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('II-rr 'QII.’ I- ti“, K n1 IIII‘II‘I'I IIII"' “I'M I; NICIIIHII-N 113"” 'IIIII III.“ “III"- ".7 .‘Ifi J‘.cause abnormal granule formation, creates a pseudodeficit of melanin by causing fewer numbers of melanosomes and/or the aggregation of melanin in larger melanosomes. This seems to be the more widely accepted theory regarding the pigmentary changes of CNS. It has also been suggested that the CH8 defect, with its alterations in melanosome structure, might actually evoke a secondary tyrosinase defect. It is well documented that an important functional relationship exists between the melanosome and tyrosinase. The significance of this relationship has been further emphasized by the recent report of Townsend et al. that c-locus mutants of C57Bl/6J mice, considered classical albinos, do not have a structural defect resulting in a decrease in the total tyrosinase activity as was previously believed3o. In this report, it was shown that the albinism of c-locus mutants was due instead to variation in the intracellular distribution of tyrosinase with a shift in activity from the melanosomal fraction to the soluble fraction. Itzis conceivable that the abnormal melanosomes, in addition to the previously mentioned pseudodeficit effect, might secondarily have decreased tyrosinase activity and that together, these might be responsible for the pigmentary dilution of CNS. 9 It has also been suggested that the abnormal CHS melanosome might be treated as a foreign body by the cell resulting in degradation of the abnormal melanosome by the cell's autolytic faculties or in the extrusion of the abnormal melanosome and subsequent degradation by phagocytes. This mechanism is supported by several features. These include the possible recognition of granules in the process of degradation27 and the report of abnormal nevus formation in some CHS human caseszo. An additional explanation has been offered by Novak et al.31. They reported finding abnormal lysosomal function associated with several different pigment mutants inrnice and they suggested that lysosomal function, and therefore also melanosomal function, were affected by abnormalities in the pigment forming apparatus causing lysosomal and melanosomal abnormalities. It is possible that the hypopigmentation of CHS is due to a combination of any or all of these phenomena. Dilution of the pigment by packaging in large granules, secondary tyrosinase hypoactivity, excessive intracellular or phagocytic degradation of the abnormal melanin granules, and defective melanosomal functions are probably not mutually exclusive. II. The Pseudohemophilia Many of the early case descriptions of CBS in humans report complications by hemorrhagic episodesl'7'12'32‘37. 10 Most of these were characterized by petechiae and ecchymoses mainly on the extremities, but occasionally over the entire body1'7'12'33'35'37. In a few cases, bleeding in the oral and nasal cavities was reported and in others there was severe gastrointestinal hemorrhage32'34'36'37. Platelet counts in most of these cases were reported to be normal; however, a terminal thrombocytopenia was sometimes noted. Other than prolonged bleeding and clotting times, no consistent abnormalities were noted in the coagulation mechanisms. Similar hemorrhagic tendencies were noted in CHS mink and cattle. In 1968, Padgett reported fatal exsanguination in 3 CBS cattle during routine surgical procedures and diffuse petechiae and ecchymoses throughout in postmortem examinations in 3 other CBS cattle38. As early as 1950, it was suggested that Aleutian mink, now known to have CHS, bled profusely at the mouth and nose following the slightest injury39. Also it was noted that the mortality rate among CHS mink, following blood collection by cardiac puncture, was higher38. Recently, prolonged bleeding was more scientifically documented in CHS mink4o. In a study of the coagulation and fibrinolytic mechanisms of CBS mink and cattle, Phillips et al. evaluated the prothrombin and partial thromboplastin times and Factor II, Factor V, Factor VIII and fibrinogen levels and no significant abnormalities were noted4l. They also reported normal platelet numbers in CHS mink and cattle. In 11 addition, thrombin times were measured in CHS mink and Styphen viper venom times were measured in CHS cattle and these were also within normal limits. Noting the similarity between the partial albinism of CH8 and the fair complexions of patients with phenylketonuria, Page et al. discovered a marked reduction in serotonin concentrations in peripheral bloods. This finding was eventually shown not to be related to defective tryptophan metabolism or to the partial albinism but became tantamount to the elicitation of the cause of the CBS bleeding problems42‘44. In 1970, Holland observed reduced concentrations of serotonin in whole blood of CHS mink and cattle42. These findings were subsequently confirmed by Meyers et al. who, in their study, also noted substantially reduced accumulations of radio-labeled serotonin in platelets from CBS cattle43. Decreased whole blood serotonin concentrations and prolonged bleeding have also been reported in beige mice44. In studies using CHS cattle and mink, Bell et al. were ableeto rule out increased vascular fragility as the cause of the excessive bleeding in CHS45. They also found no abnormalities in platelet numbers or turnover. They measured serotonin levels in platelets and confirmed earlier findings that serotonin was deficient in CHS platelets. In addition, they measured nucleotide levels of CHS platelets and they performed in vitro platelet aggregation studies. They found that platelets from CHS cattle and mink had about 12 18% of the normal nucleotide content. In the aggregation studies, they reported an essentially normal response to adenosine diphosphate (ADP) but markedly altered responses to collagen and thrombin. From these findings, they theorized that CHS platelets suffered from defective storage or uptake of serotonin and nucleotides. In 1976, the results of two similar studies were reported almost simultaneously; Buchanan and Handin studied platelet functions in two CHS human patients46 and Bell et al. reported their data on platelets from CHS cattle and additional data from studies of two CHS human patients47. These reports confirmed and reiterated several findings. They described prolonged bleeding times, normal platelet numbers, essentially normal platelet aggregation responses tn) ADP, and altered platelet aggregation responses to collagen and adrenalin / epinephrine. They also found a marked reduction in the nucleotide contents of the platelets associated with an increase in the adenosine triphosphate / adenosine diphosphate (ATP/ADP) ratios. In addithnn Buchanan and Handin reported decreased 3H—serotonin uptake and relatively normal 3H-adenine uptake and incorporation. They postulated that the reduced uptake of serotonin in CHS platelets coincided with lack of storage while the normal incorporation of 3H-adenine in CHS platelets suggested the existence of near normal activity in the metabolic nucleotide pool. These findings reinforced the 13 classification of the platelet defect in CHS as a storage pool defect. Similar studies of other CHS human cases48"so and other animal homologuesSI'54 confirmed these findings and, along with a subsequent follow-up study in CHS cattle55, further characterized the defect. In addition to the previously described findings in CHS platelets, these authors reported decreases in: the release of adenine nucleotides after stimulation48‘52, calcium (Ca++) contentso'sz, magnesium (Mg++) contentso'sz, serotonin secretion52'53, Ca++ secretion50'53, and Mg++ secretion50'53. Impaired secretion of acid hydrolases and slightly impaired conversion of nucleotides to hypoxanthines were also reported48. In the feline model of CHS, the platelet aggregation response to ADP was reportedly abnormal and the platelet Ca++ content was 100% of normalsz. In their study of CHS cattle platelets, Meyers et al. found that beta-N-acetyl- glucosaminidase release and malondialdehyde formation were normal and from this they concluded that no defect in secretory capabilities was present in CHS platelets. These findings further indicated that the reductions in secretions were due to defective storage of the various compound353. In a study using platelets from CHS humans, Corash et al. reported a normal capacity of affected platelets to concentrate and maintain non-vesicular serotonin without metabolic degradation56. They also described normal numbers 14 of alpha granules and normal release of alpha granule contents in CHS platelets. Using several concentrations of serotonin for induction, Meyers.et al.¢described a biphasic aggregation response in platelets from CHS cat554. They reported a monophasic reversible aggregation response to lesser concentrations of serotonin, a biphasic aggregation response to intermediate concentrations of serotonin, and a monophasic irreversible aggregation response to greater concentrations of serotonin. They also noted that the serotonin concentration-dependent responses were inhibited by serotonin antagonists and that the secondary aggregation wave was inhibited by aspirin and indomethacin. From these findings, they hypothesized that the secondary aggregation wave was probably caused by a serotonin induced arachidonate metabolite which most likely was thromboxane 32. In a study using CHS mink, it was later shown that in vitro treatment of affected platelets with indomethacin or aspirin compounded the defects in aggregation4o. From these findings they suggested that the cyclo-oxygenase pathway was intact in CHS platelets thus eliminating the possibility that defective arachidonic acid metabolism was responsible for the abnormal platelet function in CHS. Numerous ultrastructural studies of CHS platelets have also been completed. In 1976, Prieur et a1. studied platelets from CHS cattle and reported finding decreased numbers of non-lysosomal osmiophilic dense bodies and 15 decreased numbers of cytoplasmic vesicle857. They also noted that platelets from normal cattle developed threefold increases in their dense body populations in response to incubation with serotonin while platelets from affected cattle showed no increase. They hypothesized that these findings represented the morphologic defect in CHS platelets and, because dense bodies are non-lysosomal and because no abnormalities were noted in the alpha granules, they suggested that the CHS defect was not exclusively lysosomal in nature. In addition to confirming the finding of fewer dense bodies in CHS platelets, Costa et al.58 also reported that the dense bodies present in CHS platelets appeared to contain normal amounts of serotonin, a finding which suggested that existing granules had a normal capacity to store serotonin. In contrast to most reports, one researcher reported finding giant granules in a few of the peripheral blood platelets in a CHS child59. Abnormally large, acid phosphatase-positive granules, consistent with lysosomes, were also reported in 30% of the bone marrow megakaryocytes and in 5% of the circulating platelets in a CHS child49 and enlarged granules were again reported in