controls$(control$by$size)$during$SDS8PAGE$ (sodium$dodecyl$sulfate$polyacrylamide$gel$electrophoresis)$(218hydroxylase8GST$and$218 hydroxylase8His)$(218hydroxylase8hexa$histidine)$and$for$ELISA$(enzyme8linked$immunosorbent$ assay)$development$(218hydroxylase8His).$Since$the$requirements$and$the$amount$needed$for$ these$pure$fusion$proteins$were$different,$different$techniques$were$applied$to$purify$them.$A$ small8scale$SDS8PAGE$purification$was$performed,$followed$by$a$passive$elution$of$the$protein$ out$of$the$gel8matrix$for$proof$of$concept.$If$successful,$the$plan$was$to$scale$up$this$method$ and$to$do$an$active$elution$out$of$a$bigger$gel$to$purify$more$fusion$protein$for$the$running$ control$(by$size)$and$as$the$antigen$source$for$immunization.$The$immunization$will$be$ discussed$in$the$following$chapter$(chapter$VII).$To$create$enough$protein$for$ELISA$ ! 153$ development,$fast8protein$liquid$chromatography$(FPLC)$was$used$after$solubilization$of$the$218 hydroxylase8His$inclusion$bodies.$The$218hydroxylase8GST$was$chosen$for$injection,$because$the$ GST$tag$has$a$size$of$220$amino$acids$(roughly$25$kDa),$which$increases$the$immunogenicity$of$ the$218hydroxylase.$The$218hydroxylase8His$was$chosen$as$the$antigen$for$ELISA$development,$ since$it$is$easier$and$much$more$economical$to$purify$His8tagged$fusion$proteins$in$a$larger$ scale$than$GST8tagged$proteins.$$ $ Materials$and$methods$ $ Elution(of(the(fusion(proteins(out(of(SDS(gel2matrix:$For$the$passive$small8scale$gel$ purification,$100$mL$of$each$218hydroxylase8GST$and$218hydroxylase8His$induced$bacteria$ cultures$were$fractionated$as$described$before.$The$remaining$pellets$were$dissolved$in$3000$μL$ of$2x$SDS8PAGE$Laemmli$sample$buffer.$From$each$mixture,$100$μL$were$run$on$a$1$mm$thick$ 10$%$SDS8PAGE$gel,$using$BioRads$Protean$Mini$system$(BioRad,$Hercules,$CA,$USA)$at$constant$ 180$V$for$60$minutes.$Three$protein$staining$techniques$were$compared:$gels$were$stained$(1)$ in$0.25$M$KCl$solution,$(2)$in$Coomassie,$and$(3)$in$Coomassie,$where$only$a$cut8off$side$strip$of$ the$gel$was$stained,$followed$by$aligning$this$stained$strip$with$the$rest$of$the$gel.$After$the$ fusion$proteins$were$located,$the$218hydroxylase8GST$and$the$218hydroxylase8His$bands$were$ excised$with$a$clean$razor$blade.$When$the$bands$were$cut$out,$the$remaining$gels$were$stained$ in$Coomassie$to$determine$the$accuracy$of$the$excision.$All$cut$out$bands$were$then$carefully$ cut8up$into$small$pieces$and$transferred$into$1.5$mL$microcentrifuge$tubes$containing$500$μL$ elution$buffer$(50$mM$Tris8HCl,$150$mM$NaCl,$0.1mM$EDTA$(ethylenediaminetetraacetic$acid);$ ! 154$ pH$7.5)$and$incubated$overnight$on$a$rotary$shaker$at$30°C.$The$next$day,$the$tubes$were$ centrifuged$at$12,000$rpm$for$10$minutes$and$the$supernatants$were$carefully$pipetted$into$ clean$microcentrifuge$tubes.$An$aliquot$of$20$μL$of$each$supernatant$was$tested$for$the$ presence$of$each$fusion$protein$via$mini$10$%$SDS8PAGE$electrophoresis,$followed$by$staining$ with$Coomassie$blue.$$ $ For$the$active,$scaled8up$gel$purification,$100$mL$of$each$218hydroxylase8GST$and$218 hydroxylase8His$induced$bacteria$culture$were$fractionated$as$described$before.$The$remaining$ pellets$were$dissolved$in$3000$μL$of$2x$SDS8PAGE$Laemmli$sample$buffer$and$the$total$volume$ was$loaded$onto$a$3$mm$thick$6$%$SDS8PAGE$gel,$prepared$using$BioRad’s$Protean$II$XL$system$ (gel$size:$W$x$H$=$18.3$cm$x$20$cm),$as$shown$in$figure$VI.1.$Usually$the$gels$were$loaded$as$ follows:$molecular$marker,$small$volume$218hydroxylase$preparation,$protein$sample,$and$again$ a$small$volume$of$218hydroxylase$preparation.$For$the$small$volume$of$the$218hydroxylase$ preparation,$only$100$μL$of$the$protein$solution$were$loaded,$to$make$the$identification$and$ excision$of$the$band$of$interest$easier$by$getting$a$sharper$band.$The$gels$were$then$run$at$ constant$210$V$for$5$hours$with$cooling$with$cold$water.$The$gels$were$stained$in$Coomassie$ blue$for$30$minutes$and$then$destained$with$Coomassie$destain$solution$until$the$bands$of$ interest$were$easily$identified.$The$bands$were$then$excised$with$a$clean$razor$blade$and$ transferred$into$Coomassie$destain$solution$and$kept$at$4°C until$further$use.$$ $ For$the$electro8elution,$the$following$two$buffers$were$prepared:$soaking$buffer$(2%$ SDS$in$0.4$M$NH4HCO3),$and$elution$buffer$(0.1$%$SDS$in$0.05$M$NH4HCO3).$For$details$on$the$ ! 155$ electrophoretic$elution$tank$and$the$electrophoretic$elution$cell,$manufactured$by$C.B.S.$ Scientific$Corporation,$Del$Mar,$California,$USA,$see$figures$VI.2$and$VI.3.$A$15$kDa$cut8off$ Spectra$6$dialysis$membrane$was$soaked$in$0.1$%$NaN3$and$inserted$into$the$electrophoretic$ elution$cell.$The$previously$excised$bands$were$briefly$rinsed$with$soaking$buffer$and$cut8up$ into$1$mm$x$1$mm$cubes.$The$cubes$were$then$soaked$for$5$minutes$in$soaking$buffer$to$ remove$any$remaining$Coomassie$destain$solution$and$the$buffer$was$removed$by$suction$and$ blotting.$The$pieces$were$transferred$into$the$gel$loading$well$of$the$electrophoretic$elution$cell$ and$covered$with$soaking$buffer.$This$mixture$was$stirred$until$no$more$bubbles$would$escape$ and$DTT$(dithiothreitol)$was$added$to$a$final$concentration$of$0.1$%$(total$volume$of$the$gel$ loading$well$from$the$bottom$of$the$cell$to$the$bottom$of$the$cross$passage$is$2$mL)$and$ incubated$for$30$minutes.$After$that,$the$gel$pieces$in$soaking$buffer$were$carefully$overlaid$ with$elution$buffer$just$to$the$top$of$the$cross$passage,$which$also$filled$up$the$sample$ collection$well.$The$electrophoretic$elution$cell$was$then$inserted$into$the$elution$tank$and$ elution$buffer$was$added$to$the$elution$tank$to$a$level$just$above$the$drain$ports$in$each$ electrode$chamber$and$another$75$mL$of$elution$buffer$was$added$to$the$mixing$chamber.$A$ two8channel$peristaltic$pump$was$used$to$move$buffer$(3$mL/$min$per$line)$from$the$mixing$ chamber$to$the$electrode$chamber.$In$order$to$avoid$an$extraneous$current$path,$care$had$to$ be$taken$that$the$buffer$was$dripping$rather$than$streaming$out$of$and$into$the$electrode$ chambers.$Air$bubbles$were$removed$from$underneath$the$elution$cell$caps$by$using$a$bent$ Pasteur$pipette$and$a$constant$50$V$was$applied$for$24$hours,$cathode$near$the$gel$loading$well.$ Throughout$these$24$hours,$the$air$bubbles$underneath$the$elution$cell$caps$needed$to$be$ removed$on$a$more$or$less$regular$basis.$After$24$hours,$the$voltage$was$reversed$for$30$sec$to$ ! 156$ loosen$any$protein$that$was$stuck$to$the$dialysis$membrane$and$the$liquid$from$the$gel$loading$ well$and$the$cross$passage$was$carefully$removed$and$stored$independently.$After$that$the$ liquid$from$the$sample$collection$well$was$pipetted$up$and$down$for$10$times$to$further$loosen$ any$protein$that$was$stuck$to$the$dialysis$membrane,$transferred$into$a$clean$microcentrifuge$ tube,$and$kept$at$4°C$until$further$analysis$and$use.$The$gel$pieces$were$again$covered$with$ soaking$buffer,$containing$0.1$%$DTT,$carefully$overlaid$with$elution$buffer$to$the$top$of$the$ cross8passage$and$re8inserted$into$the$elution$tank,$containing$fresh$elution$buffer.$The$elution$ was$repeated$for$another$24$hours.$During$optimization$of$the$electro8elution,$I$also$used$a$ potential$difference$of$75$V$to$increase$protein$yield.$At$the$end$of$both$runs,$all$fractions$were$ collected$and$analyzed$via$10$%$SDS8PAGE,$loading$1$μL,$5$μL,$and$10$μL,$all$including$1x$ Laemmli$sample$buffer,$and$the$optical$density$was$compared$to$a$four8point$BSA$standard$ curve$(0.1$μg,$1$μg,$5$μg,$and$10$μg)$to$estimate$protein$concentrations,$using$the$BioRad$ ChemiDoc$XRS+$and$the$manufacturer$provided$software$Image$Lab.$ $ To$confirm$the$presence$of$218hydroxylase8GST$and$218hydroxylase8His$fusion$proteins$ in$the$electro8eluted$samples,$the$following$three$experiments$were$done:$First,$the$actual$size$ of$the$eluted$protein$was$compared$with$the$calculated$size$of$each$fusion$protein$on$SDS8 PAGE;$second,$a$standard$Western$blot$(WB)$with$both$fusion$proteins$was$carried$out,$using$ 1/10$of$the$amount$of$protein$that$was$easily$visible$on$a$Coomassie$stained$gel,$and$the$ manufacturer$recommended$concentrations$of$anti8GST$(1:200)$or$anti8His$(1:3000)$as$the$ primary$antibody$in$1$%$(w/V)$instant$nonfat$dry$milk$in$PBS8T$(1xPBS$(phosphate$buffered$ saline),$containing$1$%$Tween),$and$a$horseradish8peroxidase$labeled$anti8mouse$IgG$ ! 157$ (immunoglobulin$gamma)$antibody$in$1$%$(w/V)$instant$nonfat$dry$milk$in$PBS8T$(1:5000)$as$the$ secondary$antibody;$and$third,$the$samples$were$analyzed$using$Matrix8assisted$laser$ desorption/ionization$(MALDI).$$ 1 SDS8PAGE$was$done$according$to$Laemmli .$The$WB$technique,$immediately$following$ SDS8PAGE,$in$detail:$Polyvinylidene$fluoride$(PVDF)$membranes$were$cut$to$an$approximate$size$ of$the$SDS8PAGE$gel.$Two$Whatman$papers$per$each$PVDF$membrane$were$cut$to$a$size$slightly$ bigger$than$the$PVDF$membrane.$The$PVDF$membrane$was$then$soaked$in$100$%$methanol.$ Transfer$buffer$was$prepared$(900$mL$of$10$mM$38[cyclohexylamino]81$propane$sulfonic$acid$ (CAPS)$plus$100$mL$100$%$methanol),$which$was$poured$into$a$glass$form,$the$running$ apparatus$(BioRad)$and$a$little$bit$into$a$weight$boat.$The$PVDF$membranes$were$then$ equilibrated$in$the$weight$boat$containing$the$transfer$buffer.$The$sandwich$cast$was$then$ inspected$for$cracks$and$breaks$and$put$into$the$glass$form$containing$the$transfer$buffer,$the$ black$side$facing$down.$The$two$foam$pads$were$also$put$into$this$glass$dish$for$soaking.$The$ SDS8PAGE$gel$was$disassembled,$the$stacking$gel$was$removed$with$a$razor$blade$and$the$ remaining$gel$was$cut$on$both$sides$at$the$spacer$with$a$razor$blade.$One$of$the$soaked$foam$ pads$was$then$put$on$the$black$side$of$the$cast,$followed$by$a$Whatman$paper,$followed$by$the$ SDS8PAGE$gel.$At$this$point,$everything$was$centered$and$any$air$bubbles$were$removed.$The$ PVDF$membrane$was$then$removed$with$forceps$from$the$transfer$buffer$and$placed$on$top$of$ the$gel.$The$second$soaked$Whatman$paper$was$then$placed$on$top$of$the$membrane$and$the$ whole$sandwich$was$streaked$out$with$a$cut8off$pipette$to$increase$contact$and$to$remove$air8 bubbles.$The$second$pad$was$put$on$top,$and$the$cast$was$closed.$The$sandwich$was$then$ ! 158$ placed$into$the$transfer$apparatus,$the$black$side$facing$the$black$side$of$the$apparatus.$Each$ apparatus$could$hold$two$sandwiches.$An$ice8pack$was$then$added$and$the$whole$apparatus$ was$filled$with$transfer$buffer,$so$that$the$whole$sandwich$was$submerged$in$it.$The$transfer$ was$done$using$constant$0.51$A$for$60$min.$For$incubation$with$the$primary$antibody,$1$L$of$ 1xPBS8T,$was$prepared.$In$50$mL,$0.5$g$of$instant$nonfat$dry$milk$was$dissolved,$and$the$ antibody$was$diluted$in$it$according$to$manufacturer$recommendations$to$a$total$volume$of$1$ mL$per$membrane.$A$glass$plate$was$covered$with$stretched$parafilm.$1$mL$of$each$antibody8 containing$buffer$was$then$pipetted$onto$this$wrapped$glass$plate$and$one$membrane$was$ placed$onto$one$antibody8containing$buffer,$using$forceps,$paying$attention$not$to$introduce$ any$air8bubbles.$This$was$then$closed$with$an$old$culture8dish$lid$and$transported$into$the$cold$ room$and$incubated$overnight$at$4°C.$The$next$day,$the$membrane$was$washed$three$times$for$ 15$min$on$a$shaker$with$1xPBS8T$in$a$weight$boat.$The$horseradish$peroxidase$labeled$ secondary$antibody$was$then$diluted$in$1xPBS8T$containing$1$%$(w/v)$instant$nonfat$dry$milk$ according$to$manufacturer$recommendations$to$a$total$volume$of$10$mL$and$the$washed$ membrane$was$incubated$in$it$on$a$shaker$for$one$hour$at$room$temperature.$After$that,$the$ membrane$was$washed$three$times$as$described$previously.$The$chemiluminescence$solution$ was$then$prepared,$mixing$one$vial$of$luminol$(50$μL$of$0.44$g$of$luminol$dissolved$in$10$mL$of$ dimethyl$sulfoxide$(DMSO)),$one$vial$of$coumaric$acid$(25$μL$of$0.15$g$of$coumaric$acid$in$10$mL$ DMSO),$10$mL$of$0.1$M$Tris$(pH$8.5),$and$5$µl$of$H2O2.$The$membrane$was$then$placed$into$this$ solution$for$60$sec$and$kept$in$the$dark,$before$pictures$were$taken$on$the$ChemiDoc$XRS+.$ ! 159$ For$MALDI,$50$µl$of$the$electro8eluted$218hydroxylase8His$and$50$µl$of$the$electro8 eluted$218hydroxylase8GST$were$digested$with$1$µl$of$0.1$µg/$µl$trypsin$for$2$h$at$37°C.$The$ digested$protein$was$then$mixed$with$6$µl$of$1$%$Trifluoroacetic$acid$(TFA)$and$8.33$µl$of$6$M$ guanidine8HCl.$Three$µl$of$50$%$acetonitrile$(ACN)$in$0.1$%$TFA$in$MilliQ$H20$was$pipetted$into$a$ clean$microcentrifuge$tube.