circulating platelets by White and ClawsonGo. These findings suggest that CHS platelets, though their defect is primarily non-lysosomal, are not exempt from the lysosomal defect seen in other cell types in CHS. 16 To ascertain whether or not the lack of dense granules leCHS platelets was duelu>a shorter circulating lifespan of affected platelets, platelet circulation survival was measured and there was no difference between the control and affected values61. It was also noted that platelets from CHS cattle, incubated for 5 minutes in 0.15 M NaCl, exhibited serotonin efflux. This phenomenon was not observed in platelets from normal cattle and in platelets from affected cattle it was enhanced by imipramine. From this, the researchers concluded that the maintenance of intracellular serotonin by CHS platelets was primarily dependent upon platelet membrane uptake. In addition, they noted that thrombin induced serotonin release was slower in affected platelets and that following subcellular fractionation, most of the 5-hydroxytryptamine (5-HT) was found in the light granule fraction. The ensuing hypotheses were that either some serotonin found in platelets is stored in granules other than dense granules or that immature forms of the dense granules are present in CHS platelets and that these immature forms were found in the light granule fraction. It was noted that the non-dense granule fraction accumulation of serotonin was not affected by reserpine, a compound which inhibits accumulation of serotonin by dense granules. This would seem to indicate that the granules in question are not merely immature dense granules. Further evidence suggesting a complete absence of dense granules was provided by Meyers and Seachord62, when, using l7 monoclonal antibodies specific for antigens of bovine platelet dense granules, they reported a lack of binding in CHS bovine platelets. These data suggest that (l) neither dense granules nor remnants of dense granules are present in CHS bovine platelets or (2) if dense granules or possible remnants are present, they lack the specific antigens found on normal bovine platelet dense granules. Recently, studies of several biochemical and functional parameters of platelets from 6 CHS human patients were completed63. In many of the experiments, the results were similar to previous findings, including abnormal platelet aggregation responses, reduced levels of the dense granule constituents ATP, ADP, and serotonin, and an elevated ATP/ADP ratio. The authors also noted a difference in the kinetics of radiolabelled serotonin uptake, describing a rapid increase followed by a plateau in CHS platelets and a linear increase in normal platelets. They confirmed an earlier finding by G. Boxer et al.48 that the total calcium content was decreased in CHS platelets. They also noted that the secretable calcium in CHS platelets was 31% of normal, a finding that was not unexpected; however, approximately 67% of the intracellular calcium was secretable in CHS platelets. This compares to 61% in normal platelets and 21% in platelets affected with other storage pool diseases. They concluded that the non-secretable calcium in CHS platelets was also very low. They speculated that this had a negative influence on calcium-dependent 18 metabolic processes and therefore probably aggravated the aggregation defect of CHS platelets. A preponderance of the current data indicates that a defect in the storage of nucleotides and serotonin exists in CHS platelets and the bleeding tendencies of the syndrome can be explained, at least partially if not completely, by the associated abnormal function. It is important to note that the defective storage of nucleotides and serotonin of CHS platelets might be due secondarily to the absence of dense granules, which normally store these compounds. Further studies are needed to determine whether the dense granules are missing because of an inability to perform their normal storage or whether the(flfl3defect.causes the absence of these granules primarily resulting in defective storage of their normal contents. It is also tempting to suggest that the recently reported defect involving reduced levels of non-secretable calcium might somehow cause the absence»of the dense granules or their inability to store normal secretory components. The ramifications of the infrequently reported abnormally enlarged granules in CHS megakaryocytes and platelets is also uncertain. Kaplan et al. reported improvements in the microbicidal activity of CHS leukocytes following incubation with normal platelets or with serotonin64. They speculated that the platelet serotonin defect might also be partly responsible for the defective leukocyte function. 19 III. The Abnormal Intracytoplasmic Inclusions Abnormal intracytoplasmic inclusions have consistently been described in cells of CHS human patients and in the animal homologues of CHS. These intracytoplasmic bodies were first noted in peripheral blood leukocyteslv3' 4'14'22'60'65'79, a finding which was instrumental in establishing the entity which later became known as CHS. In leukocytes, the abnormal inclusions have been observed in neutrophi1360r69’71'73'79, eosinophils60'69'72'74r77, lymphocytes60'69'70'72'74'76, monocytes6o'69‘73'75'80, plasma ce11560'81, and basoph1135°v77. Studies of bone marrow cells have led to descriptions of abnormal granules in most of the various developmental stages of myeloid precursors 2'3'7'22'66'68'70'71'74'77‘80 and one author reports finding abnormally enlarged membrane bound strucures in erythroid cellsBz. As was noted in the discussion of the bleeding problems of CHS, megakaryocytes and platelets were consistently reported as being exempt from the problem of the abnormally enlarged granules, but more recently, several investigators have reported finding enlarged membrane bound structures in megakaryocytes and circulating plateletsso'59'60. Other cells containing the abnormal CHS granules include splenic macrophagesGB, fixed phagocytes in lymph nodes72, osteoclast383'84, histiocytic cells in various organ38'12'55'72'85'87, mast cells72'87, tissue lymphoid 20 cells65'67'72'74'88, neurons and supporting nervous system cellslz'65'78'86'89’92, gastric and small intestine mucosal ce11322'78'94'95, pancreatic acinar and islet cellszzl78'96, hepatocyteszz'78'96'97, type II pneumocytes98'99, renal tubular epithelial cellsloo'104, various ductular epithelia22'78'96, dermal and ocular melanocytesla'zo, keratinocyteslos, mink scent gland epithelium22'96, anterior pituitary ce11522196, thyroid follicular cells78, adrenal cortical ce11322'78'96 and cultured cells, including fibroblasts and blood cells76'106"111 and cells cultured from spleen and lymph nodelll. A localized non-specific membrane bound esterase activity has been described in cylindrical structures within skeletal muscle cells in beige micellz. Subsequently, the normal sarcoplasmic reticular constituents were found to be absent from these cylindrical central coresll3. Also, the central cores in skeletal muscle cells of beige mice were noted as being similar to those of humans with central core disease. Several early reports on the syndrome noted similarities between normal cytologic granules and the anomalous CHS granules7'8'12'lll. In 1966, White performed histochemical and ultrastructural studies on peripheral blood leukocytes from a CHS child and concluded that the enlarged granules were probably lysosomal in nature114. Subsequent reports on the various affected cell types have confirmed this suspicion, with the possible exceptions 21 of melanocytes, type II pneumocytes, gastric chief cells, mast cells, megakaryocytes and platelets. Subscribing to the definition of a lysosome as a granule which contains hydrolytic enzymes, it is evident that the enlarged CHS granules are not lysosomal in nature in some of the affected cells. As was noted in the section on the partial albinism, it is possible that the CHS granules in melanocytes are melanosomeszz. In type II pneumocytes, the CHS inclusions are consistent with lamellar bodiesggi99 and the CHS granules in mast cells are described as enlarged mast cell granule587. Two different CHS inclusions have been described in gastric chief cells, one of which reportedly arises by fusion of zymogen granulesg3'95. Reports of the inclusions in megakaryocytes and platelets are rare and have not included studies of their nature49'59'60. In any event, the enlarged CHS granules have consistently been shown to be altered forms of normal granule populations for the various cell types. Many investigations into the genesis of the abnormal CHS granules have been completed. Following ultrastructural studies of bone marrow granulocytes in CHS mink, Davis et a1. suggested that abnormal granules developed by unregulated fusion of primary granules79, and since then most theories on the development of the CHS inclusions indicate abnormal fusion of granules as the cause. Opinions on the exact nature of the abnormal granules differ considerably. Davis et al. found abnormalities in 22 granules which corresponded to azurophilic granules in normal granulocytes73. These included variations in size from smaller than normal to massive megagranules and variations in contents, including finding several crystalloids in some of the megagranules. They also noted that the specific granules were relatively normal in morphology and numbers, a finding which was later confirmed by FittschenllG. They concluded that the CHS granules in neutrophils formed by fusion of azurophilic granules. This would classify the CHS megabodies as primary granules which in many cell types would be primary lysosomes. This theory has been proposed for the genesis of the CHS granules in many cell types including inelanocyte326I27, granulocytes60'77‘80, monocytesBo, mast cells87'117, and type II pneumocyte887'98. Two types of abnormal granules have been described in gastric chief cells in CHS mice93. It was suggested and subsequently confirmed94 that one of these granules arose by fusion of zymogen granules via a process similar to crinophagy. These granules would also be classified as enlarged primary granules. Some authors suggest that the abnormal CHS granules arise as a result of primary granules, usually lysosomes, fusing with either autophagic or heterophagic phagosome322'27'60'93'94'100‘104'118'121'122. CHS inclusions which form in this manner would be classified as either secondary autophagic phagolysosomes or secondary 23 heterophagic phagolysosomes. Cells and tissues for which this theory has been proposed include melanocyte527, gastric chief cells and gastric parietal cells93’95, cultured fibroblastsgsills'120'121, renal tubular epithelial cellsloo‘104, neutrophils60'119'121'122, eosinophilslzz, lymphocytesGo, and monocyteslzz. Ring shaped chains of lysosomal granules, in the process of sequestering cytoplasmic constituents, were observed in circulating leukocytes and it was postulated that the formation of the abnormal CHS bodies might be related to this phenomenon7l. More specifically, materials resembling microtubules were later reported supposedly being sequestered imiaa few of the giant granules in CHS lymphocytes and monocytesGoI73'123. Fusion of azurophilic and specific granules