$The$C18$ZipTip$(Millipore/Fisher)$was$wetted$twice$with$100$%$ACN,$ the$ACN$was$discarded,$and$the$ZipTip$was$equilibrated$twice$with$0.1$%$TFA$in$MilliQ$H20,$ again$discarding$the$0.1$%$TFA$in$MilliQ$H20.$The$protein$mixture$from$above$was$then$bound$ to$the$tip$by$pipetting$up$and$down$10$times,$without$the$introduction$of$air.$After$that,$the$tips$ were$washed$three$times$with$0.1$%$TFA$in$MilliQ$H20,$by$taking$up$the$solution$and$discarding$ it.$Elution$of$the$protein$out$of$the$ZipTip$was$then$done$by$pipetting$up$and$down$four$times,$ without$the$introduction$of$air,$the$50$%$ACN$in$0.1$%$TFA$in$MilliQ$H20$that$has$previously$ been$dispensed$into$a$clean$microcentrifgue$tube.$This$cleaned8up$sample$was$then$submitted$ to$be$analyzed$with$MALDI$at$Michigan$State$University$(MSU),$East$Lansing,$MI,$USA,$and$the$ resulting$data$were$compared$with$a$theoretical$trypsin$digest$of$the$protein.$$ Purification(of(212hydroxylase2His(for(ELISA(development:$For$ELISA$development,$the$ 218hydroxylase8His$fusion$protein$inclusion$bodies$were$solubilized.$The$following$solubilization$ conditions$were$tested:$(1)$8$M$urea$in$TBS$(tris$buffered$saline)$(50$mM$Tris$HCL,$150$mM$NaCl$ (pH$8.0),$(2)$8$M$urea$and$5$mM$DTT$in$TBS,$(3)$8$M$urea,$5$mM$DTT,$and$5%$Triton$X8100$in$ TBS,$(4)$8$M$guanidine$and$5$mM$DTT$in$TBS,$(5)$50$mM$CHAPS$(38[(38Cholamidopropyl)$ dimethylammonio]818propanesulfonate)$in$TBS,$(6)$5$%$TWEEN820$in$TBS,$and$(7)$5$%$ ! 160$ polyoxyethylene898laurylether,$5$mM$DTT,$0.05$mg/mL$deoxyribonuclease,$and$0.2$mg/mL$ lysozyme$in$TBS.$Prior$to$use,$0.1$mM$of$the$protease$inhibitor$phenylmethylsulfonyl$fluoride$ (PMSF)$was$added$to$each$solution.$Seven$bacterial$pellets$from$100$ml$of$induced$218 hydroxylase$in$pET42$BL21$(DE3),$in$which$the$chaperone$proteins$were$not$induced,$were$ fractionated$and$spun$down$as$described$previously.$The$supernatants$were$discarded.$The$ resulting$pellets$were$washed$twice$with$20$mL$TBS8T$(0.05%$Tween$in$TBS).$Each$wash$was$ followed$by$centrifugation$at$20,000$x$g$(F218B$rotor)$at$4°C$for$15$min$and$the$supernatants$ were$discarded.$The$third$wash$was$done$with$TBS$without$the$addition$of$Tween.$After$ centrifugation$and$discard$of$the$supernatants,$5$mL$of$one$the$above8mentioned$solutions$was$ added$to$the$pellet,$which$was$then$dissolved$by$brief$sonification.$A$stir$bar$was$then$added$to$ each$and$the$samples$were$slowly$stirred$at$room$temperature$for$24$hours.$A$200$μL$aliquot$ from$each$sample$for$analysis$on$SDS8PAGE$was$collected$every$30$to$60$min.$All$aliquots$were$ spun$down$for$4$min$at$15,000$rpm$in$a$table8top$microcentrifuge.$All$supernatants$but$the$ ones$containing$guanidine$were$mixed$with$Laemmli$sample$buffer$to$a$final$concentration$of$ 1x.$The$pellets$that$did$not$contain$guanidine$were$dissolved$in$200$μL$2x$Laemmli$sample$ buffer.$To$100$μL$of$the$supernatants$of$the$guanidine$containing$samples,$900$μL$of$ice$cold$ ethanol$was$added$and$the$samples$were$kept$overnight$at$820°C.$The$guanidine$containing$ pellets$were$dissolved$in$450$μL$of$ice$cold$ethanol$and$were$also$kept$overnight$in$the$freezer.$ The$next$day,$these$samples$were$spun$down$at$15,000$rpm$for$15$min$at$4°C.$The$resulting$ pellets$were$washed$with$800$μL$of$90$%$ice$cold$ethanol$and$spun$at$15,000$rpm$for$15$min$at$ 4°C.$After$that,$pellets$were$allowed$to$air$dry$and$were$dissolved$in$100$μL$2x$Laemmli$sample$ ! 161$ buffer.$5$μL$of$each$sample$was$then$loaded$onto$SDS8PAGE.$As$a$size$control,$I$included$the$ previously$purified$and$electro8eluted$218hydroxylase8His$on$all$gels.$After$analysis$of$results,$ these$additional$solubilization$conditions$were$tested:$(1)$8$M$guanidine$and$10$mM$DTT$in$TBS,$ (2)$8$M$guanidine$and$4$mM$DTT$in$TBS,$and$(3)$8$M$guanidine$in$TBS.$The$experiment$was$ performed$in$triplicate,$comparing$the$effect$of$the$inclusion$body$wash$on$yield$and$purity$of$ protein$as$well$as$the$effect$of$the$previously$described$fractionation$of$the$protein$on$yield$ and$purity.$Otherwise,$the$experiment$was$done$the$same$way$as$described$above.$Results$ again$were$analyzed.$The$experiment$was$repeated$once$again,$using$8$M$guanidine$and$4$mM$ DTT$in$TBS,$taking$an$aliquot$every$20$minutes$over$5$hours.$After$that,$a$pull8down$was$ performed$as$proof$of$concept$and$to$evaluate$the$effect$of$DTT$on$the$resin,$using$HisPur$ Cobalt$resin$(Thermo$Scientific,$IL,$USA).$600$μL$slurry$was$pipetted$into$2$microcentrifuge$ tubes$and$washed$three$times$with$1$mL$of$H2O.$Each$wash$step$was$followed$by$ centrifugation$at$1,200$rpm$at$room$temperature$in$a$table8top$microcentrifuge$and$discard$of$ the$supernatant.$The$resin$was$then$washed$three$times$with$TBS,$including$8$M$guanidine,$and$ the$other$tube$with$TBS,$including$8M$guanidine$and$4$mM$DTT,$again,$setting$the$supernatant$ aside$after$each$centrifugation.$500$μL$of$the$solubilized$protein$plus$500$μL$of$either$TBS$with$ 8$M$Guanidine$or$TBS$with$8$M$Guanidine$and$4$mM$DTT$were$then$applied$and$the$tubes$were$ transferred$onto$a$rotary$shaker$in$the$cold$room$where$they$were$left$for$an$hour.$After$an$ hour,$the$tubes$were$centrifuged$as$mentioned$above$and$the$supernatants$were$set$aside.$The$ resin$was$then$washed$three$times$with$the$same$buffers$that$were$used$before,$this$time$ however$also$containing$20$mM$imidazole.$The$protein$was$then$eluted$from$the$resin,$again$ using$the$same$buffers$as$before,$this$time$however$containing$200$mM$imidazole.$The$ ! 162$ different$fractions$(SN$(supernatant)$post$incubation,$wash$fractions$post$incubation,$elution$ fractions)$were$analyzed$on$SDS8PAGE$post$ethanol$precipitation.$$ The$sonification$and$solubilization$condition$that$were$superior$to$the$others$and$which$ were$then$repeated$multiple$times,$now$using$1$L$of$induced$218hydroxylase$in$pET42$BL21$ (DE3),$in$which$the$chaperone$proteins$were$not$induced,$are$summarized$in$detail:$1$L$of$ induced$bacteria$cell$cultures$was$centrifuged$at$4,400$x$g$(F10S$rotor)$for$30$min$at$4°C$and$ the$supernatant$was$discarded.$The$resulting$pellet$was$kept$at$880°C$until$further$use.$The$ pellet$was$then$sonicated$on$ice$in$90$mL$Buffer$A$(50$mM$potassium$phosphate$(pH$7.4),$20$%$ glycerol,$0.1$mM$DTT,$0.1$mM$EDTA,$500$mM$sodium$acetate,$1.5$%$sodium$cholate,$and$1$%$ Tween),$including$freshly$added$0.1M$PMSF,$three$times$for$2$min$at$3.5$output$and$50$%$duty$ cycle.$The$sample$was$allowed$to$cool$down$on$ice$for$6$minutes$in$between$each$sonification$ step.$The$resulting$lysate$was$spun$down$in$an$ultracentrifuge$at$45,000$x$g$at$4°C$for$60$min.$ The$supernatant$was$discarded$and$the$pellet$was$frozen$at$880°C$until$further$use.$The$pellet$ was$then$resuspended$in$ice8cold$40$mL$TBS,$containing$8$M$guanidine,$4$mM$DTT,$and$0.1$mM$ freshly$added$0.1$mM$PMSF$and$stirred$at$room$temperature$for$2$h$20$min,$spun$down$at$ 23,500$x$g$(SS34$rotor)$at$4°C$for$30$min.$The$resulting$supernatant$was$diluted$1:4$in$TBS,$ containing$8$M$guanidine,$and$0.1$mM$freshly$added$PMSF,$before$being$purified$on$FPLC.$The$ pellet$was$discarded.$$ For$FPLC,$the$following$buffers$were$prepared:$Equilibration$buffer$(EB)$(8$M$guanidine$ in$TBS),$wash$buffer$(WB)$(8$M$guanidine,$and$10$mM$imidazole$in$TBS),$and$elution$buffer$ ! 163$ (ELB)$(8$M$guanidine,$and$500$mM$imidazole$in$TBS).$To$all$buffers$but$the$ELB,$0.1$mM$PMSF$ was$added$prior$to$use.$The$HisPur$Cobalt$resin$column$(1.5$cm$diameter)$was$prepared$the$ day$before$use,$containing$6$mL$bed8volume$(BV)$of$HisPur$Cobalt$resin,$and$washed$with$5$BV$ H2O$at$a$flowrate$(FR)$of$0.75$mL/$min.$The$day$of$use,$the$column$was$equilibrated$with$3$BV$ EB$at$a$FR$of$0.75$mL/$min.$The$diluted,$solubilized$sample$was$then$loaded$onto$the$column$at$ a$FR$of$0.25$mL/$min.$During$this$time,$the$column$was$closely$watched$to$avoid$compression.$If$ the$agarose$beads$compressed,$the$sample$was$further$diluted$with$EB$or$the$FR$was$slightly$ decreased.$After$loading$of$the$sample,$the$column$was$washed$with$6$BV$WB,$increasing$the$ FR$from$an$initial$0.25$mL/$min$ever$6$mL$by$0.25$mL/$min$to$a$final$FR$of$0.75$mL/$min,$again$ closely$looking$for$compression$of$the$agarose$beads.$The$218hydroxylase8His$was$then$eluted$ from$the$column$with$5$BV$of$ELB$at$a$FR$of$0.75$mL/min.$During$the$whole$column8purification,$ all$fractions$were$collected$and$were$analyzed$on$SDS8PAGE$gel,$after$protein$precipitation$with$ ethanol$as$described$above.$After$each$purification$run,$the$resin$was$washed$(up$to$three$ times)$with$10$BV$H2O$(FR$1$mL/min),$followed$by$10$BV$MES$buffer$(regeneration$MES$Buffer:$ 20$mM$28(N$8morpholine)8ethanesulfonic$acid,$0.1$M$sodium$chloride$(pH$5.0))$(FR$1$mL/min),$ followed$by$10$BV$H2O$(FR$1$mL/min),$followed$by$5$BV$of$a$solution$of$20$mM$sodium$ phosphate$supplemented$with$0.5$M$NaCl,$and$50$mM$EDTA$(pH$7.0)$(FR$1$mL/$min),$followed$ by$5$BV$H2O$(FR$1$mL/$min),$followed$by$5$BV$0.1$M$cobalt$(II)$sulfate$heptahydrate$(FR$1$mL/$ min),$and$finally$5$BV$H2O$(FR$1$mL/$min).$The$eluates$were$treated$in$different$ways:$(1)$a$salt$ exchange$against$TBS,$using$pre8packed$Sephadex$PD810$Desalting$Columns$(GE$Healthcare,$ ! 164$ Piscataway,$NJ,$USA),$following$the$manufacturers$instructions,$was$performed.$Further,$10$mL$ of$the$eluates$were$dialyzed$in$the$cold$room$for$12$h$against$1$L$of$(2)$TBS,$(3)$50$mM$ phosphate$buffer$(pH$7.4),$20$%$glycerol,$0.1$mM$DTT,$0.1$mM$EDTA,$and$1$%$sodium$cholate,$ (4)$100$mM$sodium$carbonate,$(5)$TBS$containing$1$M$guanidine,$(6)$TBS$containing$1$M$urea,$ (7)$TBS$containing$4$M$guanidine,$and$(8)$TBS$containing$4$M$urea,$with$a$buffer$exchange$after$ 6$h;$against$1$L$for$12$h$at$room$temperature$with$a$buffer$exchange$after$6$h$against$(9)$TBS$ containing$2$%$SDS,$1$%$glycerol,$and$1$%$28mercaptoethanol,$and$(10)$TBS$containing$2%$SDS,$ 1$%$glycerol,$and$5$mM$DTT.$Lastly,$(11)$the$eluate$was$diluted$1:2$with$TBS.$The$dialysates$ were$spun$down$for$15$min$at$15,000$rpm$in$a$tabletop$centrifuge$and$analyzed$on$SDS8PAGE$ gel$(ethanol$precipitation$in$guanidine$containing$samples$as$described$previously)$for$protein$ content$in$the$supernatants$versus$pellets.$The$salt$exchanged$sample$and$the$one$that$was$ diluted$1:2$were$also$analyzed$on$SDS8PAGE$gel,$followed$by$WB$to$confirm$the$presence$of$218 hydroxylase8His.$Some$samples$were$further$analyzed$for$suitability$for$ELISA$plate$coating,$ which$will$be$described$in$chapter$VIII.$$ $$ Results$ Elution(of(the(fusion(proteins(out(of(SDS(gel2matrix:(The$small8scale$purification$and$ passive$elution$was$successful$to$purify$small$amounts$of$218OH8GST$and$218OH8His.$Figure$VI.4$ shows$the$SDS8PAGE$gel$from$which$the$218hydroxylase8GST$was$excised.$Figure$VI.5$shows$the$ purified$218hydroxylase8GST$and$218hydroxylase8His$after$passive$elution$out$of$the$gel8matrix.$ The$three$staining$techniques$were$compared$in$order$to$see$what$condition$best$conserved$ ! 165$ the$protein$and$did$not$cause$any$protein$degradation.$There$was$no$difference$between$the$ three$different$staining$techniques$and$in$all$cases$the$band$of$interest$could$be$easily$and$ precisely$identified.$Therefore,$identification$of$the$correct$band$during$the$large8scale$ purifications$was$done$using$a$standard$Coomassie$staining$protocol.$The$large8scale$electro8 elution$was$also$successful.$The$BioRad$Protean$SDS8PAGE$gel$of$the$218hydroxylase8GST,$from$ which$the$band$was$cut8out$is$shown$in$VI.6.$The$excision$of$the$218hydroxylase8GST$band$could$ be$done$precisely.$The$presence$of$fusion$proteins$218hydroxylase8GST$and$218hydroxylase8His$ in$these$samples$was$confirmed$using$SDS8PAGE,$WB,$and$MALDI$as$described$in$materials$and$ methods$(figures$VI.7$to$VI.11).$The$sizes$of$the$fusion$proteins$were$as$expected,$and$there$ were$strong$signals$in$WB$with$the$anti8His$antibody$and$the$218hydroxylase8His,$and$the$anti8 GST$antibody$and$the$218hydroxylase8GST,$respectively.$With$MALDI,$the$resulting$spectra$of$ both$the$218hydroxylase8GST$and$218hydroxylase8His$clearly$showed$that$the$most$abundant$ protein$in$our$samples$was$218hydroxylase.$The$procedure$was$suitable$to$purify$sufficient$ amount$of$218hydroxylase8GST$to$use$as$the$antigen$source$for$antibody$production$in$both$ rabbits$and$dogs.$The$average$estimated$protein$concentration$of$our$purified$samples$was$ 1.27$μg/$μL,$with$a$range$of$0.32$to$3.21$μg/$μL.$There$was$no$difference$in$the$amount$of$the$ 218hydroxylase8GST$compared$to$the$218hydroxylase8His.$Increasing$the$electro$potential$to$75$ V$increased$the$yield$of$protein,$but$unfortunately$caused$the$protein$to$degrade$(figure$VI.12).