was observed in CHS neutrophils and monocytes, but the authors neglected to speculate on a classification for these unique granule5124. In 1979, ‘White and Clawson described 3 different megagranules in CHS neutrophilsso. One corresponded to the prevailing description of an enlarged azurophilic granule; the second corresponded to the inclusion described by Rausch et al.124 in which the megagranule forms by fusion of azurophilic and specific granules; and the third type resembled an autophagic vacuole involved in cytoplasmic sequestration. They also noted a redative absence of secondary granules in CHS eosinophils and speculated that 24 this suggested their involvement in the formation of the megagranules in eosinophils as well. In a subsequent report, the presence of different types of inclusions in CHS neutrophils was reemphasizedlzs. Enlarged primary granules ix: immature circulating neutrophils which reportedly develop by fusion of primary granules in the promyelocyte and early myelocyte stages were again reported. However, the authors noted that larger CHS inclusions were common in more mature neutrophils and they hypothesized that the CHS inclusions continued to develop throughout the life of the neutrophils, primarily by fusion with normal sized primary granules, specific granules, and autophagic phagosomes with sequestered contents. The megabodies found in the more mature cells were classified as secondary lysosomes. It was also noted that, contrary to the report by Rausch et alq.no monocytes were found with megagranules that developed in this manner, only those which developed through sequestration of cytoplasmic constituents as was previously reported73. Some researchers have proposed that the CHS megabodies develop by unchecked growth of granules97'118'126. In 1973, using a freeze-fracturing technique, White examined neutrophils from a CHS patient ultrastructurally and noted 2 different enlarged granules, one of which he suggested developed by unrestricted growth of azurophilic granules in early promyelocytesllg. Anomalous lysosomes have been observed arising from systems:of smooth surfaced elements 25 resembling Golgi apparatus, endoplasmic reticulum, and lysosomes (GERL) in hepatocytes of CHS mice97. Later a similar finding was noted in alveolar macrophageslze. Density gradients were used to identify granule subpopulations from neutrophils from 2 CHS patient5127. Reportedly, both types of azurophil granules normally seen in neutrophils were absent in the CHS neutrophils. In further studies, preliminary results indicate that the density of the CHS megagranules is greater than that of any normal neutrophil or eosinophil granule. The author felt that the classification of the CHS granules as enlarged azurophil granules is still appropriate because of their peroxidase positive nature. It was suggested that the absence of normal azurophil granules in CHS neutrophils is more significant to the disease process than the presence of the enlarged granules. To study lysosomal functions in renal tissues, Prieur et al. injected CHS and normal mice with horseradish peroxidase (HRP) and examined its uptake and clearance from tubular epithelial cellsgg. 131the CHS mice, some of the phagosomes containing the HRP fused with the enlarged granules in proximal convoluted tubule cells. Also the rate of digestion of the endocytosed protein was much slower in the affected mice. - Similarly, it has been shown that the uptake and degradation of exogenous proteins by the liver and kidneys in beige mice were significantly slower than normallza. The 26 conclusion drawn from this finding was that lysosomes from beige mice did not degrade these proteins identically to normal mice. In studies using CHS mice, Brandt et al. noted reduced urinary levels of several enzymes whose renal and urinary concentrations can: be eleveted artificially under the influence of androgen administrationlzg. They studied the effect of androgenic hormones on several androgen-inducible enzymes. They found marked elevations in the levels of 3 such enzymes, B-glucuronidase, B-galactosidase, and hexosaminidase, in renal tissues of beige mice. They noted that other androgen-inducible enzymes were not different in renal tissuesannithat no androgen-inducible enzymes were different in 6 other organs. They also demonstrated, both histochemically and with subcellular fractionation, that most of the increased enzyme content coincided with an accumulation of giant glucuronidase-containing lysosomes in tubular epithelial cells. They hypothesized that the CHS defect resulted in reduced lysosomal exocytosis in renal tubular cells, leading to abnormal accumulations of lysosomal enzymes. Noting no differences in the non- lysosomal androgen—inducible enzymes, they concluded that the CHS defect did not alter other organelles or other cytosolic functions. In a separate article, Brandt and Swank also reported abnormal isoproterinol—induced exocytosis of alpha amylase from parotid gland zymogen granules in CHS mice130. They suggested that the abnormal 27 CHS granules developed due to reduced exocytosis resulting from either decreased intracellular lysosomal motility or improper fusion with the plasma membrane. Subsequently, it was shown that kidney glucuronidase is secreted, both during and following testosterone administration, at a rate near threefold less than normal in beige mice131. It was also noted that following testosterone withdrawal, the loss of glucuronidase was biphasic and that the second phase was seemingly non-secretory and was specifically associated with giant lysosomes in renal proximal tubular epithelial cells near the corticomedullary junction. These findings tended to substantiate the theory that release of glucuronidase and probably other lysosomal components was abnormal in CHS. These reports also contained the first inferences that a regional increase in severity existed in relationship to the occurrence of the giant granules in renal tissues in CHS. Brandt et al. noted that there were increased numbers of the enlarged granules at the corticomedullary junctionlzg, and Swank and Brandt described an increase in severity in the megagranules in proximal tubular epithelial cells in the same area131. Exocytosis of the lysosomal enzyme myeloperoxidase has also been evaluated in CHS leukocytesl32. It was shown that the rate and extent of exocytosis was decreased in phagocytizing CHS cells and that artificially elevating the cytoplasmic Ca++ concentration had no effect on the reduced 28 exocytosis. From these data, it was concluded that the defective exocytosis of CHS probably is not related to impaired calcium mobilization. Poon et al. studied degranulation in peritoneal mast cells in response to calcium ionophore A23187 and compound 48/80 and found that mast cells from CHS mice retained their capacity to exocytose their granulesl33. They theorized that impaired degranulation was not responsible for the formation of the enlarged granules at least in mast cells in CHS. Seemingly, some characteristics of the CHS inclusions vary across species lines. While evaluating the megabodies in neutrophils from various affected species, one researcher compared such parameters as frequency of occurrence, numbers of megagranules per cell, size of the megainclusions, and the nature of the abnormal granulesgs. In this report, there was also an attempt to correlate variations in severity of the syndrome with these species oriented granule variations. Some variation also appears to exist in the frequency of occurrence and size of the abnormal granules within the same tissue in a given case. In many of the affected tissues this seems to be related to the age of the cells involved. This is evident in several studies. The CHS melanin granules were found to be larger in adults than in affected fetuses26 and the CHS inclusions in neurons and astrocytes of affected mink reportedly become progressively 29 larger with age92. Several investigators have noted that the CHS inclusions in renal tubular epithelial cells are larger and more numerous in the distal segments of the proximal convoluted tubules (S-2 segments) and in time proximal straight tubules (S-3 segments)10°'104. Increases in the size of the CHS inclusions of gastric chief cells have been associated with increased depth of the cells within the glandsg3. These progressions in the various organs were interpreted as coinciding with cell maturation93'104. Another possible explanation for the lack of CHS megagranules in some cells has come from time lapse photography and cinematography. In several studies of this nature, the investigators reported observing rapid exocytosis of the abnormal granules. The ramifications of the presence of these enlarged granules in CHS are probably extensive. There is much evidence to indicate that these inclusions are at least indirectly if not directly responsible for some of the clinical features of CHS. Many researchers have suggested that the packaging of melanin in these enlarged granules is responsible for the partial albinismlaizo. Inladdition,i1:is often theorized that the presence of these inclusions in various immune system cells is related to the increased susceptibility to disease seen in CHSl32. Thirdly, the observed reduction in the clearance of HRP and egg albumin by renal tubular 3O epithelial cells may also be due to the presence of CHS inclusionsloov102'103'135'136. Also, there is some speculation that their presence in nervous system cells may be related to the neurologic manifestations observed in some of the CHS human case88'12I86'89‘92. Their significance in other cell types is uncertain. IV. The Increased Susceptibility to Disease Probably the most devastating of the clinical features of CHS is the increased susceptibility to diseases. A propensity toward frequent infections was noted in nearly all the early CHS cases, many of which were diagnosed in retrospect38'137. Primarily this manifested itself in the forms of tonsillitis, rhinopharyngitis, otitis media, boils, abscesses, and other pyogenic infections38'137. In the animal homologues of CHS, similar increased incidences of disease are reported137‘139, and somewhat uniquely, the increased susceptibility is also manifested in mink by an increased incidence of a viral disease which slightly misleadingly is called Aleutian Disease136'139. In addition, increased bone resorption and gingival inflammation were described in CHS mink and mice. From the findings, this was speculated to be related to the increased incidence of periodontal disease observed in the affected speciesl4o. It has also been reported that beige mice, as a 31 result of experimental cytomegalovirus infection, had increased tissue damage141. Most of the early studies of immunologic functions, including serum protein electrophoresis, postvaccination antibody production, and leukocyte motility, failed to disclose any significant alterationsa'15'33'68'142'144. In fact, one author observed increased phagocytic activity by neutrophils from a CHS patient in response to an antityphoid-paratyphoid vaccination68 and another reported getting similar reactions to experimental allergic encephalomyelitis in affected and control mink145. Also, no differences were