$ Purification(of(212hydroxylase2His(for(ELISA(development:(For$the$purification$of$218 hydroxylase8His$for$ELISA$development,$the$inclusion$bodies$needed$to$be$solubilized$and$the$ purification$was$done$under$denaturing$conditions.$The$protein$precipitation,$using$ice8cold$ ethanol,$was$suitable$to$remove$the$guanidine$out$of$the$samples$to$be$able$to$analyze$them$on$ ! 166$ SDS8PAGE.$Guanidine$containing$samples$can$otherwise$not$be$analyzed$on$SDS8PAGE.$After$ analyzing$all$SDS8PAGE$gels,$it$became$apparent$that$with$TBS$containing$8$M$guanidine$and$5$ mM$DTT,$some$of$the$inclusion$body$protein$was$solubilized$(figure$VI.13)$whereas$none$of$the$ other$conditions$was$successful.$As$a$representative$example,$figure$VI.14$shows$the$negative$ attempt$to$solubilize$the$218hydroxylase8His$with$TBS$containing$8$M$urea,$5$mM$DTT,$and$5%$ Triton$X8100.$The$effect$of$different$DTT$concentrations$was$then$tested,$using$TBS$containing$8$ M$guanidine$and$0$mM$DTT,$4$mM$DTT,$and$10$mM$DTT.$These$three$conditions$were$tested$in$ triplicates,$also$testing$the$need$to$fractionate$the$protein$and$to$wash$the$inclusion$bodies$ prior$to$solubilization.$The$extra$condition$with$4$mM$DTT$was$included,$because$HisPur$Cobalt$ resin,$which$was$needed$for$the$large8scale$column$purification,$is$very$sensitive$to$reducing$ agents,$and$only$concentrations$up$to$5$mM$can$be$used.$The$data$showed$that$it$was$not$ necessary$to$fractionate$the$protein$or$to$wash$the$inclusion$bodies$prior$to$solubilization,$but$ including$DTT$in$the$solubilization$buffer$was$necessary$to$solubilize$the$218hydroxylase8His$and$ 4$mM$was$an$adequate$concentration$(figure$VI.15).$The$optimal$length$of$the$solubilization$ step$was$established$to$be$2$h$20$min.$$ In$order$to$find$the$best$conditions$for$the$column$purification,$a$pull8down$with$the$ same$resin$was$performed.$In$particular,$the$binding$between$the$solubilized$218hydroxylase8 His$and$the$HisPur$Cobalt$resin$and$the$effects$of$DTT$were$examined.$The$results$showed$that$ the$addition$of$extra$DTT$during$pull8down$was$not$necessary$(figures$VI.16).$Having$ determined$the$best$conditions,$the$purification$of$218hydroxylase8His$was$scaled$up.$During$ FPLC,$unfortunately$some$218hydroxylase8His$did$not$stick$to$the$column$and$was$lost$during$ the$loading$phase.$However,$most$protein$was$bound$to$the$HisPur$Cobalt$resin$and$washing$of$ ! 167$ the$resin$removed$most$contaminating$proteins,$resulting$in$an$apparently$pure$218 hydroxylase8His$protein$upon$elution$as$visualized$by$SDS8PAGE$after$ethanol$precipitation$of$ samples$(figures$VI.17$and$VI.18).$Unfortunately,$the$purified$218hydroxylase8His$precipitated$ and$buffer$conditions$for$storage$had$to$be$determined.$The$use$of$PD810$Desalting$columns$ resulted$in$protein$loss.$Nine$different$dialysis$conditions$were$therefore$tested,$as$described$in$ materials$and$methods.$After$12$hours$of$dialysis,$the$samples$were$collected$and$analyzed.$All$ samples$but$those$that$were$dialyzed$against$TBS$containing$4$M$urea$or$TBS$containing$4$M$ guanidine$were$cloudy,$showing$that$at$least$some$of$the$protein$was$precipitated.$All$samples$ were$spun$down$and$the$protein$concentration$in$the$pellets$and$supernatants$were$compared$ to$each$other.$The$macroscopic$observations$were$confirmed,$meaning$that$most$of$the$protein$ was$in$the$pellet$except$for$the$samples$dialyzed$against$TBS$containing$4$M$urea$or$the$TBS$ containing$4$M$guanidine$(figures$VI.19$and$VI.20).$Alternatively,$the$guanidine$samples$were$ diluted$1:2$with$TBS,$resulting$in$4$M$guanidine$and$250$mM$imidazole$as$final$concentrations.$ No$precipitation$occurred,$indicating$that$the$protein$remained$in$solution.$Looking$at$the$ intensity$of$bands$on$SDS8PAGE,$this$condition$appeared$to$have$given$the$highest$amount$of$ purified$fusion$protein$218hydroxylase8His.$Further,$this$condition$is$also$suitable$for$a$protein$ concentration$assay$(Bradford$or$bicinchoninic$acid$(BCA)),$which$is$necessary$for$estimation$of$ protein$concentration$for$ELISA$plate$coating.$Further,$it$is$acceptable$to$use$protein$mixtures$ that$contain$4$M$guanidine$and$250$mM$imidazole$for$ELISA$plate$coating.$Protein$ concentration$measurement$and$coating$of$ELISA$plates$will$be$discussed$in$chapter$VIII.$Based$ on$all$findings,$the$following$218hydroxylase8His$preparations$were$submitted$to$Oxford$ Biomedical$Research$for$evaluation$for$ELISA:$218hydroxylase8His$in$TBS$(salt$exchanged),$218 ! 168$ hydroxylase8His$in$TBS$containing$4$M$urea$(dialyzed),$218hydroxylase8His$in$TBS$containing$4$M$ guanidine$(dialyzed),$and$218hydroxylase8His$in$TBS$containing$4$M$guanidine$and$250$mM$ imidazole$(diluted).$$ $ Discussion$ $ Usually,$SDS8PAGE$is$used$as$an$analytical$tool$to$assess$protein$purification.$In$this$ experiment,$SDS8PAGE$was$used$as$an$active$step$in$the$purification$process,$because$of$its$ excellent$ability$to$resolve$individual$components$of$complex$mixtures.$After$resolving$the$ individual$components$of$the$complete$protein$mixture$via$SDS8PAGE,$the$proteins$of$interest$ were$identified$by$negative$stain$with$0.25$mM$KCl,$side$strip$staining$with$Coomassie$blue$ stain,$or$stain$of$the$entire$gel$with$Coomassie.$The$purified$proteins$were$then$passively$and$ actively$eluted$from$the$gel$matrix.$The$resulting$purified$proteins$were$identified$by$size,$WB,$ and$MALDI,$and$the$218hydroxylase8GST$fusion$protein$was$prepared$for$antibody$production$ in$two$dogs$and$two$rabbits.$The$218hydroxylase8GST$was$chosen$for$injection,$because$of$its$ size,$which$increases$the$immunogenicity$of$the$fusion$protein.$GST$has$been$shown$to$induce$ an$antibody$response$in$experimental$animals,$sometimes$to$such$a$strength$where$it$provides$ 2 partial$protection$against$a$number$of$parasitic$worms,$including$Schistosoma$and$Fasciola .$In$ addition,$the$218hydroxylase8GST$and$the$218hydroxylase8His$fusion$proteins$were$used$as$ standards$during$SDS8PAGE.$The$218hydroxylase8His$was$chosen$as$the$antigen$for$further$assay$ development,$since$it$is$easier$and$more$economical$to$purify$His8tagged$fusion$proteins$in$a$ ! 169$ larger$scale.$The$histidine$tag$consists$of$six$histidine$residues$linked$to$the$protein$and$was$ 3 invented$by$Roche .$Polyhistidine$tags$are$a$common$method$for$affinity$purification$of$ 4 recombinant$proteins$expressed$in$E.#coli .$Affinity$columns$contain$bound$metal$ions$such$as$ nickel$or$cobalt,$to$which$the$polyhistidine$tag$binds$with$high$affinity.$The$resin$is$washed$with$ buffers$containing$low$concentrations$of$imidazole$to$remove$non8specifically$bound$proteins$ and$the$protein$of$interest$is$then$eluted$with$a$buffer$containing$high$imidazole$ concentrations.$The$main$advantage$of$His8tag$affinity$purification$is$the$purity$of$the$protein$ especially$when$expressed$in$a$prokaryotic$organism.$The$purification$from$an$expression$ 5 system$in$a$eukaryotic$organism$may$require$a$tandem$affinity$purification$with$two$tags .$ Using$polyhistidine$tagged$proteins$is$also$the$option$of$choice$for$purifying$recombinant$ proteins$under$denaturing$conditions,$because$its$mode$of$action$is$dependent$only$on$the$ primary$structure$of$proteins,$whereas$GST$purification$requires$the$protein$to$be$properly$ folded.$$ $ Cholesterol$is$the$parent$compound$from$which$all$steroid$classes$are$produced.$The$ process$is$complex$and$is$mediated$by$enzymes$from$the$cytochrome$P450$family.$P450$ enzymes$are$membrane$bound,$contain$heme$and$serve$as$terminal$oxidases$in$electron8 transfer$chains$originating$in$NADPH$(nicotinamide$adenine$dinucleotide$phosphate),$or$the$ 6 hydroxysteroid$dehydrogenase$superfamily .$The$predominant$glucocorticoid$and$ mineralocorticoid$in$several$mammals,$including$the$dog,$are$cortisol$and$aldosterone,$ respectively.$These$are$synthesized$by$the$adrenal$cortex$and$characterized$by$a$hydroxyl$group$ ! 170$ at$position$C21$of$the$steroid$ring.$The$microsomal$steroid$218hydroxylase$cytochrome$P450c21$ enzyme$catalyzes$the$hydroxylation$of$progesterone$or$deoxycorticosterone$at$position$C21.$ The$deficiency$of$this$enzyme$in$humans$is$of$great$clinical$importance$and$is$called$congenital$ adrenal$hyperplasia.$It$has$also$been$recognized$that$this$enzyme$is$the$main$target$in$the$ development$of$autoimmune$Addison’s$disease$in$humans.$To$prove$whether$this$enzyme$plays$ the$same$important$role$in$Addison’s$disease$in$dogs,$the$canine$218hydroxylase$needed$to$be$ cloned,$overexpressed$in$E.#coli$and$purified.$The$canine$218hydroxylase$is$structurally$closely$ 7 related$to$the$P450c21$from$other$species$and$is$only$found$in$the$adrenals .$Because$of$the$ structural$similarities$to$the$bovine$enzyme,$we$expected$that$the$modifications$suggested$by$ 8 Arase$and$Waterman $would$yield$similar$results$for$the$expression$of$canine$218hydroxylase$in$ E.#coli.$In$this$study,$the$anchor$and$basic$region$of$the$218hydroxylase$was$replaced$and$the$ 8 protein$was$co8expressed$with$chaperone$proteins$significantly$increasing$the$overproduction .$$ Unfortunately,$the$same$strategy$did$not$result$in$increased$overexpression$of$the$ canine$218hydroxylase.$In$short,$the$results$remained$unsatisfactory$and$the$protein$was$ primarily$found$in$the$insoluble$fraction$independent$of$the$tag$and$the$condition$used.$P450$ 8816 enzymes$are$known$to$be$notoriously$difficult$to$overexpress .$For$assay$development,$it$is$ not$absolutely$necessary$to$obtain$a$protein$in$its$native$form,$even$though$sometimes$ preferred$for$ELISAs.$I$therefore$decided$to$solubilize$the$protein$from$the$inclusion$bodies$and$ purify$it$under$denaturing$conditions.$To$solubilize$the$inclusion$bodies,$a$variety$of$anionic,$ cationic,$and$zwitterionic$detergents,$as$well$as$ethoxylates$were$tested,$and$only$8$M$ ! 171$ guanidine$in$the$presence$of$4$mM$DTT$gave$the$desired$results.$After$the$optimal$conditions$ were$found,$the$purification$was$relatively$straight8forward,$even$though$very$time8consuming$ and$expensive.$$ For$the$FPLC$purificartion,$HisPur$Cobalt$resin$was$chosen$rather$than$a$Ni(2+)8based$ resin,$because$although$Ni(2+)$chelate$resins$achieve$high$protein$yields,$proteins$from$E.#coli$ can$bind$indiscriminantly,$resulting$in$suboptimal$purity$and$the$requirement$of$additional$ 4 cleanup$steps ,$whereas$cobalt$maximizes$protein$purity$without$sacrificing$protein$yield.$ HisPur$Cobalt$Resin$binds$fewer$contaminating$proteins,$such$as$FKBP8type$peptidyl$prolyl$ 3 isomerase$from$E.#coli .$Cobalt$also$displays$less$metal$leaching$and$enables$less$stringent$ elution$conditions$than$nickel$resins.$However,$the$present$purification$scheme$is$time8$and$ labor$consuming$because$the$column$has$to$be$constantly$watched$during$the$purification$to$ avoid$damages$due$to$overpressure.$The$viscosity$of$the$solubilized$inclusion$bodies$required$ dilution$of$the$sample$by$at$least$1:4$in$connection$with$a$FR$of$no$more$than$0.25$mL/min,$ which$meant$that$the$loading$of$the$protein$onto$the$column$alone$took$at$least$10.5$h.$Minor$ compression$of$the$resin$was$reversible$and$could$be$counteracted$with$further$dilution$of$the$ sample$or$with$decreasing$the$FR.$If$the$compression$got$too$severe$however,$the$resin$was$ damaged,$dramatically$decreasing$its$binding$capacity.$The$addition$of$8$M$guanidine$to$all$ buffers$adds$to$the$costs$and$has$to$be$taken$into$account$when$scaling$the$procedure$up.$ Approximately$500$g$of$guanidine$for$every$1$L$of$induced$bacteria$culture$was$needed.$$ ! 172$ To$summarize,$the$protocol$developed$here$results$in$an$apparently$pure$canine8218 hydroxylase,$is$reproducible$and$produced$sufficient$amounts$for$the$development$of$an$ELISA.$ ! 173$ APPENDIX ! 174$ Appendix$ Figure$VI.1:$BioRad’s$Protean$II$XL$system$ $ $ ! $ 175$ Figure$VI.2:$Electrophoretic$elution$tank.$(a)$Top$view;$(b)$side$view;$(c)$end$view;$(d)$ port$connector$for$pump$tubing.$A,$terminal$lug;$B,$platinum$wire$electrode;$C,$baffle$plate;$D,$ separation$plate$for$electrode$chambers;$E,$slot$for$elution$cell;$F,$drain$trough;$G,$mirrored$ surface;$H,$to$pump$tubing;$I,$to$tank$chamber;$J,$set$screw.$Plexiglas$covers$for$tank$chambers$ are$not$shown.$(from:$Methods$in$Enzymology,$Volume$91,$Enzyme$Structure$Part$1,$1983,$page$ 229).$ $ ! 176$ Figure$VI.3:$Electrophoretic$elution$cell.$A,$gel$loading$well;$B,$sample$collection$well;$C,$ Spectra/Por$disk;$D,$silicon$rubber$washer;$E.$screw$cap$with$open$top;$F,$peg$for$holding$slot$in$ elution$tank;$G,$cross$passage.$(from:$Methods$in$Enzymology,$Volume$91,$Enzyme$Structure$ Part$1,$1983,$page$229).