found in the cytopathic effects or in virus replication in cultured leukocytes from CHS and normal cattle which were exposed to bovine enterovirus and to pseudorabies virusl46'147. Studying neutrophils from CHS mink and cattle, Padgett found no difference in the content of several enzymes, the motility of peripheral blood and peritoneal exudate neutrophils in Pierce-Martin chambers, the amount of bactericidal cationic proteins, the intracellular killing of bacteria, the oxygen (02) consumption of resting and phagocytizing cells, and the percentage of neutrophils from peripheral blood and peritoneal exudates which contained abnormal granules148. However, it was noted that the abnormal lysosomes failed to lyse and this led to speculation that these granules were more stable than normal lysosomes. This finding would prove to be very significant 32 in that subsequent studies described defective degranulation in CHS ce113129'132. Elevated serum muramidase activities were reported associated with bone marrow hyperplasia in 4 granulocytopenic CHS patients amd it was hypothesized that this was indicative of increased granulocyte turnover149. However, an editorial in the same journal aptly pointed out the possibility that other tissues could be the source of the excessive muramidase and that some CHS patients with pancytopenia had hypoplastic bone marrowlso. Later, excessive autophagia was described in myeloid precursors and mature neutrophils and the author theorized that this could be representative of intramedullary destruction of these cellslSI. Many of the investigations into the immunologic abnormalities of CHS have centered around peripheral blood leukocytes. Mainly, these studies involved light microscopic and ultrastructural evaluations of the cellular changes associated with CHS, primarily the nature and genesis of the enlarged granules. These authors could only offer speculation as to the relationship of their findings to the immune system defect. These studies are discussed in the section on the abnormal intracytoplasmic inclusions. Functional and biochemical studies were also completed. Alterations in the content and the metabolism of sphingolipids have been reported in leukocytes from CHS patient3152'153. Granulocyte chemotaxis was studied using 33 Boyden chambers and a significantly diminished chemotactic capacity was found in cells from CHS patient8154. It was also noted that the defect was magnified by reductions in the incubation time and by reduction of the pore size. Similar studies on granulocytes from CHS mink were performed and decreased in vivo and in vitro chemotactic responses were again reported, in light of normal capacities to generate chemotactic factorslss. Marked reductions in the activities of 4 lysosomal enzymes, acid hydrolases, acid phosphatase, B-glucuronidase, and muramidase, were reported in granulocytes from CHS patientslSG. Subsequently, similar reductions III the activities of myeloperoxidase and B-glucuronidase as well as an increase in the activity of alkaline phosphatase were described in 4 CHS human3157. Increases in the activity of several hydrolases were also noted in that part of a centrifugation gradient that corresponds to the cytoplasmic component with corresponding decreases in the part that corresponds to the granular components. From these findings it was theorized that the abnormal CHS granules tended to leak their contents into the cytosol. Elastase activities in neutrophils from CHS humans and mice were reportedlsg. The authors did not speculate on the reason for their finding but they suggested that it might be related to the susceptibility problem. Reaction products specific for acid phosphatase and myeloperoxidase have also been described outside the 34 confines of the giant granuleslzs. It was further suggested that the adjacent more normal granules were affected adversely by the cytosolic presence of these compounds. These findings along with the findings reiterating the phenomenon of sequestration of cytoplasmic constituents were interpreted as manifestations of cytoplasmic injury, findings which somewhat coincided with those of OberlinngI. Studying leukocytes from affected cattle, Davis et al. described defective bactericidal capacity in CHS cells159. Similar results have been obtained from studies on neutrophils from CHS humanle6'160'161, CHS mice162, and a similar study of CHS cattle neutrophi15163. Also a similar defect in mononuclear cell chemotaxis has been reported in CHS humans, mink, and cattle164. Some alterations in metabolic functions of leukocytes were also reported132'163. Increased oxidation of glucose- 1-14C by nonphagocytizing cells and increased iodination of intracellular protein were noted132. The author also noted that nearly all of the peroxidase activity was localized in the giant CHS granules and that there was a failure of delivery of the enzyme to the phagosome. This finding coincided with Padgett's speculation that the enlarged granules were more stable148, and led to the hypothesis that degranulation was defective in CHS. It has been reported that, though the enlarged CHS granules failed to empty their contents into phagosomes (degranulation), they did sometimes incorporate 35 phagosomeslzs. The authors speculated that the defect in degranulation was probably due to the fact that most of the enlarged granules in circulating neutrophils were already secondary lysosomes. Cellular and humoral antibody responses of normal pastel and sapphire mink (one of the phenotypes of CHS mink) to intraperitoneal inoculation with goat erythrocytes were studied by Lodmell et al. and they found that fewer plaque forming cells developed in the spleens and lymph nodes from the CHS mink165. They noted that total hemolysin and 2— mercaptoethanol-resistant hemolysin titers were higher in the nonaffected mink. Each of these differences was magnified by booster inoculations. Along the same line, CHS and control mice were challenged with Candida albicans, Staphylococcus aureus, and pneumococci and significantly increased mortality rates were noted in the affected groupsl66. It was also noted that responses to these challenges by affected mice were characterized tur significantly elevated serum immunoglobulin-M and immunoglobulin-A concentrations. These findings corroborated the existence of the CHS immunologic problem in beige mice and suggested that the problem was not due to hypogammaglobulinemia. Both the C57Bl/6J (bg/bg) and the C3H/HeJ (bg/bg) strains of CHS mice were evaluated for their susceptibility to infection with Cryptococcus neoformansl67. There was increased susceptibility in both strains and the C57Bl/6J 36 (bg/bg) mice had more severe infections than did the C3H/HeJ (bg/bg) mice. In studies using CHS mice168 and a CHS human patient169, an abnormally frequent occurrence of the capped labelling pattern of Concanavalin-A (Con-A) on the surface of affected neutrophils was reported. Because of the similarity of this capping of Con-A in CHS with what is seen on normal cells whose microtubules have been disrupted, the author hypothesized that a defect involving microtubules existed in CHS. In support of this theory, subsequent studies report finding elevated intracellular cyclic Adenosine Monophosphate (cAMP) and reduced intracellular cyclic Guanosine Monophosphate (cGMP) concentrations, findings which might explain the defective function of microtubulesl7o. It was also noted that treatment of CHS cells and individuals with compounds known to elevate cGMP levels corrected many of the structural and functional abnormalities of CHSl70‘176. It is conceivable that such a defect could be responsible for the problems of CHS. These findings will be discussed in more detail in the section on the abnormal biochemistry. Other biochemical differences have been described in CHS immune system cells. These will be discussed in the section on the abnormal biochemistry. It has been demonstrated that the increased susceptibility to infections can be reversed in CHS mice by 37 bone marrow transplantation177, thus indicating that the deficient cells largely responsible for time increased susceptibility are primarily of bone marrow origin. It has also been shown that in vitro exposure to normal platelets or in vitro treatment with serotonin generated improvements in leukocyte function in CH864. Using a 5 micron filter pore size, Clawson et al. confirmed the abnormal chemotaxis of CHS neutrophils in Boyden chambers but they reported near normal chemotaxis of affected neutrophils when 8 micron filter pore size was used178. From this discovery, they hypothesized that the defective chemotaxis was due to impedance of migration caused by the presence of the abnormal granules. Somewhat conversely, the migration of CHS neutrophils through filters of 8 and 12 micron pore sizes and in chambers (Sykes-Moore) which impose no spatial constraints was more recently described as being markedly impaired. The authors suggested that the abnormal chemotaxis was due minimally at best to the presence of the enlarged granulesl79. They also reported normal polarization and motility in suspended CHS neutrophils; however, they noted that following contact with the substratum, CHS neutrophils formed large contact areas, suggesting hyperadhesiveness, and then experienced reductions in polarity and motility. They hypothesized that the reduced chemotaxis of CHS cells might be due to this hyperadhesiveness. Defective 38 locomotion has also been reported in monocyteslso‘182 and lymphocyteslaz. Using increased activity of aa bacterial cell cytoplasmic enzyme as an indication that the bacterial envelope was perforated, Hamers et al. reported normal ingestion and perforation of the bacteria by CHS neutrophils, but retarded inactivation of the enzyme183. The abnormality in the inactivation of the exzyme was attributed to reported reduced myeloperoxidase activity of CHS. The ability of CHS neutrophils to phagocytize latex particles was assessed and acflunniluminescence assay was used to evaluate CHS neutrophil function inlcells from CHS mink184. In the results of this report, it was again suggested that neutrophils from CHS mink were not defective in their ability'to phagocytize'but had ainarked reduction in the capacity to generate high energy antimicrobial oxygen species. For the most part, early studies of lymphocyte functions failed to disclose any significant alterations. This was indicated in normal antibody responses to vaccinationslas, normal lymphotoxin production185, normal B and T cell responses to mitogensla7'188, and normal capacities in: develop delayed hypersensitivity reactionsl85'187. While studying lymphoid cells from beige mice, Roder and Duwe described a complete and selective impairment of 39 natural killinglae. The described defect reportedly was not altered by prolonged incubation times, high effector to target ratios, or interferon or interferon inducing agents. They also reported that the cytotoxic capacities of other antitumor cells were apparently normal, leading to the characterization of the natural killer (NK) cell defect as being selective. This finding was reiterated in a more indepth report190 and was confirmed, both in mice and human patients, by other