$ $ ! 177$ Figure$VI.4:$SDS8PAGE,$showing$the$218hydroxylase8GST$prior$to$gel8purification$and$ small$scale$passive$elution.$ $ $ $ $ $ $ $ $ ! $ 178$ Figure$VI.5:$SDS8PAGE,$showing$the$purified$218hydroxylase8His$and$218hydroxylase8GST$ post$gel$purification$and$passive$elution$out$of$the$gel8matrix.$$ $ $ $ $ $ ! 179$ Figure$VI.6:$SDS8PAGE$maxi$gel,$after$the$218hydroxylase8GST$was$excised.$Using$ Coomassie$stain,$it$was$possible$to$accurately$identify$the$band$of$interest.$$ $ $ $ $ $ ! 180$ Figure$VI.7:$SDS8PAGE,$showing$the$gel8purified$and$electro8eluted$218hydroxylase8His$in$ comparison$to$BSA.$The$known$concentrations$of$BSA$were$used$to$estimate$the$concentration$ of$the$electro8eluted$218hydroxylase8His.$$ $ ! 181$ Figure$VI.8:$SDS8PAGE,$showing$the$gel8purified$and$electro8eluted$218hydroxylase8GST$ in$comparison$to$BSA.$The$known$concentrations$of$BSA$were$used$to$estimate$the$ concentration$of$the$electro8eluted$218hydroxylase8GST.$$ $ $ ! $ 182$ Figure$VI.9:$WB$of$the$electro8eluted$218hydroxylase8His,$showing$that$the$purified$ protein$reacts$strongly$with$an$antibody$against$His.$(From$left$to$right:$lane$1:$marker,$lanes$2$ through$5:$218hydroxylase8His$from$different$electro8elution$runs).$ $ ! 183$ $ Figure$VI.10:$WB$of$the$electro8eluted$218hydroxylase8GST,$showing$that$the$purified$ protein$reacts$strongly$with$an$antibody$against$GST.$(From$left$to$right:$lane$1:$Marker,$lanes$2$ through$4:$218hydroxylase8GST$from$different$electro8elution$runs).$ $ ! 184$ Figure$VI.11:$MALDI$spectra$of$the$218hydroxylase8GST.$The$peaks$are$similar$to$the$ ones$of$the$theoretical$trypsin$digest$of$the$218hydroxylase8GST.$$ $ $ $ $ $ ! $ 185$ Figure$VI.12:$SDS8PAGE,$showing$the$gel8purified$and$degraded$electro8eluted$218 hydroxylase8GST,$after$increasing$the$electro8potential$during$electro8elution$from$50$V$to$75$V.$$ $ ! 186$ Figure$VI.13:$Solubilization$of$218hydroxylase8His$inclusion$bodies$with$TBS$containing$8$ M$guanidine,$and$5$mM$DTT$over$24$hours.$Some$of$the$218hydroxylase8His$is$found$in$the$ supernatant.$(SN:$supernatant;$P:$pellet).$$ $ $ $ $ $ $ $ ! $ 187$ Figure$VI.14:$Solubilization$of$218hydroxylase8His$inclusion$bodies$with$TBS$containing$8$ M$urea,$5$mM$DTT,$and$5%$Triton$X8100$over$24$hours.$The$218hydroxylase8His$remains$in$the$ pellet.$(SN:$supernatant;$P$=$pellet).$$ $ $ $ $ $ $ ! $ 188$ Figure$VI.15:$Solubilization$of$218hydroxylase8His$inclusion$bodies$with$TBS$containing$8$ M$guanidine$versus$TBS$containing$8$M$guanidine,$and$4$mM$DTT$after$2$h$20$min.$The$yield$of$ 218hydroxylase8His$in$the$supernatant$is$increased$with$DTT.$(SN:$supernatant;$P:$pellet).$$ $ ! 189$ $ Figure$VI.16:$Pulldown$of$solubilized$218hydroxylase8His$in$TBS$containing$8$M$guanidine$ with$HisPur$cobalt$resin,$without$the$addition$of$additional$DTT.$The$218hydroxylase8His$was$ solubilized$in$TBS,$containing$8$M$guanidine$and$4$mM$DTT.$The$sample$was$diluted$1:2$with$ TBS$prior$to$pulldown,$decreasing$the$final$DTT$concentration$to$2$mM.$(SN:$supernatant$ (unbound$protein$post$incubation$with$HisPur$resin);$W:$wash$fractions;$E:$elution$fractions).$ $ $ $ $ $ ! 190$ Figure$VI.17:$FPLC$of$218hydroxylase8His,$loading$and$wash$fractions.$Contaminating$ proteins$do$not$bind$to$the$HisPur$cobalt$resin$and$are$washed$off$during$washing.$Some$of$the$ 218hydroxylase8His$however$is$lost$during$loading$and$washing.$(SN,$induced:$induced$bacteria$ culture$post$sonification$and$ultracentrifugation,$supernatant;$P,$uninduced:$uninduced$ bacteria$culture$post$sonification$and$ultracentrifugation,$pellet$(negative$control);$P,$induced:$ induced$bacteria$culture$post$sonification$and$ultracentrifugation,$pellet$(positive$control);$L:$ loading$fraction,$number$corresponds$to$the$collected$fraction$number;$W:$wash$fraction,$ number$corresponds$to$the$collected$fraction$number).$ $ $ ! 191$ Figure$VI.18:$FPLC$of$218hydroxylase8His,$wash$and$elution$fractions.$Some$of$the$218 hydroxylase8His$was$lost$during$washing,$but$most$was$eluted$with$high$imidazole$ concentration.$(W:$wash$fraction,$number$corresponds$to$the$collected$fraction$number;$E:$ elution$fraction,$number$corresponds$to$the$collected$fraction$number).$ $ $ $ $ $ ! 192$ Figure$VI.19:$Localization$of$the$purified$218hydroxylase8His$post$dialysis$in$7$different$ conditions.$Most$of$the$218hydroxylase8his$remains$in$the$pellet,$except$for$TBS$containing$4$M$ guanidine,$TBS$containing$4$M$urea,$and$TBS$containing$2$%$SDS,$1$%$glycerol,$and$1$%$28 mercaptoethanol.$‘Pool’$is$the$pool$of$fractions$that$were$used$for$the$dialysis.$(P:$pellet$post$ centrifugation$of$the$dialysates).$ $ $ $ $ $ ! 193$ Figure$VI.20:$Localization$of$the$purified$218hydroxylase8His$post$dialysis$in$7$different$ conditions.$Most$of$the$218hydroxylase8his$remains$in$the$pellet,$except$for$TBS$containing$4$M$ guanidine,$TBS$containing$4$M$urea,$and$TBS$containing$2$%$SDS,$1$%$glycerol,$and$1$%$28 mercaptoethanol,$in$which$the$majority$was$found$in$the$SN.$‘Pool’$is$the$pool$of$fractions$that$ were$used$for$the$dialysis.$(SN:$supernatant$post$centrifugation$of$the$dialysates).$ $ ! 194$ REFERENCES ! 195$ References$ $ 1.$ Laemmli$UK.$Cleavage$of$structural$proteins$during$the$assembly$of$the$head$of$ bacteriophage$T4.$Nature$1970;227:6808685.$ $ 2.$ Pillai$S,$Dermody$K,$Metcalf$B.$Immunogenicity$of$genetically$engineered$glutathione$ S8transferase$fusion$proteins$containing$a$T8cell$epitope$from$diphtheria$toxin.$Infect#Immun$ 1995;63:153581540.$ $ 3.$ Hochuli$E.$Purification$of$recombinant$proteins$with$metal$chelate$adsorbent.$Genet# Eng#(N#Y)$1990;12:87898.$ $ 4.$ Hengen$P.$Purification$of$His8Tag$fusion$proteins$from$Escherichia#coli.$Trends# Biochem#Sci$1995;20:2858286.$ $ 5.$ Gavin$AC,$Bosche$M,$Krause$R,$et$al.$Functional$organization$of$the$yeast$proteome$ by$systematic$analysis$of$protein$complexes.$Nature$2002;415:1418147.$ $ 318.$ 6.$ Miller$WL.$Molecular$biology$of$steroid$hormone$synthesis.$Endocr#Rev$1988;9:2958 $ 7.$ Martineau$I,$Belanger$A,$Tchernof$A,$et$al.$Molecular$cloning$and$expression$of$ guinea$pig$cytochrome$P450c21$cDNA$(steroid$218hydroxylase)$isolated$from$the$adrenals.$J#Steroid# Biochem#Mol#Biol$2003;86:1238132.$ $ 8.$ Arase$M,$Waterman$MR,$Kagawa$N.$Purification$and$characterization$of$bovine$ steroid$218hydroxylase$(P450c21)$efficiently$expressed$in$Escherichia#coli.$Biochem#Biophys#Res# Commun$2006;344:4008405.$ $ 9.$ Saribas$AS,$Gruenke$L,$Waskell$L.$Overexpression$and$purification$of$the$membrane8 bound$cytochrome$P450$2B4.$Protein#Expr#Purif$2001;21:3038309.$ $ 10.$ Kagawa$N,$Cao$Q,$Kusano$K.$Expression$of$human$aromatase$(CYP19)$in$Escherichia# coli$by$N8terminal$replacement$and$induction$of$cold$stress$response.$Steroids$2003;68:2058209.$ $ 11.$ Kagawa$N,$Hori$H,$Waterman$MR,$et$al.$Characterization$of$stable$human$aromatase$ expressed$in$Escherichia#coli.$Steroids$2004;69:2358243.$ ! 196$ $ 12.$ Uchida$E,$Kagawa$N,$Sakaki$T,$et$al.$Purification$and$characterization$of$mouse$ CYP27B1$overproduced$by$an$Escherichia#coli#system$coexpressing$molecular$chaperonins$GroEL/ES.$ Biochem#Biophys#Res#Commun$2004;323:5058511.$ $ 13.$ von$Wachenfeldt$C,$Richardson$TH,$Cosme$J,$et$al.$Microsomal$P450$2C3$is$ expressed$as$a$soluble$dimer$in$Escherichia#coli$following$modification$of$its$N8terminus.$Arch# Biochem#Biophys$1997;339:1078114.$ $ 14.$ Scott$EE,$Spatzenegger$M,$Halpert$JR.$A$truncation$of$2B$subfamily$cytochromes$ P450$yields$increased$expression$levels,$increased$solubility,$and$decreased$aggregation$while$ retaining$function.$Arch#Biochem#Biophys$2001;395:57868.$ $ 15.$ Iwata$H,$Fujita$K,$Kushida$H,$et$al.$High$catalytic$activity$of$human$cytochrome$P450$ co8expressed$with$human$NADPH8cytochrome$P450$reductase$in$Escherichia#coli.$Biochem# Pharmacol$1998;55:131581325.$ $ 16.$ Cosme$J,$Johnson$EF.$Engineering$microsomal$cytochrome$P450$2C5$to$be$a$soluble,$ monomeric$enzyme.$Mutations$that$alter$aggregation,$phospholipid$dependence$of$catalysis,$and$ membrane$binding.$J#Biol#Chem$2000;275:254582553.$ $ ! 197$ Chapter$VII:$Creation$of$positive$control$sera$for$assay$development$ $ Introduction$ $ As$demonstrated$during$preliminary$experiments,$progress$was$impeded$by$the$lack$of$ positive$control$serum$for$assay$development.$Two$dogs$were$therefore$immunized$with$ modified$canine$21Fhydroxylase,$which$was$expressed$in$E.#coli.$I$was$also$attempting$to$induce$ autoimmuneFinduced$adrenal$inflammation$and$dysfunction,$which$would$be$demonstrated$via$ ACTH$(adrenocorticotropic$hormone)$stimulation$tests$and$histopathology$of$the$adrenal$ glands$at$the$end$of$this$experiment.$The$Institutional$Animal$Care$and$Use$Committee$at$ Michigan$State$University$(MSU),$East$Lansing,$MI,$USA,$approval$number$#02/08F017F00,$ approved$this$part$of$the$project.$Protein$was$also$provided$to$Oxford$Biomedical$Research,$ Rochester,$MI,$USA,$who$outsourced$the$immunization$of$two$rabbits.$The$immunization$of$ these$two$rabbits$was$done$using$standard$procedures$and$will$not$be$further$discussed.$The$ decision$to$immunize$two$rabbits$was$made$because$of$the$uncertainty$that$the$two$dogs$ would$effectively$mount$an$immune$response.$The$obtained$preF$and$postF$immunization$sera$ however$were$analyzed$for$antibody$content,$which$will$be$discussed$in$the$following$chapter.$$ $ $ $ ! 198$ Materials$and$methods$ $ Three$healthy$dogs$were$obtained$from$other$researchers$at$MSU$and$housed$at$the$ Vivarium$at$the$College$of$Veterinary$Medicine$at$MSU.$The$three$dogs$had$access$to$ad$libitum$ water,$were$fed$a$commercially$available$diet$once$a$day$in$the$morning,$and$were$dewormed$ and$vaccinated$on$a$regular$basis.$Blanket,$an$intact$male$BeagleFBriardFCorgi$mix,$was$born$in$ January$of$2007.$His$weight$throughout$the$study$was$approximately$17$kg.$Lola,$a$spayed$ female$Beagle,$was$born$in$July$of$2006.$Her$weight$throughout$the$study$was$approximately$8$ kg.$Lucy,$another$spayed$female$Beagle,$was$the$negative$control$dog$that$did$not$get$ immunized$with$recombinant$modified$canine$21FhydroxylaseFGST.$She$was$born$in$February$of$ 2006.$Her$weight$throughout$the$study$was$approximately$7$kg.$A$control$dog$that$did$not$get$ immunized$with$21FhydroxylaseFGST$was$included$to$rule$out$biopsyFinduced$inflammation$and$ dysfunction$of$the$adrenal$glands.$$ The$timeline$of$this$complete$experiment$is$summarized$in$table$VII.1.$The$different$ steps$done$are$described$in$detail$below.$In$order$to$monitor$antibody$production,$progression$ of$adrenal$inflammation$and$potential$development$of$hypoadrenocorticism,$we$collected$ adrenal$biopsies$and$were$performing$ACTH$stimulation$tests.$$ Blood%collection:$Early$experiments$by$Meek$and$Eyster$in$1921,$testing$acute$ hemorrhage$in$dogs,$determined$that$loss$of$2%$of$body$weight$(400$mL$for$a$20$kg$dog),$which$ 1 is$about$25%$of$blood$volume,$“usually$causes$no$serious$physiologic$effect ”.$$Experiences$of$ veterinarians$collecting$blood$from$blood$donors$in$the$literature$and$at$MSU$have$also$been$ ! 199$ helpful.$They$report$that$dogs$weighing$>$27$kg$have$donated$450$mL$of$blood$at$3$week$ 2 intervals$for$over$a$2$year$period$without$adverse$effects .$Our$dogs$weight$was$between$6$kg$ and$17$kg,$and$no$more$than$50$mL$of$blood$were$collected$from$the$two$smaller$dogs$and$no$ more$than$100$mL$of$blood$from$the$larger$dog$at$any$given$time.$Blood$was$collected$in$the$ morning,$after$an$overnight$fast.$Blood$was$collected$via$venipuncture$from$the$jugular$veins,$ into$10$mL$vacutainer$tubes.$Prior$to$collection,$capillary$refill$time$and$mucous$membranes$ were$checked.$The$tubes$were$left$at$room$temperature$for$30$minutes$to$allow$for$clotting,$ before$being$spun$down$in$a$bloodFcentrifuge$for$20$minutes.$The$serum$was$then$separated$ and$frozen$at$F80°C$until$use.$$ A$final$blood$collection$was$done$under$deep$standard$anesthesia$prior$to$euthanasia.$ Blood$was$collected$from$the$jugular$vein$as$well,$and$we$collected$approximately$5%$of$body$ weight$of$blood$before$the$dogs$were$euthanized.$Blood$was$then$treated$as$described$above.$$ ACTH%stimulation%test:$Approximately$10$ml$of$blood$was$collected$as$described$above$ and$5$μg/$kg$of$synthetic$ACTH$was$administered$intravenously.$Exactly$one$hour$later,$another$ blood$sample$was$collected$and$cortisol$and$aldosterone$concentrations$were$measured$via$ commercially$available$and$validated$test$at$the$Endocrinology$Laboratory,$Diagnostic$Center$ for$Population$and$Animal$Health$(DCPAH),$MSU.$The$ACTH$stimulation$test$was$performed$to$ demonstrate$adrenal$function$and$was$always$done$between$9$am$and$11$am.$$ Immunization:$Lola$and$Blanket$were$immunized$once$with$125$μg$of$the$previously$ described$purified$recombinant$modified$canine$21FhydroxylaseFGST$fusion$protein,$mixed$with$ ! 200$ complete$Freund’s$adjuvants,$and$four$additional$times$with$the$same$protein,$but$mixed$with$ incomplete$Freund’s$adjuvants,$over$a$time$of$approximately$4$months.$The$complete$and$ incomplete$adjuvants$were$mixed$1$to$1$(v/v)$with$the$antigen$and$the$final$volume$for$each$ injection$was$then$brought$up$to$1$mL$with$sterile$0.9$%$NaCl$(sodium$chloride)$solution.$The$ mixture$was$injected$subcutaneously$and$the$inoculation$sites$were$separated$between$each$ injection$(first$injection:$left$dorsal$ribcage;$second$injection:$right$dorsal$ribcage;$third$ injection:$left$dorsal$abdomen;$fourth$injection:$right$dorsal$abdomen;$fifth$injection:$left$ ventral$abdomen).$Lucy,$the$negative$control$dog,$did$not$get$immunized.$Over$the$next$two$ weeks$post$each$injection,$the$injection$site$was$observed$daily$for$swelling,$and$the$dogs$ overall$clinical$wellFbeing$was$observed$and$recorded.$$ Laparoscopic%adrenal%biopsy:%Prior$to$surgery,$the$dogs$were$induced$with$intravenous$ thiopental.$The$dogs$were$then$intubated$and$anesthesia$maintenance$was$achieved$with$ Isoflurane$in$a$semi$closed$circle$system.$The$anesthetized$dogs$were$placed$in$dorsal$ recumbency$and$the$ventral$abdomen$was$clipped$from$the$xiphoid$to$the$pubis$and$each$ lateral$flank.$The$exposed$skin$was$cleaned$with$alternating$skin$prep$agents$(alcohol$and$dilute$ chlorhexidine$surgical$scrub).$$Standard$sterile$draping$techniques$were$employed.$A$6$mm$skin$ incision$was$made$immediately$caudal$to$the$umbilicus$on$midline.$Using$blunt$dissection,$the$ linea$alba$was$then$identified.$Two$stay$sutures$were$placed$in$the$linea$alba$and$a$sharp$tipped$ 5$mm$trocar/$cannula$was$inserted$into$the$peritoneal$cavity.$A$Storz$CO2$(carbon$dioxide)$ insufflating$device$was$connected$to$the$5$mm$cannula$and$CO2$was$infused$into$the$abdomen$ at$a$rate$of$1$L/$minute$until$intraabdominal$pressure$was$12$mmHg.$A$5$mm$0$degree$rigid$ ! 201$ telescope$was$then$placed$into$the$midline$cannula$and$the$abdomen$was$explored.$Under$ direct$visualization$of$the$endoscopic$camera,$two$5$mm$instrument$cannulas$were$placed$on$ ventral$midline$3F5$cm$cranial$and$caudal$to$the$first$cannula.$To$place$these$portals,$a$2F3$mm$ stab$incision$into$the$skin$was$made$using$a$sharp$trocar$tip$to$penetrate$into$the$peritoneal$ cavity.$Once$all$three$portals$were$in$place$and$secured,$the$animal$was$placed$in$right$lateral$ recumbency.$$ A$5$mm$bluntFtipped$fan$retractor$was$then$used$to$displace$the$spleen$and$stomach$to$ the$right$side$of$the$abdomen;$this$exposed$the$left$kidney$and$left$adrenal$gland.$Once$the$left$ adrenal$gland$was$identified,$a$5$mm$laparoscopic$punch$biopsy$instrument$was$inserted$into$ the$abdomen$and$a$biopsy$of$the$adrenal$gland$was$taken$immediately$caudal$to$the$ phrenicoabdominal$vein.$Hemorrhage$was$controlled$by$direct$pressure$from$a$5$mm$blunt$ probe,$and$a$hemostatic$agent$(Gelfoam)$was$placed$on$the$biopsy$site.$Once$any$hemorrhage$ was$controlled,$the$patient$was$turned$into$left$lateral$recumbency$and$a$fan$retractor$was$ used$to$displace$the$caudate$lobe$of$the$liver$and$the$proximal$duodenum$in$a$cranialFleftF lateral$direction$to$expose$the$right$kidney$and$right$adrenal$gland.$Once$the$right$adrenal$ gland$was$identified,$the$same$biopsy$technique$was$employed.$$ The$patient$was$then$placed$back$into$dorsal$recumbency$and$the$pneumoperitoneum$ was$released$by$removing$all$three$portals.$Once$the$CO2$was$evacuated,$two$small$sutures$of$ 3F0$PDS$(polydioxanone)$were$used$in$an$interrupted$pattern$to$close$the$5$mm$defects$in$the$ linea$alba$and$3F0$Nylon$was$used$to$close$the$skin.$ ! 202$ Flank%approach%adrenal%biopsy:$Dogs$were$induced$with$intravenous$Propofol.$The$dogs$ were$then$intubated$and$anesthesia$maintenance$was$achieved$with$Isoflurane$in$a$semiFclosed$ circle$system.$The$anesthetized$dogs$were$then$placed$in$dorsal$recumbency$and$the$ventral$ abdomen$was$clipped$from$the$xiphoid$to$the$pubis$and$along$each$flank.$The$exposed$skin$was$ then$cleaned$with$alternating$antiseptics$(alcohol$and$dilute$chlorhexidine$surgical$scrub).$For$ left$adrenal$biopsy,$the$dog$was$positioned$in$right$lateral$recumbency.$Standard$sterile$draping$ techniques$were$employed.$A$10$cm$incision$was$made$caudal$to$the$last$rib,$extending$through$ skin$and$subcutaneous$tissues.$The$abdominal$musculature$was$separated$with$sharp$and$blunt$ dissection$to$allow$entry$of$the$peritoneal$cavity.$Gelpi$or$Nelson$retractors$were$used$to$ improve$exposure$through$the$incision.$The$left$adrenal$gland$was$then$identified$on$the$cranial$ pole$of$the$left$kidney.$Adhesions$from$previous$adrenal$biopsy$procedures$were$removed$with$ electrocautery$and$an$adrenal$biopsy$sample$was$obtained$with$5$mm$laparoscopic$punch$ biopsy$forceps.$Hemorrhage$from$the$biopsy$site$was$controlled$with$direct$pressure,$ electrocautery,$a$topical$hemostatic$sponge$(Gelfoam),$and$vascular$ligatures$as$needed.$The$ incision$was$closed$with$3F0$PDS$suture$in$three$layers$(body$wall,$subcutaneous$tissue,$and$ skin).$For$right$adrenal$biopsy,$the$same$technique$was$employed$with$the$dog$in$left$lateral$ recumbency.$ Necropsy:$Dogs$were$induced$with$intravenous$Propofol.$Dogs$were$then$intubated$and$ anesthesia$maintenance$was$achieved$with$Isoflurane$in$a$semiFclosed$circle$system.$After$ collecting$blood$as$described$above,$the$dogs$were$euthanized$using$an$overdose$of$ pentobarbital.$After$opening$the$dogs$up$in$midline,$both$adrenal$glands,$and$a$section$from$ the$thyroid$glands,$spleen,$kidneys,$and$liver$were$harvested$and$immediately$immersed$into$ ! 203$ 10%$neutral$buffered$formalin$for$fixation.$The$tissues$were$submitted$to$the$Histology$ Laboratory$at$the$DCPAH,$MSU,$where$they$were$embedded$in$paraffin$and$routinely$ processed$and$stained$with$hematoxylin$and$eosin$for$evaluation.$Additional$sections$of$the$ right$and$left$adrenal$glands$were$assessed$for$the$presence$of$lymphocytes,$using$ immunohistochemistry$to$identify$T$cells$and$B$cells$using$antibodies$directed$against$cell$typeF specific$antigens$(CD3F$(cluster$of$differentiation$3)$T$cells;$CD79aF$B$cells).$$ $ Results$ $ Blood%collection:$The$harvested$serum$was$clear$and$amber$in$color.$There$were$no$ negative$side$effects$for$the$dogs$from$the$blood$collection.$The$serum$was$aliquoted$and$ stored$at$F80°C$until$further$analysis$as$described$in$chapter$VIII.$$ ACTH%stimulation%test:$The$cortisol$and$aldosterone$results$are$summarized$in$tables$ VII.2$and$VII.3.$The$cortisol$and$aldosterone$concentrations$post$ACTH$are$graphed$in$figures$ VII.1$through$VII.4.$Given$only$two$treatment$animals$and$one$control$animal,$it$was$not$ appropriate$to$perform$statistical$tests$for$differences$overFtime$or$by$treatment.$Since$there$is$ variation$in$the$baseline$levels,$the$decision$was$made$to$graph$postFACTH$cortisol$and$postF ACTH$aldosterone$percentage$difference$over$time$so$that$the$control$dog$(biopsies$but$no$ injections)$could$be$compared$to$the$treated$dogs$on$the$same$basis.$The$data$indicates$that$ there$are$no$differences$attributable$to$the$immunizations$when$compared$to$the$control$dog.$ There$did$not$appear$to$be$a$consistent$effect$over$time$(multiple$biopsies)$although$ ! 204$ subjectively$cortisol$response$may$have$been$lower$on$the$final$day$01/18/2011$and$ aldosterone$response$reduced$on$11/14/2011.$Almost$all$values$remained$within$the$reference$ range,$and$Lola’s$slightly$decreased$aldosterone$concentrations$preFACTH$on$11/14/2010,$ 12/5/2010$and$1/18/2011$are$of$questionable$clinical$significance,$based$on$the$normal$ aldosterone$response$postFACTH.$The$slightly$increased$cortisol$concentrations$preFACTH$on$ 10/23/2010$and$10/23/2010,$11/14/2010,$and$12/5/2010$in$Lola$and$Lucy,$respectively,$again$ are$of$questionable$clinical$significance$and$are$most$likely$related$to$stress.$$$ % Immunization:%Other$than$minor$swelling$at$the$injection$site$for$an$average$of$8$days,$ there$were$no$negative$side$effects$for$the$dogs$from$the$immunizations.$Each$immunization$ was$given$in$a$small$final$volume$of$1$ml,$injected$into$five$different$sites$on$the$flank$of$the$ dogs.$Many$of$the$undesirable$side$effects$of$Freund’s$adjuvant$were$eliminated$through$ refinements$in$its$use.$Special$care$was$taken$in$mixing$the$adjuvants$with$the$protein$ preparation$to$get$an$emulsion,$which$decreases$side$effects.$$ Laparoscopic%adrenal%biopsy:$The$first$laparoscopic$adrenal$biopsies$went$well$and$a$ biopsy$sample$from$all$adrenal$glands$from$all$three$dogs$was$obtained.$The$dogs$recovered$ well$and$there$were$no$complications.$During$the$second$attempt,$significant$adhesions$and$ scar$tissues$were$encountered.$When$Lola$was$biopsied$a$second$time$via$laparoscopic$surgery,$ no$sample$was$obtained$from$the$left$adrenal$gland.$The$right$adrenal$gland$was$adhered$ underneath$the$vena$cava$and$retroperitoneal$fat$and$no$sample$was$obtainable.$Laparoscopic$ adrenal$biopsy$was$therefore$only$performed$in$all$dogs$to$obtain$baseline$histopathology.$The$ adrenal$glands$are$small,$vascular$structures$located$near$the$vena$cava,$aorta,$renal$veins,$and$ ! 205$ lie$dorsal$to$the$phrenicoabdominal$veins.$When$anatomy$cannot$be$adequately$visualized$in$ this$area,$minimally$invasive$biopsy$methods$risk$damage$to$surrounding$vascular$structures,$ potentially$causing$lifeFthreatening$hemorrhage.$For$subsequent$adrenal$biopsies,$the$Animal$ Use$Form$was$amended$to$do$flank$approach$adrenal$biopsies.$This$approach$provides$direct$ access$to$the$adrenal$glands$through$a$smaller$incision$than$that$necessary$with$a$more$ conventional$midline$incision,$allowing$excellent$access$to$the$glands$for$sampling,$ease$of$ releasing$adhesions,$and$better$control$of$hemostasis$with$postoperative$discomfort$similar$to$ that$with$a$laparoscopic$procedure.$$$ $ The$results$of$the$histopathology$will$be$discussed$together$with$the$biopsies$obtained$ during$flank$approach$and$during$necropsy$later$on$in$this$chapter.$$$ $ Flank%approach%adrenal%biopsy:$Obtaining$adrenal$biopsies$via$flank$approach$as$ described$in$materials$and$methods$was$very$easy$and$straightforward.$The$dogs$recovered$ quickly$and$there$were$no$complications.$Adrenal$biopsies$from$both$adrenal$glands$from$all$ three$dogs$were$obtained.$The$results$of$the$histopathology$will$be$discussed$together$with$the$ biopsies$obtained$during$laparoscopic$approach$and$during$necropsy$later$on$in$this$chapter.$$$ $ Necropsy:$The$adrenal$gland$biopsies$taken$throughout$the$experiment$were$ histologically$normal$and$no$adrenal$inflammation$could$be$demonstrated.$The$adrenal$glands$ of$the$study$dogs$were$indiscernible$from$the$control$dog$adrenal$glands,$and$no$infiltration$of$ T$(CD3$+)$or$B$lymphocytes$(CD79a)$were$identified$immunohistochemically.$$ The$zona$glomerulosa,$which$lies$just$beneath$the$adrenal$capsule,$was$composed$of$ small$clusters$and$short$trabeculae$of$relatively$small,$well$defined$cells.$These$cells$had$less$ ! 206$ cytoplasm$than$the$other$cortical$cells.$The$zona$glomerulosa$accounted$for$approximately$ 15$%$of$the$cortical$volume.$The$zona$fasciculate$was$formed$by$a$broad$band$of$large$cells$with$ distinct$membranes$that$were$arranged$in$cords,$usually$two$cells$wide.$The$cytoplasm$had$ numerous$small$lipid$vacuoles,$which$sometimes$indented$the$central$nucleus$and$resembled$ lipoblasts.$The$zona$fasciculata$accounted$for$approximately$75$%$of$the$cortical$volume.$The$ zona$reticularis$was$grossly$browner$than$the$other$two$layers.$Cells,$smaller$than$the$cells$of$ the$zona$fasciculata,$were$randomly$arranged,$with$granular$and$eosinophilic$cytoplasm$with$ lipofsucin$but$minimal$lipid.$This$layer$was$thinner$than$the$zona$glomerulosa$or$zona$ fasciculata.$$ $ Discussion$ $ Throughout$this$experiment,$adrenal$inflammation$was$not$observed$by$the$injection$of$ the$purified$21Fhydroxylase.$The$histology$of$the$adrenal$glands$remained$completely$normal,$ adrenal$function$remained$within$the$physiologic$limit$and$no$treatment$effect$was$found.$ Based$on$this,$I$concluded$that$hypoadrenocorticism$was$not$induced.$These$findings$were$not$ unexpected$since$previous$studies$showed$that$the$immunization$of$animals$with$homologous$ adrenal$did$not$result$in$histopathologic$changes;$whereas$only$the$immunization$with$ heterologous$adrenal$resulted$in$adrenalitis$with$predominantly$polymorphonuclear$cells$in$ 3F6 some$glands$and$round$cells$in$others ! .