researcher5188'191‘193. In addition, Roder et al. showed that there was essentially normal tissue distribution of NK cells and that the defect was not related to age-dependent maturation or a shift in target selectivity. They concluded that the described defect in NK cell function was probably due to an intrinsic failure in the lytic machinery of the cells and it became apparent that the increased susceptibility to disease in CHS was not limited to bacterial and/or viral infections. In their study, Haliotis et al. showed that the use of enriched NK cell populations failed to eliminate the defect indicating that the reduced NK cell activity was not the result of supressor cells or factorslgl. In a subsequent report, the defect in NK cell activity was described as being predetermined at the level of the progenitor cells in the bone marrow and that it was also characterized by a normal capacity to interact with target cells and a normal frequency of target-binding cellslg4. This further supports the hypothesis that the defect is 40 intrinsic, probably in the lytic mechanism, in the NK cells. It was noted that partial correction of the defect was achieved with interferon,auiimportant contradiction from the first report. Numerous investigations have corroborated the reports of low NK cell activity using in vitro and in vivo testslaa‘zos; however, it has since been shown that the deficiency in NK cell activity was not absolute194'196. One report emphasized finding adequate numbers of potentially cytotoxic target binding cells, but significantly reduced numbers of active NK cellszoz. It was also noted that the activelfltcells that were present were capable of recycling and lysing multiple target cells and that in vitro interferon treatment of effector cells increased thelnaximum NK cell capacity, the percentage of active NK cells, the maximum recycling capacity, and the rate of lysis in both the control and the affected cells. From these findings, it was hypothesized that the reduction in the NK cell activity in CHS is actually due, secondarily, to a reduction in the number of active NK cells. The authors referred to the inactive target binding cells as pre-NK cells and emphasized the potential importance of interferon as an agent capable of inducing pre-NK cells to become active. Comparable replication of lymphocytic Choriomeningitis virus (LCMV) and interferon synthesis has been reported in experimental infections in beige and control mice195. From these data, it was suggested that the role of NK cells in 41 curtailing viral synthesis may be minimal and that the defect in NK cell function was not due to the lack of interferon production. Interferon and several other agents have been shown to augment NK cell activity or to induce near normal NK cell activity in CHS. Increased NK cell activity in response to in vivo treatment with polyinosinic:polycytidylic acid has been described in CHSl97. It has also been reported that infection with LCMV or exposure to UV-inactivated vesicular stomatitis virus enhances in vitro NK cell activity in affected mice196'198'200. Augmentation of NK activity has also been described in beige mice with low doses of syngeneic tumor cells201 and following exposure to such compounds in; Bacillus Calmette Guerin or tilorone hydrochloridelgg. More recently, it was demonstrated that prolonged interaction of endogenous CHS NK cells with appropriate target cells or in vitro incubation of CHS NK cells with mitomycin-treated B cells would enhance near normal.NK cell activity207h In each of these studies, the cytolytic activity of the stimulated CHS cells was significantly less than the cytolytic activity of the stimulated control cells. In other words, augmentation of the NK cell activity was evident in the affected and the control groups and the difference between the two was not eliminated. In studies using cells from affected and control mice, no difference in the splenic adherent cell populations were 42 noted, a finding which further indicated the presence of an intrinsic defect in NK cellslg9. These authors speculated that NK cells might be refractory to the stimuli necessary to convert them to their active forms. Using the frequency of occurrence of several cell surface markers and morphologic characteristics to identify NK cells, Roder et al. again reported depressed cytotoxic activity associated with normal target recognition and binding capacitie5204. They' also noted a: normal postactivation burst of oxygen intermediates by NK cells using a chemiluminescence assay. From this observation, it was hypothesized that the intrinsic cellular defect, responsible for the NK cell deficiency, involved a post— recognition, post-activation phase of the cytolytic mechanism. In a study of 4 Venezuelan patients with CHS, Merino et al. reported finding extremely high antibody titers to Epstein-Barr virus (EBV) specific viral capsid antigen (VCA), to the restricted (R) component of the EBV induced early antigen complex, and to the EBV—associated nuclear antigen (EBNA) in 2 patientszos. Reportedly these 2 patients had enlarged livers, spleens, and lymph nodes suggesting the presence of the lymphoproliferative phase. The other 2 patients subsequently became seropositive for VGA and R and also developed features of the lymphoproliferative phase. Because of this and because high anti-R titers are frequently encountered in Burkitt's 43 lymphoma, they speculated that the lymphoproliferative phase of CHS might be attributed to the development of a lymphoma- like condition as a result of EBV infection. They also suggested that the defects in NK cell and antibody-dependent cellular cytotoxicity (ADCC) activity allowed maximal viral productivity of the infected B lymphocytes because of their probable delayed elimination. After studying EBV serology in 3 human immunodeficiencies, Vilmer et al. reached a similar conclusionzos. They reported a frequent absence of anti- EBNA antibodies in patients with either Wiskott-Aldrich syndrome or ataxia telangiectasia but high persistent EBNA titers in 2 of the 3 CHS patients associated with normal allogenic cell-mediated lympholysis. From these data, they concluded that the defective NK cell functions of CHS allowed the viral development to progress to the stage at which virally infected cells could release mature virus particles. They also speculated that, because of the virtual absence of clinical EBV infection, NK cell activity is not exclusively essential in controlling latent EBV infection. In a study utilizing calcium pulse, 51Cr-release assays, Targan et al. reported finding an unaltered capacity of CHS NK cells to generate NK cell soluble cytotoxic factors and normal kinetics of lysis of CHS NK cellsZO7. From these findings, they reemphasized the hypothesis that the CHS NK cell defect was intrinsic and probably involved 44 the cellular machinery required for early activation of the cytolytic pathway. They suggested that the lytic machinery was intact but that CHS NK cells had a relative refractoriness to respond to normal levels of cytotoxic triggering and activating stimuli. In vivo tumor susceptibility tests have been completed in CHS mice as we11198'201. Following exposure of CHS mice and syngeneic normal mice to NK resistant and NK sensitive variants of the B16 malignant lymphoma, Talmadge et al. described slower growth, longer induction times, and fewer metastases of the NK sensitive tumors in the normal mice198. In addition, Karre et al. found that CHS mice developed progressive tumors faster and more frequently than do their normal littermateSZOI. Recent findings suggest that the defect in the cytotoxicity is not limited to NK ce115188'193'205'208'211. Several authors have shown that ADCC against solid tumor cells is also depressed in CH8188'193'205. After evaluating the lymphocyte subpopulations in 6 CHS human patients, Merino et al. noted an increase in the numbers of supressor/cytotoxic cells and a decrease in the number of helper ce113208. Also, a marked reduction in the generation of cytotoxic T lymphocytes in response to alloimmune challenge has been described in beige micezog. In a recent study, Goldfarb et al. found that large granular lymphocytes had reduced plasminogen activator activity in addition to altered morphology and decreased NK 45 cell killingZIO. Also, subpopulations of CHS human lymphoid cells were recently evaluated for functional, cytochemical and morphologic defect5211. Reportedly, B cells, NK cells, normal allogenic T cells, and monocytes all express morphologic and functional characteristics consistent with reported CHS abnormalities. However,aaT‘cell population that displays Gall bodies and lacks NK cell lineage characteristics was not morphologically or functionally abnormal. The researchers' conclusions were that this population of T cells was developmentally divergent from the allogenh: T cell and the NK cell populations and that lysosomal integrity was important in cell mediated cytotoxicity. In studies using CHS mice, Mahoney et al. reported normal Inobilization of Inacrophages iJ) response to intraperitoneal inoculation with Egrynebacterhmg 212,213. parvum They also described normal immunoglobulin-G Fc- and C3b-mediated rosette formation and phagocytosis. In addition, they noted that, in spite of normal endpoint antitumor activity of beige mouse macrophages, defective macrophage-mediated tumor cell cytotoxicity was evident in the kinetic studies characterized by marked delays in cytotoxic activity and in macrophage-mediated cytostatic activity. They did note that in vivo resistance in the CH3 mice was not markedly impaired. Diminished eosinophil responses to experimental Schistosoma mansoni infections have also been documented in beige mice214. 46 It is apparent that the increased susceptibility to disease is due to defective functional capabilities in cells throughout the immunologic system. These various functional defects result most prominently in increased susceptibility to bacterial and viral diseases but probably also can be related ix) other increases :hi disease frequencies and severities in CHS. V. The Pathobiochemistry Mudh of the pathobiochemistry has been discussed, whenever appropriate, in previous chapters in this text. Early studies centered around defining the nature and contents of the enlarged intracytoplasmic inclusions. In 1954, Higashi described a congenital abnormality which was manifested by enlargement of peroxidase granules in leukocytes7. Later, Higashi et al. reported finding marked variation ill the intracellular localization of alkaline phosphatase in CHS cells, some of which was found in the enlarged granule3215, a finding which was later confirmed by Sadan et al.70. As was discussed in the section on the intracytoplasmic inclusions, the CHS megagranules are mainly abnormally enlarged variants of normal granule populations of the affected cells and the contents of the enlarged granules apparently mimic the contents of the granules from which they develop. Functional and biochemical abnormalities 47 associated with the CHS inclusions have been discussed in the sections on the intracytoplasmic inclusions and the increased susceptibility to disease. Serotonin was described as being virtually absent from the peripheral blood of 2 CHS human patientsB, a finding which was traced to the absence of dense granules in CHS platelets. As was discussed in the section on the pseudohemophilia, the dense granules normally store serotonin and ATP and ADP and their absence in CHS platelets is related to defective storage of these compounds, which is believed to be responsible for the bleeding tendencies of CHS. Adenine nucleotide concentrations of leukocytes from CHS mink and cattle have also been described as being abnormally low216. Etonithese findings,i¢:was suggested that the defective storage of adenine nucleotides, which has been well documented in platelets from CHS humans and animals, also exists in leukocytes and is related to the basic problem of CHS. Several authors have described hyperlipemia in CHS patientslz'217, the significance of which is uncertain. Others have reported marked reductions in the sphingolipid contents152 and alterations in the sphingolipid metabolism153 in CHS leukocytes. The rate of iodination of intracellular proteins in resting and phagocytizing CHS leukocytes has been documented as being abnormally fast134. In addition, the rate of 48 oxidation of glucose-l—14C by resting CHS granulocytes and monocytes and was found to be increased. In monocytes, this increased rate of oxidation of glucose-l-14C was also significantly higher in the period immediately following phagocytosis. The authors felt that the rates of oxidation of formate-14C and glucose-l-14C were higher than normal in resting CHS cells but noted that the differences were not statistically significant. Other metabolic functions that were reportedly normal :hi CHS leukocytes included the postphagocytizing burst in oxygen consumption and the postphagocytizing oxidation of glucose-l-14C, glucose-6-14C, and formate-14C. It was felt that these findings coincided with the earlier observations of altered sphingolipid metabolism and that they could be explained by increased peroxide production. The functional capabilities cu? several lysosomal enzymes were evaluated kinetically in intact lysosomes from affected cellsl36. It was shown that CHS lysosomes are much less efficient at degrading substrates. While studying neutrophils from beige mice,.L.Oliver et al. noted a marked elevation in the number of neutrophils which spontaneously formed caps in response to Con-A168. They described 3 labelling patterns of ConmA on mouse neutrophils: random or diffuse, capped, and patchy or clumped. They reported an increase in the percentage of spontaneously capped neutrophils of affected mice and a converse reduction in the percentage of neutrophils with the 49 random pattern. They also noted a similarity between the percentages of randomly labelled and capped colchicine treated neutrophils from normal mice, a process which presumably disrupts microtubules, and untreated neutrophils from CHS mice. In addition, they found that preincubation of either the colchicine treated neutrophils from normal mice or the untreated neutrophils from CHS mice with cGMP or carbamylcholine or phorbol myristate acetate, agents which elevate cGMP levels, prior to Con-A exposure would result in normal Con-A labelling patterns in both groups. From these data they concluded that the basic defect in CHS might be an abnormality in microtubular polymerization and/or in the interaction between membranes and microtubules. They also felt that the antagonistic properties of cGMP to the abnormal pattern of Con-A cap formation in colchicine treated neutrophils was a further indication that cGMP promotes microtubule polymerization. With this in mind, they theorized that the CHS neutrophils had a diminished capacity to generate cGMP in response to Con-A. These findings were subsequently duplicated in neutrophils from CHS humansng. This work has been extended to include correction of defects in other cell types, correction of morphologic as well as functional defects, and correction by other compounds. In one report, it was shown that agents which elevate cGMP levels prevented the formation of giant granules in cultured fibroblasts from CHS mice17o. It has 50 also been described that treatment with carbamyl B- methylcholine, as well as cGMP or carbamylcholine, causes a reduction in the spontaneous Con—A capping in CHS human neutrophils and an increase in the number of cultured CHS human macrophages which had morphologically normal granulesl73. Similar results were attained with in vivo administrations of these agents in studies using CHS mice. Ultrastructural evidence indicating that microtubular assembly is absent in CHS neutrophils was also noted174. Following in vitro supplementation of leukocytes from a CHS infant with cGMP, the problems of impaired degranulation and diminished bactericidal capacity were reportedly corrected218. Subsequently, the concentrations of cGMP and cAMP were measured in CHS neutrophils and increased concentrations of cAMP and decreased concentrations of cGMP were noted171. It was also found that in vitro treatment with ascorbic acid generated normal bacterial killing in CHS neutrophils and that treatment of the patient with ascorbic acid normalized the neutrophilic defects in chemotaxis, degranulation, and bactericidal capacity. In a more indepth report, it was shown that in vitro supplementation with ascorbic acid resulted in a reduction in the cAMP concentrations in CHS neutrophils to near normal levelsl72. In a similar study, Kaplan et al. treated leukocytes from beige mice with lithium chloride and found that the defect involving bactericidal capacity was normalized219. It was also noted that parenteral administration of lithium 51 at normal therapeutic levels resulted in reductions in leukocyte cAMP levels and in spontaneous Con—A capping. Because lithium is known to inhibit adenyl cyclase, they hypothesized that these results strengthened the theory that a defect involving cyclic nucleotides is central in CHS. Reductions in cAMP concentrations to near normal levels have also been reported in neutrophils from a CHS human patient following in vitro and in vivo treatment with ascorbic acidzzo. In the same study, ascorbic acid therapy reportedly resulted in normalization of the bactericidal activity of affected neutrophils and a significant reduction in the number of infectious episodes but it was noted that the chemotactic response remained abnormally low. Also, no noticeable improvements in the problem of the bleeding tendencies or' the defect :hn antibody-dependent lymphocytotoxicity of CHS were noted. There has also been considerable evidence that would tend to disprove the theory that a microtubular defect, either primary or secondary to an abnormality in cyclic nucleotides, exists in CHS. Normalization of lysosomal morphology has been described in cultured skin fibroblasts grown on modified Eagle's medium containing human AB serum176. The report emphasized the finding that cells cultured in the presence of either carbachol, ascorbic acid, or cyclic nucleotides had no improvement in the abnormal lysosomal morphology. The authors could only speculate as 52 to the nature of the agent in the human AB serum that was responsible for the improvements. In ultrastructural studies of CHS platelets White et al. described circumferential bundles (ME microtubules identical to those of normal plateletssg. They also noted that the microtubules of CHS platelets responded similarly to those of normal platelets when exposed to collagen, cold, or chilling and rewarming. It was their opinion that these findings were inconsistent with the concept that a defect in microtubular assembly was basically responsible for the problems of CHS. The numbers and distribution of microtubules and actin cables have also been described as being essentially normal in CHS fibroblasts and macrophages221'222. In their study, Ostlund et al. also described normal microtubule numbers and morphology but noted that CHS lysosomes seemed to interact abnormally with the cytoskeletonzzz. From this observation it was speculated that CHS is associated with either a defect in the lysosomal membrane or a defect in the interaction between the lysosomal membrane and microtubules. The effect of ascorbic acid treatment was studied in 2 CHS human patients and in beige mice and the results added to the controversylaz. Reportedly, ascorbic acid therapy had no effect on the clinical course of the syndrome, the abnormal leukocyte morphology, the defective chemotaxis, the reduced bacterial killing, or the lymphocyte dysfunctions. No abnormalities in leukocyte cAMP*or cGMP concentrations 53 were observed and no changes were noted in the cyclic nucleotide concentrations during ascorbic acid therapy. Reportedly, treatment with ascorbic acid did promote improved survival in the mice exposed to lethal Candida albicans infections. ‘It also improved chemotaxis and bactericidal capacity in neutrophils from CHS mice; however, the abnormal morphology was unchanged. Similar to the results of the studies using human cells, no abnormalities were observed in the basal leukocyte cyclic nucleotide concentrations and no changes in those concentrations were recorded following the ascorbic acid treatment. Several researchers have described abnormalities in the metabolism of various proteins, some of which were enzymes, by different organsloo'102'128‘131'135'136. In each report, abnormal storage of the protein in the CHS granules was incriminated. These were discussed in detail in the section on the abnormal intracytoplasmic inclusions. Abnormal accumulations of a ceroid-like pigment have been described in hepatocytes, in renal proximal tubule cells, and in splenic macrophages in beige mice and were associated with increased age of the anima1223. In the same report, abnormal accumulations of hemosiderin were also noted in splenic cells from CHS mice. Other abnormalities have been documented in cell membranes in CHS. In a study of leukocyte membranes, Haak et al. reported an increase in the fluidity of the membranes that was evident near the cell surface, deep in the lipid 54 bilayer, and in isolated plasma membrane fraction5224. They did note that the order parameters were normalized in CHS mouse leukocytes following havitro treatment with either ascorbate or glucose oxidase, agents which alter the environmental oxidationzreduction potential,tnn:not with dibutyryl cGMP. Increased fluidity, which reportedly was correctable with ascorbic acid treatment, has also been described in erythrocyte membranes of affected humans and micezzs. In this study, it was also found that membranes from CHS erythrocytes had a greater number of unsaturated fatty acids. It was concluded that the abnormal fluidity of membranes from leukocytes and erythrocytes indicated the CH8 pathophysiology may be related to a general membrane disorder and that failure of cGMP to improve thelnembrane order parameters suggests the proposed nficmotubular defect coexists but is not directly related. Membrane glycolipids have also been examined in CHS cells. The membrane glycolipid asialo gangliosideMl (61.41) was found to be unaltered in biege mouse thymocytes but is found at reduced levels in splenic T cell preparations in 5 to 15 week old affected mice226. It was also shown