$$ 207$ Because$of$the$use$of$adjuvants,$I$was$still$expecting$that$the$two$injected$dogs$ developed$antibodies$against$21Fhydroxylase,$which$will$be$tested$and$discussed$in$the$ following$chapter.$The$rabbit$sera$were$tested$as$well$and$also$discussed$in$the$following$ chapter.$$ $ In$order$to$obtain$a$strong$immune$response$to$the$injected$21FhydroxylaseFGST$fusion$ protein,$I$used$Freund’s$adjuvants.$Freund’s$adjuvant$is$an$immunopotentiator$and$is$named$ after$Jules$T.$Freund.$Freund’s$complete$adjuvant$contains$inactivated$and$dried$ Mycobacterium#tuberculosis$in$mineral$oil,$whereas$the$incomplete$form$only$contains$water$in$ mineral$oil.$Due$to$its$painful$reactions$and$potential$for$tissue$damage$including$necrosis,$the$ use$of$Freund’s$adjuvants$is$strictly$regulated$and$only$subcutaneous$or$intraperitoneal$ injections$are$allowed.$Freund’s$adjuvant$has$been$investigated$in$the$prevention$of$juvenile$ 7F10 diabetes$and$in$an$animal$model$of$Parkinson’s$disease .$Even$though$other$adjuvants$are$ nowadays$available,$Freund’s$adjuvant$was$chosen$for$my$experiment$since$the$production$of$ 11F15 antibodies$in$dogs$is$best$described$using$Freund’s$adjuvants $ .$$ An$adjuvant$may$stimulate$the$immune$system$without$having$any$specific$antigenic$ effect$itself.$An$adjuvant$is$defined$as$“any$substance$that$acts$to$accelerate,$prolong,$or$ enhance$antigenFspecific$immune$responses$when$used$in$combination$with$specific$ 16 antigens” .$An$adjuvant$stimulates$cellFmediated$immunity$and$leads$to$the$potentiation$and$ production$of$immunoglobulins.$The$potentiation$is$accomplished$by$mimicking$sets$of$ evolutionary$conserved$molecules$(PAMPs),$including$liposomes,$lipopolysaccharide,$molecular$ ! 208$ 17 cages$for$antigen,$components$of$cell$walls,$aluminum$salts,$and$endocytosed$nucleic$acids .$ These$specific$antigenic$moieties$are$recognized$by$the$immune$system,$and$the$recognition$in$ conjunction$with$another$antigen$greatly$increases$the$innate$immune$response$to$the$antigen$ by$augmenting$the$activities$of$dendritic$cells,$lymphocytes,$and$macrophages$by$mimicking$a$ natural$infection.$Since$there$are$many$links$between$the$innate$immune$system$and$the$ adaptive$immune$system,$an$adjuvantFenhanced$innate$immune$response$results$in$an$ enhanced$adaptive$immune$response.$Specifically,$a$recent$study$showed$that$adjuvants$may$ 18 exert$their$immuneFenhancing$effects$by$five$immuneFfunctional$activities :$First,$adjuvants$ help$in$the$translocation$of$antigens$to$the$lymph$nodes.$In$the$lymph$node,$they$can$be$ recognized$by$T$cells,$ultimately$resulting$in$greater$T$cell$activity.$Second,$adjuvants$physically$ protect$antigens,$which$increases$the$exposure$duration,$upregulating$the$production$of$B$and$ T$cells.$Third,$adjuvants$cause$local$reactions$at$the$injection$site,$inducing$a$greater$release$of$ chemokines,$which$in$turn$increases$the$release$of$helper$T$cells$and$mast$cells.$Fourth,$ adjuvants$induce$the$release$of$inflammatory$cytokines,$which$recruit$B$and$T$cells,$and$ increase$transcriptional$events,$leading$to$a$net$increase$of$immune$cells$as$a$whole.$Fifth,$ adjuvants$increase$the$innate$immune$response$to$the$antigen$by$interacting$with$pattern$ recognition$receptors,$including$TollFlike$receptors.$$ $ My$hope$was$to$achieve$a$robust$immune$response$in$the$injected$animals.$This$would$ enable$me$to$use$their$sera$as$positive$and$negative$(Lucy)$controls$during$assay$development.$ Positive$and$negative$controls$are$needed$during$ELISA$development$to$eliminate$alternate$ explanations$of$test$results.$ ! 209$ APPENDIX ! 210$ Appendix$ $ Table$VII.1:$Immunization$timeline.$ Antibody$production$schedule:$ 6/20/2010$ ACTH$stimulation$ #1F$Baseline$ACTH$stimulation$ 6/21/2010$$ Bleed$ #1F$PreFimmune$bleed$ 6/28/2010$ Laparoscopic$adrenal$biopsy$ #1F$Baseline$adrenal$biopsy$ 7/4/2010$ Bleed$ #2F$Production$bleed$ 7/11/2010$$ Immunization$ #1F$Complete$Freund’s$Adjuvant$$ 8/1/2010$$ Immunization$ #2F$Incomplete$Freund’s$Adjuvant$$ 8/8/2010$ Bleed$ #3F$Production$bleed$ 8/20/2010$ ACTH$stimulation$ #2F$ACTH$stimulation$ 8/20/2010$ Laparoscopic$adrenal$biopsy$$ (unsuccessful,$see$text)$ 8/30/2010$ Immunization$ #3F$Incomplete$Freund’s$Adjuvant.$ 9/5/2010$ Bleed$ #4F$Production$bleed$ 9/19/2010$ Immunization$ #4F$Incomplete$Freund’s$Adjuvant.$ $ ! 211$ Table$VII.1$(cont’d):$ 9/26/2010$ Bleed$ #5F$Production$bleed$ 10/23/2010$ ACTH$stimulation$ #3F$ACTH$stimulation$ 10/27/2010$ Adrenal$biopsy$through$flank$ #2F$Adrenal$biopsy$ 11/7/2010$ Immunization$ #5F$Incomplete$Freund’s$Adjuvant.$ 11/14/2010$ Bleed$ #6F$Production$bleed$ 11/14/2010$ ACTH$stimulation$ #4F$ACTH$stimulation$ 12/5/2010$ Bleed$ #7F$Production$bleed$ 12/5/2010$ ACTH$stimulation$ #5FACTH$stimulation$ 1/18/2010$ Bleed$ #8F$Production$bleed$ 1/18/2011$ ACTH$stimulation$ #6FACTH$stimulation$ 1/20/2011$ Euthanasia$and$necropsy$ #7FProduction$bleed;$#3F$Adrenal$biopsy$ $ $ ! $ 212$ Table$VII.2:$ACTH$stimulation$test,$cortisol$concentrations.$ Cortisol$(normal):$$ post:$220F550$nmol/L$ 10/23/2010$ 11/14/2010$ 12/5/2010$ 1/18/2011$ 27$ 10$ 35$ 40$ 89$ 92$ $$$$$$$$$$$$$$$$$post:$ 352$ 316$ 326$ 342$ 340$ 286$ Lola$$$$$$$$$$pre:$ 61$ 37$ 164$ 81$ 70$ 91$ $$$$$$$$$$$$$$$$$post:$ 480$ 334$ 486$ 488$ 444$ 245$ Lucy$$$$$$$$$pre:$ 120$ 52$ 294$ 167$ 128$ 51$ $$$$$$$$$$$$$$$$$post:$ ! 8/20/2010$ Blanket$$pre:$ $ Baseline$ pre:$$$$$15F110$nmol/L$ 450$ 335$ 459$ 449$ 378$ 314$ $ 213$ Table$VII.3:$ACTH$stimulation$test,$aldosterone$concentrations.$ Aldosterone$(normal):$$ post:$197F2103$nmol/L$ Baseline$ 8/20/2010$ 10/23/2010$ 11/14/2010$ 12/5/2010$ 1/18/2011$ pre:$$$$$14F957$$$nmol/L$ Blanket$$pre:$ 55$ 32$ 65$ 39$ 134$ 67$ $$$$$$$$$$$$$$$$$post:$ 499$ 308$ 514$ 286$ 535$ 431$ Lola$$$$$$$$$$pre:$ 38$ 109$ 22$ 0$ 5$ 11$ $$$$$$$$$$$$$$$$$post:$ 475$ 392$ 489$ 223$ 439$ 280$ Lucy$$$$$$$$pre:$ 156$ 135$ 459$ 77$ 257$ 151$ $$$$$$$$$$$$$$$$post:$ 897$ 851$ 792$ 469$ 704$ 611$ $ $ ! $ 214$ Figure$VII.1:$Cortisol$concentrations$post$ACTH$[%$of$baseline].$ $ $ Figure$VII.2:$Cortisol$concentrations$post$ACTH.$ $ ! 215$ Figure$VII.3:$Aldosterone$concentrations$post$ACTH$[%$of$baseline].$ $ $ Figure$VII.4:$Aldosterone$concentrations$post$ACTH.$ ! 216$ REFERENCES ! 217$ References$ $ 1.$ Meek$WJ,$Eyster$JAE.$Reactions$to$hemorrhage.$Am#J#Physiol$1921;56:1F15.$ $ 2.$ Potkay$S,$Zinn$RD.$Effects$of$collection$interval,$body$weight,$and$season$on$the$ hemograms$of$canine$blood$donors.$Lab#Anim#Care$1969;19:192F198.$ $ 3.$ Steiner$JW,$Langer$B,$Schatz$DL,$et$al.$Experimental$Immunologic$Adrenal$Injury$:$ A$Response$to$Injections$of$Autologous$and$Homologous$Adrenal$Antigens$in$Adjuvant.$J#Exp# Med$1960;112:187F202.$ $ 4.$ Milgrom$F,$Witebsky$E.$Immunological$studies$on$adrenal$glands.$I.$Immunization$ with$adrenals$of$foreign$species.$Immunology$1962;5:46F66.$ $ 5.$ Witebsky$E,$Milgrom$F.$Immunological$studies$on$adrenal$glands.$II.$ Immunization$with$adrenals$of$the$same$species.$Immunology$1962;5:67F78.$ $ 6.$ Barnett$EV,$Dumonde$DC,$Glynn$LE.$Induction$of$Autoimmunity$to$Adrenal$Gland.$ Immunology$1963;6:382F402.$ $ 7.$ Suri$A,$Calderon$B,$Esparza$TJ,$et$al.$Immunological$reversal$of$autoimmune$ diabetes$without$hematopoietic$replacement$of$beta$cells.$Science$2006;311:1778F1780.$ $ 8.$ Sadelain$MW,$Qin$HY,$Lauzon$J,$et$al.$Prevention$of$type$I$diabetes$in$NOD$mice$ by$adjuvant$immunotherapy.$Diabetes$1990;39:583F589.$ $ 9.$ Qin$HY,$Sadelain$MW,$Hitchon$C,$et$al.$Complete$Freund's$adjuvantFinduced$T$ cells$prevent$the$development$and$adoptive$transfer$of$diabetes$in$nonobese$diabetic$mice.$J# Immunol$1993;150:2072F2080.$ $ 10.$ Armentero$MT,$Levandis$G,$Nappi$G,$et$al.$Peripheral$inflammation$and$ neuroprotection:$systemic$pretreatment$with$complete$Freund's$adjuvant$reduces$6F hydroxydopamine$toxicity$in$a$rodent$model$of$Parkinson's$disease.$Neurobiol#Dis$2006;24:492F 505.$ ! 218$ $ 11.$ Graves$SS,$Stone$D,$Loretz$C,$et$al.$Establishment$of$longFterm$tolerance$to$SRBC$ in$dogs$by$recombinant$canine$CTLA4FIg.$Transplantation$2009;88:317F322.$ $ 12.$ Szabo$MP,$Bechara$GH.$Immunization$of$dogs$and$guinea$pigs$against$ Rhipicephalus#sanguineus$ticks$using$gut$extract.$Vet#Parasitol$1997;68:283F294.$ $ 13.$ Haak$T,$Delverdier$M,$Amardeilh$MF,$et$al.$Pathologic$study$of$an$experimental$ canine$arthritis$induced$with$complete$Freund's$adjuvant.$Clin#Exp#Rheumatol$1996;14:633F641.$ $ 14.$ Haines$DM,$Penhale$WJ.$Experimental$thyroid$autoimmunity$in$the$dog.$Vet# Immunol#Immunopathol$1985;9:221F238.$ $ 15.$ Neubauer$HP,$Schone$HH.$The$immunogenicity$of$different$insulins$in$several$ animal$species.$Diabetes$1978;27:8F15.$ $ 16.$ Sasaki$S,$Okud$K.$The#Use#of#Conventional#Immunologic#Adjuvants#in#DNA# Vaccine#Preparations.$DNA$Vaccines:$Methodes$and$Protocols:$Humana$Press,$2000.$ $ 17.$ Gavin$AL,$Hoebe$K,$Duong$B,$et$al.$AdjuvantFenhanced$antibody$responses$in$the$ absence$of$tollFlike$receptor$signaling.$Science$2006;314:1936F1938.$ $ 18.$ Schijns$VE.$Immunological$concepts$of$vaccine$adjuvant$activity.$Curr#Opin# Immunol$2000;12:456F463.$ $ ! 219$ Chapter#VIII:#Evaluation#of#immunoreactivity#of#canine#and#rabbit#sera# with#the#purified#21>hydroxylase>His# # Introduction## In#this#part#of#the#project,#the#immunoreactivity#of#the#dog#and#rabbit#sera#pre>#and# post>immunization#against#the#purified#21>hydroxylase>His#was#evaluated#using#Western#blot# (WB).#The#immunoreactivity#of#sera#from#dogs#with#Addison’s#disease#(collected#at#the# Endocrinology#Section,#Diagnostic#Center#for#Population#and#Animal#Health#(DCPAH),#Michigan# State#University#(MSU),#East#Lansing,#MI,#USA)#against#the#purified#21>hydroxylase>His#was#also# evaluated#using#WB.#Further#analyzed#were#study#dog#sera#pre#and#post#immunization#with# equine#adrenal#glands#using#immunohistochemistry#(IHC).#Lastly,#the#immunoreactivity#of#the# dog#sera#pre>#and#post>immunization#and#sera#from#dogs#with#Addison’s#disease#(collected#at# the#Endocrinology#Section,#DCPAH,#MSU)#against#purified#21>hydroxylase>His#was#analyzed# using#enzyme>linked#immunosorbent#assay#(ELISA).## # Materials#and#methods# # For#the#three#proposed#techniques,#the#protein#concentration#of#the#21>hydroxylase>His# in#TBS,#the#21>hydroxylase>His#in#TBS#containing#4#M#urea,#the#21>hydroxylase>His#in#TBS# containing#4#M#guanidine,#and#the#21>hydroxylase>His#in#TBS#containing#4#M#guanidine,#plus# 250#mM#imidazole#was#measured#via#bicinchoninic#acid#(BCA)#protein#assay,#following#the# ! 220# manufacturer#instructions.#The#appropriate#amount#of#urea,#guanidine#and/#or#imidazole#was# added#to#prepare#the#standards#for#the#BCA#assay#to#adjust#for#interferences#from#these# chemicals.#The#optical#density#was#then#read#at#562#nm#and#the#concentrations#were#calculated.# Western'Blot:#Approximately#200#ng#of#21>hydroxylase>His#was#run#on#SDS>PAGE# (sodium#dodecyl#sulfate#polyacrylamide#gel#electrophoresis)#and#transferred#to#polyvinylidene# difluoride#(PVDF)#membranes#in#transfer#buffer#(10#mM#CAPS#(3>[cyclohexylamino]>1#propane# sulfonic#acid),#pH#11#containing#10%#methanol)#for#one#hour#at#0.51#A.#The#membranes#were# then#incubated#with#the#primary#antibody#(canine#and#rabbit#serum)#in#1xPBS#(phosphate# buffered#saline),#containing#1#%#Tween#(PBS>T),#and#1#%#(w/V)#instant#nonfat#dry#milk#milk#at# 4°C#overnight#at#a#dilution#of#1:1,000.#The#membranes#were#washed#three#times#with#PBS>T#for# 15#min#each#at#room#temperature#and#incubated#with#the#secondary#antibody#(anti>canine#or# anti>rabbit#IgG/#horseradish#peroxidase,#1:5,000#in#PBS>T,#containing#1#%#(w/V)#instant#nonfat# dry#milk).#After#the#wash#step,#chemiluminescence#solution#was#added#for#about#one#minute# and#the#signals#detected#using#the#ChemiDoc#XRS+.## Immunohistochemistry:#Normal#equine#adrenal#gland#collected#served#as#control#tissue# to#test#for#the#presence#of#21>hydroxylase#autoantibody.#5>6#µm#paraffin#sections#were#placed# on#glass#slides.#To#perform#the#IHC,#the#sections#were#deparaffinized#in#xylene#and#dehydrated# in#a#graded#series#of#ethanol.#Non>specific#protein#binding#sites#were#blocked#using#bovine# serum#albumin#in#PBS.#Serum#from#the#experimental#dogs#(pre>#and#post>vaccination)#as#well#as# known#positive#serum#from#a#human#Addison’s#disease#patient#and#normal#human#serum# served#as#positive#and#negative#controls.#All#serum#samples#(primary#antibody)#were#applied#to# ! 221# the#equine#adrenal#sections#at#1:50,#1:#1,000,#and#1:10,000#concentrations.#Negative#controls# for#the#primary#antibody#consisted#of#PBS#substitution#for#the#primary#antibody#or#application# of#normal#mouse#serum#(nonsense#control).#The#secondary#antibodies#consisted#of#horseradish# peroxidase#labeled#rabbit#anti>canine#IgG#(immunoglobulin#gamma)#and#rabbit#anti>human#IgG.# Nova#red#served#as#the#chromagen#substrate#for#the#horseradish#peroxidase#catalyzed#reaction# at#sites#of#antibody#binding.#Each#slide#was#lightly#counterstained#with#hematoxylin.# ' ELISA:#Coating#buffer#was#prepared#(0.1#M#Na2CO3;#pH#9.6)#and#the#wells#of#a#96>well# high>binding#microplate#were#coated#with#100#µL#of#21>hydroxylase>His#in#TBS,#21>hydroxylase> His#in#TBS#containing#4#M#guanidine,#and#21>hydroxylase>His#in#TBS#containing#4#M#urea,#diluted# in#coating#buffer#at#a#concentration#of#0.01#mg/#mL.#Each#well#contained#1#µg#of#antigen# obtained#from#three#different#preparations#as#described#in#chapter#VI.#A#second#plate#was# coated#with#just#coating#buffer#to#measure#non>specific#binding#(NSB)#of#the#samples.#The# plates#were#then#incubated#at#4°C#overnight#(approx.#16>20#hours).' Upon#removal#from#refrigeration,#the#wells#were#blocked#by#adding#300#µL#of#blocking# buffer#(obtained#from#Oxford#Laboratories,#Rochester,#MI,#USA)#to#each#well.#The#blocking# buffer#was#added#directly#over#the#coating#solution#and#allowed#to#incubate#at#room# temperature#(RT)#for#one#hour.# After#the#blocking#incubation,#the#plate#was#washed#using#the#following#procedure:#1)# decant#the#contents#of#the#plate;#2)#add#300#µL#of#wash#buffer#(Oxford#Laboratories)#to#each# well.#Let#stand#for#2#minutes#at#RT,#then#decant.#This#was#repeated#two#more#times#for#a#total# ! 222# of#three#washes;#and#3)#the#plates#were#inverted#and#blotted#to#remove#any#residual#moisture# from#the#wells.# To#perform#the#assay,#canine#serum#was#diluted#1:100#in#enzyme#immunoassay#(EIA)#buffer# (Oxford#Laboratories)#and#100#µL#of#the#diluted#serum#in#duplicate#wells#of#both#antigen>coated# and#NSB#plates#was#added.#The#plates#were#incubated#at#RT#for#2#hours#and#then#washed#as# described#in#steps#1>3#above.# 100#µL#of#affinity#purified#horseradish#peroxidase#(HRP)>conjugated#goat#anti>Dog#IgG# (heavy#and#light#chain)#(Kirkegaard#&#Perry#Laboratories,#Maryland,#USA)#(final#dilution#of#1:70,000# in#EIA#buffer)#was#added#to#wells#(antigen>coated#and#NSB)#and#incubated#at#RT#for#one#hour.#The# plates#were#then#washed#as#in#steps#1>3#above.# 150#µL#of#TMB#(3,3′,5,5′>tetramethylbenzidine)#(obtained#from#Oxford#Laboratories)# substrate#was#transferred#to#each#well#and#incubated#at#RT#for#30#minutes#or#until#an# appreciable#blue#color#had#formed.#Color#development#was#terminated#by#adding#50#µL#of#3N# H2SO4#to#the#wells.#Plates#were#read#on#a#spectrophotometric#plate#reader#at#450#nm.## In#the#first#experiment,#three#different#antigen#preparations#of#the#purified#21> hydroxylase>His#were#compared#with#one#another,#using#serum#from#the#two#study#dogs#(Lola# and#Blanket)#pre>#and#post>immunization,#from#our#control#dog#(Lucy),#and#from#two#young#and# healthy#dogs.#In#one#well,#only#buffer#was#added.#In#the#second#experiment,#the#21> hydroxylase>His#in#TBS#was#used#for#plate#coating,#and#sera#from#our#three#dogs,#from#10# healthy#dogs,#and#from#12#dogs#diagnosed#with#hypoadrenocorticism#were#evaluated#for# immunoreactivity#with#the#purified#21>hydroxylase>His.## ! 223# #Results# After#one#FPLC#(fast>protein#liquid#chromatography)#purification#run,#the#elution# fractions#were#pooled.#After#that,#an#aliquot#was#either#salt>exchanged#against#TBS,#using#PD>10# desalting#columns#(GE#Healthcare,#Piscataway,#NJ,#USA),#dialyzed#against#TBS#containing#4#M# guanidine#or#TBS#containing#4#M#urea,#or#diluted#1:2#with#TBS.#The#concentration#of#the#21> hydroxylase>His#was#then#measured#as#described#in#materials#and#methods.#The#results#are# shown#in#table#VIII.1,#clearly#showing#that#the#highest#amount#of#purified#21>hydroxylase>His# was#present#in#the#sample#that#was#diluted#1:2#with#TBS.#The#sample#that#was#salt#exchanged,# using#a#PD>10#desalting#column#had#the#lowest#concentration.## Western'Blot:'The#samples#from#the#immunized#dogs#and#rabbits#showed# immunoreactivity#with#the#21>hydroxylase#post>immunization,#but#not#before#(figure#VIII.1#and# figure#VIII.2).#Out#of#13#analyzed#samples#from#dogs#with#hypoadrenocorticism,#four#tested# positive#(31%)#using#WB.## Immunohistochemistry:'No#specific#labeling#for#21>hydroxylase#could#be#identified#in# the#equine#adrenal#glands#using#either#the#pre>#or#post>vaccination#canine#serum#or#the#two# different#(known#positive#and#known#negative)#human#sera.#There#was#extensive#diffuse# background#labeling#of#all#of#the#equine#adrenal#gland#sections,#including#the#negative#controls,# thus#I#could#not#identify#the#presence#of#21>hydroxylase#auto>antibodies#using# immunohistochemistry.' ELISA:'Results#of#experiment#1#are#shown#in#figure#VIII.3.#The#three#compared#21> hydroxylase>His#preparations#gave#similar#results.#The#optical#density#is#significantly#increased# in#the#two#immunized#dogs#post>immunization#(2#to#more#than#3#fold),#demonstrating# ! 224# immunoreactivity#between#the#purified#21>hydroxylase>His#and#the#sera#from#these#animals.#In# contrast,#Lucy#(our#negative#control),#Aerdenbouts,#and#Partydoll#are#considered#to#be#healthy# and#all#have#a#low#optical#density.#In#the#second#experiment,#3#out#of#the#12#dogs#that#have# been#diagnosed#with#hypoadrenocorticism#showed#a#high#optical#density,#as#well#as#did#Blanket# and#Lola#post#immunization#(figure#VIII.4).#Due#to#time>constraints#of#our#collaborators,#the#21> hydroxylase>His#in#TBS#containing#4#M#guanidine,#and#250#mM#imidazole#has#not#yet#been# tested.## # Discussion# The#results#of#WB#are#encouraging,#showing#that#the#immunized#dogs#mounted#a# measurable#immune#response#against#the#21>hydroxylase>GST#(21>hydroxylase>glutathione#S> transferase).#The#serum#of#the#immunized#dogs#could#therefore#be#used#for#adjusting#IHC# techniques#and#for#ELISA#development.#The#results#of#IHC#remain#unsatisfactory,#having#to#deal# with#an#immense#amount#of#background#staining.#The#preliminary#results#of#ELISA#are#very# promising,#even#though#further#work#needs#to#be#done.#Ideally,#more#clinical#samples#as#well#as# samples#from#certain#breeds#that#are#known#to#be#at#high#risk#to#develop#hypoadrenocorticism# should#be#analyzed.#Up#to#this#point,#some#dogs#clearly#show#immunoreactivity#with#the# purified#21>hydroxylase>His,#which#is#promising,#but#not#all#analyzed#dogs#with#Addison's# disease#show#this#kind#of#immunoreactivity.#Further,#some#outwardly#healthy#dogs,#including# Blanket#pre>immunization,#have#a#relatively#high#result#in#ELISA.#The#fact#that#some#healthy# dogs#have#a#high#result#in#ELISA#was#repeatable#and#cannot#be#explained.## Non>specific#reactions#in#ELISAs#are#a#known#problem,#and#from#my#own#experience#at# ! 225# the#Endocrinology#Laboratory#at#the#DCPAH,#MSU,#especially#tricky#when#used#with#canine# serum.#In#order#to#decrease#non>specific#reactions#and#to#increase#specificity#and#sensitivity#of# an#ELISA,#primarily#two#steps#should#be#taken.#First,#care#should#be#taken#during#the#purification# of#the#21>hydroxylase>His#to#have#an#as#pure#as#possible#protein#as#an#antigen#source.#This#is# the#most#important#factor#during#ELISA#development#to#minimize#background#reactions#or# 1>3 nonspecific#reactions .#Second,#the#optimal#working#conditions#for#each#component#of#an# ELISA#need#to#be#found#to#increase#specificity#and#decrease#background#readings,#including#but# not#limited#to#the#determination#of#antigen#concentrations,#test#sera#dilutions,#blocking#agents,# incubation#times#and#washing#steps.#As#an#example,#if#the#antigen#concentration#is#too#high,#it# might#inhibit#the#binding#of#the#antibody#through#steric#inhibition.#Further,#it#may#increase# stacking#or#layering#of#antigen,#which#may#lead#to#an#unstable#interaction#of#subsequent# reagent.#In#contrast,#low>density#binding#of#antigen#may#result#in#an#unevenly#coated#plate,#and# 1,4 unoccupied#spaces#might#then#bind#sera#protein#or#other#contaminating#proteins .#The# conditions#of#the#developed#ELISA#in#its#current#form#have#not#been#optimized#yet#and#further# work#needs#to#be#done.#In#the#literature,#especially#the#use#of#Tween#20#in#the#washing#buffers# and#the#use#of#bovine#serum#albumin#as#a#blocking#agent#in#any#buffers#have#been#shown#to# 4 inhibit#nonspecific#reactions .## Even#not#optimized,#the#ELISA#in#its#current#form#is#highly#reproducible,#showing# minimal#intra#and#inter>assay#variation#of#less#than#10%.#Because#of#the#low#number#of# repeated#testing#as#of#yet,#statistical#analysis#could#not#be#performed.#Even#though#the# interpretation#of#the#less#than#10%#of#intra>#and#inter>assay#variation#should#be#done#cautiously# ! 226# because#of#the#low#numbers#of#testing,#both#values#are#clearly#within#the#standard#repeatability# 5 criterion#for#ELISA#validation .## Another#critical#step#in#the#standardization#of#an#ELISA#and#the#determination#of# diagnostic#usefulness#of#a#serologic#test#is#the#determination#of#the#cutoff#value#to#separate# positive#from#negative#results.#At#present,#different#methods#are#used#to#set#these#cutoff# values 6>9 .#The#most#commonly#used#method#for#cutoff#determination#is#adding#two#or#three,# and#occasionally,#especially#for#in>house#ELISAs,#four#standard#deviations#to#the#mean#of#the# 10 negative#control#sera .#Depending#on#the#purpose#of#the#test,#two#or#three#standard# deviations#might#be#more#appropriate,#with#two#preferred#for#screening#purposes#and#three# 11 appropriate#for#diagnostic#purposes .#Once#the#cutoff#has#been#set,#the#specificity,#sensitivity,# and#reproducibility#can#be#assessed.#Therefore,#future#evaluation#and#optimization#of#the#ELISA# is#needed.## For#further#discussion#of#results#obtained#in#this#chapter,#also#see#chapter#IX.## ! 227# APPENDIX ! 228# Appendix# Table#VIII.1:#Comparison#of#the#21>hydroxylase>His#concentrations#in#the#four#different# preparations,#clearly#showing#that#the#highest#concentration#was#found#in#TBS,#containing#4#M# guanidine,#and#250#mM#imidazole.### 21#OH#in# Concentration#[mg/ml]# Total#amount#of#21>OH>His# from#2.5#mL#starting#volume# [mg]# TBS# 0.09# 0.315# TBS,#containing#4#M#guanidine# 0.18# 0.45# TBS,#containing#4#M#urea# 0.16# 0.4# TBS,#containing#4#M#guanidine,# 0.15# 0.75# and#250#mM#imidazole# # # ! # 229# # Figure#VIII.1:#Western#blotting#of#the#rabbit#sera,#clearly#showing#immunoreactivity#with# the#expressed#21>hydroxyalse>His#after#immunization,#but#not#before.## # ! 230# # Figure#VIII.2:#Western#blotting#of#the#dog#sera,#clearly#showing#immunoreactivity# between#the#expressed#21>hydroxylase>His#and#21>hydroxylase>GST#and#the#two#study#dogs# post#immunization,#but#not#before.#Lucy’s#serum#does#not#show#immunoreactivity.## # # # ! # 231# Figure#VIII.3:#Comparison#of#the#three#21>hydroxylase>His#preparations#in#ELISA,#using# sera#from#the#study#dogs,#two#healthy#dogs,#and#buffer#alone.#All#three#preparations#gave# similar#results.#The#optical#density#is#significantly#increased#in#the#two#immunized#dogs#post> immunization#(2#to#more#than#3#fold),#demonstrating#immunoreactivity#between#the#purified# 21>hydroxylase>His#and#the#sera#from#these#animals.## # # ! # 232# Figure#VIII.4:#Results#of#the#second#ELISA#experiment,#testing#canine#serum#from#healthy# dogs,#dogs#diagnosed#with#Addison’s#disease,#and#from#our#two#positive#control#study#and#dogs.# (1:#Blanket#pre#immunization;#2:#Blanket#post#immunization;#3:#Lola#pre#immunization;#4:#Lola# post#immunization;#5#to#14:#healthy#dogs;#15#to#26:#dogs#diagnosed#with#hypoadrenocorticism# (see#table#VIII.2#for#details)).#Three#out#of#the#12#dogs#that#have#been#diagnosed#with# hypoadrenocorticism#show#a#high#optical#density,#as#well#as#Blanket#and#Lola#post#immunization.# # # ! # 233# Table#VIII.2:#Information#on#the#Addisonian#dogs#that#were#tested#using#the#newly# developed#ELISA.#A#German#Shepherd#Mix,#a#Rottweiler,#and#a#Jack#Russell#tested#positive#for# the#presence#of#21>hydroxylase#antibodies.## Sample#ID# Age#[years]# Sex# Sample#type# Breed# 15# 1.8# MC# Plasma# German#Shorthaired#Pointer# 16# 12# FS# Serum# Chow#Mix# 17# 5# MC# Plasma# Australian#Terrier# 18# 2# FS# Plasma# Golden#Retriever# 19# 2# FS# Plasma# West#Highland#White#Terrier# 20# 5# M# Plasma# German#Shepherd#Mix# 21# 4# FS# Plasma# Schnoodle# 22# 2# FS# Plasma# English#Mastiff# 23# 1# FS# Serum# West#Highland#White#Terrier# 24# 3# MC# Plasma# Shih#Tzu# 25# 2# MC# Plasma# Rottweiler# 26# 7# FS# Plasma# Jack#Russell#Terrier# # ! 