that a sialidase sensitive sialosylated derivative of asialo GMl is increased postnatally but also falls to below normal levels in both splenic and thymic lymphocytes in 5tx>15 week old CHS mice. They concluded that several possible deletions in 55 the gangliosides exist in beige mouse thymic and splenic lymphocytes. The post-translational incorporation of tyrosine into tubulin alpha-chains was described as being two- to threefold higher in both resting and formyl-methionyl- leucyl-phenylalanine (FMLP)-stimulated polymorphonuclear leukocytes from CHS human patient3227. Reportedly, this defect was also corrected by in vitro and in vivo administration of ascorbate. The in vitro addition of ascorbate was also found to inhibit FMLP-induced stimulation of tyrosine incorporation in both control and affected cells. Similar improvements were also noted with other reducing agents such as glutathione, cysteine, and dithiothreitol, which suggested the existence of a possible relationship between the cellular redox and tubulin tyrosinolation in neutrophils. Recently, it was reported that EBV transformed B cell lines from CHS individuals generated the oxygen radical superoxide more rapidly than did cells from similarly transformed normal B cell lineszza. Since that report, Carter completed studies on neutrophils from CHS and normal mink184. He found that the latex induced, luminal enhanced chemiluminescence in CHS neutrophils was significantly less than normal. He also found, from subjective evaluations of neutrophils in electron micrographs, a similar capacity of affected neutrophils to phagocytize latex particles. From this he concluded that CHS neutrophils were not defective in 56 their ability to phagocytize but had a pronounced defect in the ability to generate high energy antimicrobial oxygen species. VI. Miscellaneous Reports and Experimental Uses of the CHS Defect The morphologic and functional defects of CHS have been exploited in various experiments. Also, there have been reports of supposedly different syndromes with similarities to CHS. Studying leishmania infections, Kirkpatrick and Farrell found that beige mice had normal humoral and cellular responses to Leishmania tropica and the courses of the infections following either primary or challenge inoculations were similar to those in normal mice229. They did note that in spite of similar anti—leishmanial antibody titers and similar responses to footpad injections of Leishmania donovani antigens, beige mice failed to eliminate amastigotes of Leishmania donovani from their spleens. With the then current understanding that the CHS defect in cytotoxicity was selective for NK cells, they concluded that NK cells were of major significance in the immunologic response to Leishmania donovani but not in the response to Leishmania tropica. Cases of acute myeloid leukemia have been reported in which enlarged intracytoplasmic inclusions resembling those seen their whicl neut1 tubul Clinf infec staph noted cases of e OCCUI 57 seen in CHS were observed230'231. These authors refer to their findings as Pseudo-Chediak-Higashi anomaly. In one report of a human patient, enlarged granules, which proved to be enlarged lysosomes, were described in neutrophils, eosinophils, basophils, melanocytes, renal tubular epithelial cells, thyroid cells, and neuron3232. Clinically, the patient had recurrent, ultimately fatal infections and severe neurologic impairment. Defective staphylococcal bactericidal activity by neutrophils was also noted. The striking similarities between this case and cases of CHS were mentioned but it was felt that the absence of enlarged granules in lymphocytes and their rare occurrence in monocytes as well as the finding of normal cAMP levels in neutrophils, normal peripheral blood neutrophil counts, and normal exocytic degranulation, distinguished it from CHS. It should be noted that each of these characteristics is either extremely variable or is very controversial in reports of CHS. The CHS megabodies have been used asrnarkers to trace the origin of some cell lines. In this manner, it was demonstrated that osteoclasts are of hematopoietic origin233 and that precursors of tissue mast cells can be transferred from one mouse to another by bone marrow transplantation234. VII 85‘ nu: CE ll'dC UDC fa: pm 58 VII. Hypothesis The basic defect underlying the cellular abnormalities of CHS has not been defined. In this study, we attempted to ascertain whether or not the defect in storage of adenine nucleotides seen in platelets in CHS might also exist in other cells. Currently, it is generally accepted that cells, other than platelets, do not store adenine nucleotides; however, my experience with the literature has uncovered no scientific evidence to support this belief. In fact, several authors have shown that ATP and 5-HT have a propensity'to form micelles in artificial mixtures and in storage organelles235 and they have documented this tendency, not only' in platelets, but also in megakaryocytes236 and in cells of the adrenal medulla237. Serotonin storage has also been documented in neurons238 and some authors have suggested that the neuronal amine storage resembles that of platelets enough to warrant using platelets as a model for aminergic neuron8239. This would tend to support a hypothesis that other cells might also store amine nucleotide complexes. In addition, it has been shown that treatment of CHS neutrophils with serotonin achieved partial correction of the bactericidal defect64. Also, our initial studies215 showed that leukocytes of CHS mink and cattle had significantly lower levels of ATP and ADP than normal. It was hypothesized that, if an undefined sstorage pool for adenine nucleotides existed in cells, the 59 CHS defect might be manifested, similarly to platelets, by defective storage of these nucleotides. Adenine nucleotides were measured in beige mouse kidneys and in CHS mink peripheral blood leukocytes. VIII. Rationale for Chosen Procedure Because of equipment availability and technique viability, the firefly luciferase technique was chosen to measure the nucleotides in our initial leukocyte studies and the results of those studies suggested validity in the hypothesis. To strengthen the hypothesis, it was necessary to demonstrate that the proposed defect was consistent in other affected species and tissues. Kidney tissue was chosen becauee it has well documented morphologic and functional defects. Because of expense of experimental subjects, ease of tissue sampling, and relative ease of maintenance and care, beige mice were used. The concentrations of ATP and ADP were of primary interest in this study. Because of the extremely short half life in tissues, liquid nitrogen freeze clamping was used to inactivate potential catabolic processes as rapidly as possible. Also, ice cold acid extraction was used to denature enzymes that might degrade the nucleotides during the extraction procedure. Though the primary focus of the study was to again eavaluate ATP and ADP concentrations in the selected tissue, 60 the prospects of also evaluating cAMP concentrations and the adenylate energy charge (AEC) as well were inviting. The concentrations of ATP, ADP and AMP are required to calculate the AEC; therefore,aihigh pressure liquid chromatography (HPLC) procedure was selected because of its capacity to quantitate each ATP, ADP, adenosine monophosphate (AMP), and cAMP simultaneously. The AEC is a measurement of the extent that the ATP- ADP-AMP system is filled with high energy phosphate bonds. It is calculated using the formula described in the Materials and Methods (see "Formula for calculating the AEC" page 76). An AEC of 1.0 indicates that the ATP-ADP-AMP systenlis entirely in the form of ATP; Conversely, if the system is dominated by AMP, the AEC would be near OJL In intact cells, an ABC of 0.85 or less will activate the ATP- generating sequences. Following completion of the study on kidney tissue from beige mice” it was deemed necessary to repeat the study on leukocytes. Mink were chosen because of availability, ease of housing, and experimental sample volume requirements. ATP, ADP, AMP, and cAMP were measured in peripheral blood leukocytes as well as in granulocyte and non-granulocyte fractions of peripheral blood leukocytes. Again acid extraction was used to inactivate enzymes that might possibly catabolize the nucleotides. MATERI ALS AND METHODS I. Mouse Renal Studies A. Animals Three breeding pairs each of CHS mice (C57 Bl/6J bg/bg) and control mice (C57 Bl/6J) were purchased from Jackson Laboratory, Bar Harbor, ME. The colonies were established from these breeding pairs and they were housed in the Laboratory Animal Care Service Center at Michigan State University. Age and sex matching were considered in selecting the experimental animals. B. Preparation of Reagents 1. Nucleotide assays a. 7% V/V perchloric acid The 7% perchloric acid was prepared by diluting 7 m1 of concentrated perchloric acid to a final volume of 100 ml with distilled water. b. Potassium carbonate (K2CO3) Potassium carbonate was prepared in 5.0 normal (N), 1.0 N and 0.1 N concentrations. The 5.0 N K2C03 was prepared by solubilizing 17.28 g of K2C03 to a final volume of 50 ml. The 1.0 N solution was prepared by diluting 10.0 ml of the 5.0 N solution to a final volume of 61 62 50 ml. The Ofilhlsolution was prepared by diluting 5.01n1 of the 1.0 N solution to a final volume of 50 ml. c. 0.02 M Tris HCl The 0.02 M Tris HCl was prepared by solubilizing 1.58 9 of trizma—HCl in approximately .475 liter of distilled water. The pH was then adjusted to 7M4 with l N NaOH and the solution was diluted to a final volume of 0.5 liter. The solution was stored at l.0°C. d. Nucleotide standards Cocktails of phosphoadenylates were prepared in 1 mM, 0.5 mM, 0.2 mM, and 0.1 mM concentrations of each ATP, ADP, AMP, and cAMP. {Nualuo mM cocktail was prepared by solubilizing 0.055599 ATP, 0.042379 ADP, 0.034509 AMP, and 0.035519 cAMP in deionized distilled water to a final volume of 100 ml. The 0.5 mM cocktail was prepared by diluting 50 ml of the 1.0 mM cocktail to a final volume of 100 ml. The 0.2 mM cocktail was prepared by diluting 40 ml of the 0.51nM cocktail to a final volume of 100 ml. The 0.1 mM cocktail was prepared by diluting 20 ml of the 0.5 mM cocktail to a final volume of 100 ml. The cocktails were frozen in 5.0 ml aliquots for storage and thawed when needed. e. HPLC Solution A (0.007 M NH4H2PO4) The 0.007 M solution of NH4H2PO4 was prepared by solubilizing 1.6104 g of NH4H2PO4 in deionized, organopure distilled water to a volume of 1.990 liters. The 5%! was then adjusted to 4.0 with dilute phosphoric acid and 63 the solution was brought to a final volume of 2.0 liters. The solution was then filtered through a 0.45 micron filter. f. HPLC Solution B (0.6 M NH4H2PO4) The 0.6 M NH4H2PO4 was prepared by solubilizing 69.04 9 of NH4H2PO4 in 0.990 liters of deionized, organopure distilled water. The pH was then adjusted to 4.5 with concentrated ammonium hydroxide and then brought to a final volume of 1.00 liter. The solution was then filtered through a 0.45 micron filter. 