234# REFERENCES ! 235# References# # 1.# Chard#T.#An#Introduction#to#Radioimmunoassay#and#Related#Techniques.# Laboratory#Techniques#in#Biochemistry#and#Molecular#Biology,#5th#ed.#St.#Louis,#Missouri:# Elsevier#Science,#1995:5.# # 2.# Albina#E,#Leforban#Y,#Baron#T,#et#al.#An#enzyme#linked#immunosorbent#assay# (ELISA)#for#the#detection#of#antibodies#to#the#porcine#reproductive#and#respiratory#syndrome# (PRRS)#virus.#Ann#Rech#Vet#1992;23:167>176.# # 3.# Marquardt#WW,#Johnson#RB,#Odenwald#WF,#et#al.#An#indirect#enzyme>linked# immunosorbent#assay#(ELISA)#for#measuring#antibodies#in#chickens#infected#with#infectious# bursal#disease#virus.#Avian#Dis#1980;24:375>385.# # 4.# Li#C,#Cheng#A,#Wang#M,#et#al.#Development#and#validation#of#an#indirect#enzyme> linked#immunosorbent#assay#for#the#detection#of#antibodies#against#duck#swollen#head# hemorrhagic#disease#virus.#Avian#Dis#2010;54:1270>1274.# # 5.# Jacobson#RH.#Validation#of#serological#assays#for#diagnosis#of#infectious#diseases.# Rev#Sci#Tech#1998;17:469>526.# # 6.# Greiner#M.#Two>graph#receiver#operating#characteristic#(TG>ROC):#a#Microsoft> EXCEL#template#for#the#selection#of#cut>off#values#in#diagnostic#tests.#J#Immunol#Methods# 1995;185:145>146.# # 7.# Barajas>Rojas#JA,#Riemann#HP,#Franti#CE.#Notes#about#determining#the#cut>off# value#in#enzyme#linked#immunosorbent#assay#(ELISA).#Prev#Vet#Med#1993;15:231>233.# # 8.# Funk#ND,#Tabatabai#LB,#Elzer#PH,#et#al.#Indirect#enzyme>linked#immunosorbent# assay#for#detection#of#Brucella#melitensis>specific#antibodies#in#goat#milk.#J#Clin#Microbiol# 2005;43:721>725.# # 9.# Zhao#XL,#Phillips#RM,#Li#GD,#et#al.#Studies#on#the#detection#of#antibody#to#duck# hepatitis#virus#by#enzyme>linked#immunosorbent#assay.#Avian#Dis#1991;35:778>782.# ! 236# # 10.# Kich#JD,#Schwarz#P,#Eduardo#Silva#L,#et#al.#Development#and#application#of#an# enzyme>linked#immunosorbent#assay#to#detect#antibodies#against#prevalent#Salmonella# serovars#in#swine#in#southern#Brazil.#J#Vet#Diagn#Invest#2007;19:510>517.# # 11.# Pinto#PS,#Vaz#AJ,#Germano#PM,#et#al.#ELISA#test#for#the#diagnosis#of#cysticercosis# in#pigs#using#antigens#of#Taenia#solium#and#Taenia#crassiceps#cysticerci.#Rev#Inst#Med#Trop#Sao# Paulo#2000;42:71>79.# # ! 237# Chapter$IX:$Discussion$and$future$research$ $ The$overarching$goal$of$my$dissertation$research$was$to$develop$a$diagnostic$test$to$ facilitate$early$diagnosis$of$hypoadrenocorticism$in$dogs$before$they$develop$more$serious$life@ threatening$clinical$signs.$The$test,$when$developed,$will$also$help$to$better$understand$the$ pathogenesis$and$epidemiology$of$the$disease$and$has$the$potential$to$assist$in$future$ molecular$genetic$research.$$ The$data$showed$that$dogs$with$naturally$occurring$primary$Addison’s$disease$produce$ 21@hydroxylase$antibodies.$This$is$consistent$with$the$human$form$of$the$disease.$What$ remains$unknown$is$whether$autoantibody$production$precedes$clinical$disease,$implying$a$role$ in$the$pathogenesis$of$the$disease;$if$it$is,$then$the$presence$of$21@hydroxylase$autoantibodies$ would$be$expected$to$be$more$prevalent$in$dog$breeds$susceptible$to$developing$ hypoadrenocorticism.$The$specific$aims$were$to$establish$a$diagnostic$test$to$detect$canine$anti@ adrenal$autoantibodies,$and$to$determine$whether$such$autoantibodies$are$present$in$dogs$ with$hypoadrenocorticism;$both$aims$were$successfully$established.$Development$of$the$test$to$ detect$autoantibodies$would$enable$epidemiologic$studies$to$address$specific$aim$3$“to$ determine$whether$development$of$anti@adrenal$autoantibodies$has$breed,$sex,$and$age@based$ predispositions”.$ The$canine$21@hydroxylase$was$expressed$in$E.#coli,$using$standard$techniques.$First,$RNA$was$ obtained$from$freshly$harvested$canine$adrenal$glands.$The$expression$sequence$of$the$21@ hydroxylase$gene$was$then$cloned$into$expression$vectors$and$21@hydroxylase@fusion$protein$ production$was$induced$with$isopropyl$β@D@1@thiogalactopyranoside$(IPTG).$$ ! 238$ Unfortunately,$even$though$the$anchor$of$the$protein$was$modified$to$make$it$more$ soluble,$the$protein$was$almost$exclusively$expressed$into$inclusion$bodies.$The$purification$of$ the$protein$was$therefore$very$difficult$and$had$to$be$done$under$denaturing$conditions.$Since$ the$purification$was$so$difficult$and$time@intensive,$I$decided$to$describe$it$in$more$detail$than$ what$is$usually$expected$in$a$dissertation.$My$goal$was$that$anybody$who$would$be$interested$ in$optimizing$the$purification$of$the$21@hydroxylase@fusion$proteins$would$be$able$to$do$so$with$ this$dissertation,$and$would$know$exactly$what$had$been$tried$before.$I$also$strongly$believe$ that$purification$conditions$could$further$be$optimized$to$increase$protein$yield.$$ The$results$of$Western$blotting$(WB)$suggested$that$the$immunized$dogs$mounted$an$ immune$response$to$the$denatured$21@hydroxylase@glutathione$S@transferase$(GST)$when$ injected$with$adjuvants.$$We$therefore$were$able$to$use$their$sera$for$the$development$of$an$ ELISA$(enzyme@linked$immunosorbent$assay)$that$would$be$able$to$detect$antibodies$in$clinical$ samples.$Using$the$same$conditions$that$gave$us$positive$signals$in$WB$with$the$serum$of$our$ immunized$dogs,$an$array$of$sera$from$dogs$with$and$without$Addison’s$disease$was$also$ examined.$Out$of$13$analyzed$samples$from$dogs$diagnosed$with$hypoadrenocorticism,$four$ tested$positive$(31%).$The$results$strongly$suggested$the$presence$of$circulating$21@hydroxylase$ antibodies$in$dogs$that$were$diagnosed$with$hypoadrenocorticism.$$ Using$the$positive$control$sera,$an$ELISA$was$developed$and$partially$optimized.$Thus$far$ 12$clinical$samples$from$dogs$with$hypoadrenocorticism$have$been$tested.$Of$the$12$samples$ tested,$three$(25%)$tested$positive$for$the$presence$of$21@hydroxylase$antibodies.$As$a$control,$ 10$healthy$samples$were$tested.$None$of$10$healthy$control$samples$tested$positive.$Taken$ ! 239$ together$with$the$results$from$WB,$I$have$shown$that$21@hydroxylase$antibodies$are$present$in$ Addison’s$disease$in$dogs.$$ In$humans,$up$to$90$%$of$newly$diagnosed$patients$with$Addison’s$disease$have$ detectable$circulating$21@hydroxylase$autoantibodies.$The$relatively$low$percentage$of$antibody$ positive$samples$obtained$from$dogs$with$Addison’s$disease$compared$to$humans$may$be$ explained$by$several$different$mechanisms.$First,$other$antibodies,$for$example$17@hydroxylase$ antibodies$or$antibodies$against$cholesterol$side$chain@cleaving$enzyme$may$play$a$prominent$ role$in$the$pathogenesis$of$hypoadrenocorticism$in$dogs.$Second,$antibody$titers$might$ disappear$once$a$dog$develops$clinical$signs.$In$humans,$it$has$been$observed$that$only$78%$of$ individuals$with$chronic$Addison’s$disease$remain$antibody$positive.$This$phenomenon$is$also$ observed$in$dogs$that$develop$autoimmune$hypothyroidism;$many$dogs,$over$time$and$once$ they$receive$adequate$thyroid$replacement$therapy,$become$thyroglobulin$antibody$negative$ even$though$they$tested$positive$upon$or$prior$to$diagnosis.$In$addition,$clinical$Addison’s$ disease$does$not$develop$until$at$least$90$%$of$the$adrenal$gland$is$destroyed.$Once$the$organ$is$ completely$destroyed,$the$offending$antigens$disappear$and$the$antibody$titer$many$become$ undetectable.$$ Third,$the$conditions$for$the$ELISA$need$to$be$optimized.$Different$blocking$and$ incubation$conditions$for$the$ELISA$need$to$be$evaluated.$Luckily,$our$collaborator$throughout$ this$study,$Oxford$Biomedical$Research,$Rochester,$MI,$USA,$has$extensive$expertise$in$the$ development$of$ELISAs$for$the$use$in$dogs.$ELISAs$appear$to$be$very$sensitive$to$non@specific$ binding$of$serum$of$dogs;$this$is$a$common$problem$when$working$with$canine$serum$in$such$ ! 240$ laboratory$settings.$This$characteristic$of$canine$serum$may$have$contributed$to$the$unusually$ high$background.$Currently,$Oxford$Biomedical$Research$is$the$only$company$that$markets$a$ useful$enzyme$immunoassay$for$the$detection$of$thyroglobulin$autoantibodies$(TgAA)$in$dog$ serum.$The$TgAA$is$a$marker$for$the$early$development$of$hypothyroidism$in$dogs,$long$before$ clinical$signs$appear.$Many$other$companies$tried$to$develop$a$similar$assay,$but$their$success$ was$hampered$by$non@specific$binding$of$canine$serum.$Oxford$Biomedical$Research$developed$ a$proprietary$blocking$buffer$that$took$care$of$this$non@specific$binding.$Their$expertise$will$help$ us$in$the$further$development$of$the$21@hydroxylase@antibody$assay.$On$the$other$hand,$the$ ELISA$format$might$not$be$the$preferred$method$to$detect$21@hydroxylase$antibodies,$as$ currently$all$of$the$commercially$available$test$kits$to$detect$21@hydroxylase$antibodies$in$ humans$are$radioimmunoassays.$An$iodination$of$the$purified$21@hydroxylase@His$should$ therefore$be$considered$and$evaluated.$$ Lastly,$the$low$percentage$of$detected$dogs$might$be$related$to$the$fact$that$the$ purification$of$the$21@hydroxylase@His$was$done$under$denaturing$conditions$and$the$purified$ denatured$21@hydroxylase$might$not$react$with$the$21@hydroxylase$antibody$present$in$dogs$ with$Addison’s$disease.$$ At$this$point,$we$have$only$tested$a$small$number$of$samples$from$dogs$with$confirmed$ Addison’s$disease,$i.e.$dogs$with$advanced$disease.$More$clinical$samples$need$to$be$analyzed,$ especially$samples$from$currently$healthy$dogs$from$pedigrees$or$breeds$that$are$known$to$be$ at$high$risk$to$develop$hypoadrenocorticism$(Bearded$Collies,$West$Highland$White$Terriers,$ Standard$Poodles,$Portuguese$Water$Dogs,$Leonbergers,$Great$Danes,$Airedale$Terriers,$Basset$ ! 241$ Hounds,$Wheaten$Terriers,$Rottweilers,$Springer$Spaniels,$Great$Pyrenees,$and$Nova$Scotia$ Duck$Tolling$Retrievers).$I$would$like$to$attend$breed$shows$to$collect$serum$samples$from$ these$breeds$of$dogs.$These$and$individual$dogs$should$be$followed$in$long@term$prospective$ studies$to$ascertain$changes$in$21@hydroxylase$autoantibody$levels$over$time$as$dogs$go$on$to$ develop$clinical$Addison’s$disease.$Further,$samples$that$are$sent$to$the$laboratory$at$the$ Diagnostic$Center$for$Population$and$Animal$Health$(DCPAH),$Michigan$State$University$(MSU),$ East$Lansing,$MI,$USA,$for$the$thyroid$registration$through$the$Orthopedic$Foundation$for$ Animals$should$be$analyzed$for$the$presence$of$21@hydroxylase$antibodies.$Received$are$ samples$from$healthy,$purebred$dogs.$The$submitting$veterinarian$has$to$sign$a$statement$that$ the$dog$is$healthy$at$the$time$the$sample$was$drawn.$Samples$are$only$accepted$if$they$were$ shipped$to$the$laboratory$on$ice$within$two$days,$which$means$they$are$very$fresh$and$high$in$ quality.$Collecting$data$from$these$two$groups$will$help$us$validate$and$improve$the$test$and$to$ better$understand$the$epidemiology$and$pathogenesis$of$the$disease.$$ In$summary,$the$21@hydroxylase@autoantibody$ELISA,$which$was$developed$during$this$ study$will$prove$to$be$a$useful$tool$for$the$early$detection$of$Addison’s$disease$in$dogs.$The$test$ will$improve$the$health$and$welfare$of$dogs$with$adrenal$disease$by$improving$our$ understanding$of$its$pathogenesis$and$the$value$of$early$detection.$Hypoadrenocorticism$is$ perceived$as$a$significant$problem,$especially$in$the$purebred$dog$population,$with$a$large$ genetic$component$in$several$breeds,$but$progress$towards$understanding$and$elimination$of$ the$disorder$has$been$hampered$by$lack$of$diagnostics$for$the$condition$before$onset$of$clinical$ signs.$Diagnostic$tests$currently$exist$to$confidently$document$the$presence$of$ hypoadrenocorticism$only$after$adrenal$function$is$compromised$and$clinical$signs$are$present,$ ! 242$ but$not$before.$The$technique$developed$in$this$study$is$likely$to$be$both$life@$and$money@saving$ by$making$an$early$diagnosis,$and$could$conceivably$assist$in$making$decisions$about$which$ dogs$to$breed$in$susceptible$breeds,$and$provide$a$framework$around$future$studies$into$the$ molecular$pathogenesis$of$this$important$canine$disease.$Tests$to$document$preclinical$ hypothyroidism,$for$example,$have$been$well$received$by$breeders$of$dogs$and$are$used$on$a$ regular$basis.$As$a$long@term$outcome,$the$assay$for$the$detection$of$21@hydroxylase$antibodies$ can$be$used$to$calculate$the$annual$progression$rate$from$preclinical$hypoadrenocorticism$to$ clinical$hypoadrenocorticism$and$will$help$to$assess$the$value$of$early$detection$of$adrenal$ disease.$$ Overall,$I$am$satisfied$with$the$obtained$results$and$very$optimistic$that$the$21@ hydroxylase$antibody$ELISA$will$be$a$valuable$tool$for$the$early$discovery$of$Addison’s$disease$in$ dogs.$ ! 243$