2. Enzyme assays (ATPase) a. 0.01 M Tris HCl The stock solution of 0.01 M Tris HCl was prepared by solubilizing 0.79 9 of Trizma-HCl in distilled water to an approximate volume of 0.48 liter. The pH was then adjusted to 7.5 and the solution was brought to a final volume of 0.5 liter. The solution was stored at l.0°C. b. 0.25 M Tris HCl The stock solution of 0.25 M Tris HCl was prepared by solubilizing 19.7 9 of Trizma-HCl in distilled water to an approximate volume of 0.48 liter. The pH was then adjusted to 7.5 and the solution was then brought to a final volume of OJSliter. The solution was stored atJHOOC. c. 0.05 M MgC12 The 0.05 M M9C12 was prepared by 64 solubilizing 0.476 9 of MgClz in distilled water to a final volume of 100 ml. The solution was stored at l.0°C. d. 1.0 M NaCl The luo b4 NaCl was prepared by solubilizing 5.84 g of NaCl in distilled water to a final volume of 100 ml. The solution was stored at l.0°C. e. 0.15 M KCl The 0.15 M KCl was prepared by solubilizing 1.118 9 of KCl in distilled water to a final volume of 100 ml. The solution was stored at l.0°C. f. 0.05 M Tris ATP The 0.05 M Tris ATP was prepared by solubilizing 0.1659 of Tris-ATP in distilled water to a final volume of 50 ml. The solution was then frozen in 2.5 ml aliquots for storage and thawed when needed. 9. 0.001 M Ouabain The 0.001 M ouabain was prepared by solubilizing 0.0369 of ouabain in distilled water to a final volume of 50 ml. The solution was stored at l.0°C. h. Color Reagent The color reagent was prepared by solubilizing 1.00 9 of (NH4)6M07024 (ammonium molybdate) in 80 ml of distilled water. Then 3.3 ml of concentrated H2304 was added and 4.0 9 of crystalline FeSO4 was added. The solution was then brought to a final volume of 100 ml. This reagent must be mixed daily. 65 i. 0.8 N HClO4 The 0.8 N HClO4 was prepared by adding 4.0 ml of 70% HClO4 to 41.6 ml of H20. The solution was stored at IJPC. j. 0.0002 M KH2P04 The 0.0002 M KH2P04 was prepared by solubilizing 0.0027 g of KH2P04 in distilled water to a final volume of 1.0 liter. k. Sigma Diagnostics Total Protein Kit C. Procedure 1. Nucleotide assays a. Tissue sampling Two sampling techniques were used. Initially the mice were euthanized by cervical dislocation and the kidneys were immediately removed. Secondarily, the Inice were anesthetized by ether inhalation and the kidneys were surgically removed. Immediately after either dissection or surgical removal, the kidneys were placed in labelled polyethylene freezer bags and were quickly frozen to -l96°C. by freeze clamping as described by Giesy et al.239. The process of freeze clamping involves the use of an aluminum clamping device with opposing flattened surfaces, which.is placed in liquid nitrogen.and cooled to -l96°C. It simultaneously and instantaneously crushes and freezes .pa the tissue. The crushed kidneys are then stored in liquid nitrogen until extracted. 66 b. Extraction A hand held mortar and pestle stainless steel tissue grinder,eadrill press held mortar and pestle stainless steel tissue grinder, polycarbonate centrifuge tubes,anuia funnel were all placed in liquid nitrogen and maintained at -196°C. until needed. One milliliter of the 7% perchloric acid was pipetted into the mortar of the hand held tissue grinder and allowed to freeze. It was then ground to a powder using the liquid nitrogen cooled pestle of the hand held tissue grinder. The mortar containing the ground frozen perchloric acid was then rested in liquid nitrogen until the tissue was ground. The crushed kidneys were placed in the liquid nitrogen cooled drill press mortar and then ground to a fine powder. This ground tissue was poured, using the liquid nitrogen cooled funnel, into the Inortar containing the ground frozen perchloric acid. The tissue and the perchloric acid were mixed by regrinding and the mixture was funneled, again using the liquid nitrogen cooled funnel, into a polycarbonate centrifuge tube. iflmz centrifuge tube containing the mixture was placed in an ice- acetone bath for storage. This procedure was repeated for each sample and after all samples were completed, they were transferred to an ice-water bath and allowed to thaw. The samples were centrifuged at 12,000 G - 0°C. for 20 minutes and the supernatants were removed. They were placed, iruiividually, in labelled polycarbonate centrifuge tubes. The tissue pellets were allowed to dry at room temperature 67 for 24 hours and the tubes and pellets were weighed. The tubes were washed, allowed to dry and reweighed. The dry weight of the pellets was established by calculating the difference of the 2 weights. The supernatants were maintained in an ice-water bath. The pH of each supernant was adjusted to 7.4 using first the 5.0 N K2C03 for approximation, and sequentially the 1.0 N and the 0.1 N solutions for precise adjustments. The samples were immediately returned to the ice-water bath. They were then centrifuged at 12,000 G — 0°C. for 20 minutes and the supernatants were removed and placed in an ice-water bath. Approximately 5.0 ml of the 0.02 M Tris HCl was added to each pellet and the tubes were vortexed. They were then recentrifuged at 12,000 G - 0°C. for 20 minutes and the supernatants were removed. The corresponding supernatants were combined and then each was brought to a final volume of 3.0 ml with ice cold 0.02 M Tris HCl. The samples were frozen for storage and then thawed when needed. c. Measurement The samples were measured with HPLC. The mobile phase was driven by 2 LDC constametric HPLC pumps. Samples were injected in precise 10.0 microliter volumes and the nucleotides were separated with a Partisil- 10 strong anion exchange (SAX) column. The nucleotides were detected withaulultraviolet ISCO V-4 variable wavelength detector at a wavelength of 258 nanometers. A Hewlett Packard recording integrator was used to record the data. A 68 gradient system, controlled by an LDC gradient master, was used for the mobile phase. The flow rate was maintained at 1.0 ml per minute throughout the gradient. The initial conditions were 100% solution A and the gradient proceeded non-linearly (mode 3) to 85% solution B in 45 minutes. The final conditions were maintained for 15 minutes and then the gradient was automatically reset. The column was allowed to reequilibrate between runs by making injections at precise 1.5 hour intervals. Nucleotide values were corrected based on the dry weight of the tissue pellets. 2. Enzyme assays a. Tissue sampling The mice were euthanized by cervical dislocation andlxnfllkidneys were immediately removed and placed in a beaker on ice. b. Extraction A 15 ml Corning glass tissue grinder was placed on ice and about 5.0 ml of the 0.01 M Tris HCl was pipetted into the tissue grinder. A mouse was then selected and euthanized by cervical dislocation. Both kidneys were :hnmediately removed and placed on ice. One kidney from each mouse was weighed and assayed for its protein content using a Sigma Diagnostics Total Protein Kit. The other kidney was placed in the tissue grinder and the grinder was shaken. The 0.01 M Tris HCl was poured off, retaining the kidney, and ice cold 0.01 M Tris HCl was added to a final volume of 8.0 ml. The kidney-Tris HCl mixture was then ground thoroughly 69 while holding the tissue grinder in ice. The homogenate from each mouse was assayed for protein content with a Sigma Diagnostics Total Protein Kit and was then adjusted to 1A) mg-protein/ml. c. Preparation of reaction mixtures A blank, a total ATPase, and a ouabain inhibited ATPase reaction tube were prepared for each sample. The reaction mixture for the blank and the total ATPase assays were prepared together. The reaction mixtures were prepared in 2 beakers and at the same time they were maintained at 0°C. by being held on ice. The ingredients of the reaction mixtures are listed in Table 1. TABLE 1. REACTION MIXTURES FOR ATPase ACTIVITY STUDY TOTAL AND OUABAIN REAGENT BLANK INHIBITED H20 6.0 ml 3.0 ml .25 M Tris HCl 4.0 ml 2.0 ml .05 M MgCl 2.0 ml 1.0 ml 1.0 M NaCl 2.0 ml ______ .15 M KCl 2.0 ml ------ .05 M Tris ATP 2.0 ml 1.0 ml .001 M Ouabain ------ 2.0 ml 70 This provided enough of each reaction mixture to do approximately 10 samples; however, because of the time regimen in the actual assay, it was very difficult to complete more than 6 samples at a time. The standard curve was prepared by setting up centrifuge tubes with 0.00, 0.05, 0.1, 0.15, and 0.2 mM concentrations of KH2P04. The 0.0002 M stock solution was used and Table 2 depicts the preparation of the tubes. TABLE 2. PREPARATION OF STANDARD CURVE TUBES FOR SPECTROSCOPY OF PHOSPHATE CONCENTRATIONS KH2P04(ml) 0.0 0.5 1.0 1.5 2.0 d. Measurement A water bath with a shaker was set at 37°C. Exactly 0.9 ml of the respective reaction mixtures was pipetted into labelled centrifuge tubes and maintained on ice. iReading tubes were then prepared by labelling one for each reaction tube and then pipetting 1.0 ml of distilled water into each. The tubes for the standard curve were prepared as described and the reading tubes and the standard curve tubes were also placed on ice. The 0.8 N HClO4 was placed on ice as well. 71 Each reaction tube was preincubated for 51nin.iJlthe 37°C. water bath shaker, taking care to begin each preincubation at exactly 30 sec. intervals. The reactions were initiated by adding 0.1 ml of water to the blank tubes and 0.1 m1 of the tissue homogenates to the total ATPase and ouabain inhibited tubes while maintaining the same 30 sec. intervals. The reactions were halted after 10 min. incubation periods by adding 1.0 ml of ice cold 0.8 N HClO4, vortexing, and placing the tube in an ice-water bath. The same 30 sec. intervaLs were maintained throughout the cessation of the reactions as well. The reaction tubes were centrifuged at 1000 G - 0.0°C. for 15 min. and then returned to the ice-water bath to await spectrosc0py. The color reagent was then prepared as described. The color reagent was pipetted, in 1J)nu.aliquots, into each standard curve tube and into each reading tube. Maintaining the same 30 sec. intervals, 1J)nd.of each reaction tube was pipetted into the corresponding reading tubes. The reading tubes were then vortexed and maintained in the ice-water bath for 20 min. The absorbance of each reading tube was measured at (a wavelength of 700 nanometers, starting with the standard curve tubes and maintaining the 30 sec. intervals. II. Mink Leukocyte Studies A. Animals The mink were purchased from farms with no recent history of Aleutian Disease and were kept for a period known to exceed the incubation period for Aleutian Disease. They 72 were housed outside at the Poultry-Fur Bearing Animal Farm at Michigan State University. Forty adult males, half CHS with the genotype (aa) homozygous recessive and half phenotypically normal mink with either genotype (aA) or (AA) for the Aleutian gene were used for the experiments. B. Preparation of Reagents 1. 7% V/V perchloric acid (See Materials and Methods Section I B l a.) 2. 0.02 M Tris HCl (See Materials and Methods Section I B l c.) 3. Nucleotide standards (See Materials and Methods Section I B l d.) 4. HPLC Solution A (See Materials and Methods Mection I B 1 e.) 5. HPLC Solution B (See Materials and Methods Section I B l f.) 6. 0.18 M (K3)EDTA The 0.18 M (K3)ethylene diamine tetraacetic acid [(K3)EDTA] was prepared by solubilizing 7.5 g of (K3)EDTA in approximately 95 m1 of distilled water. The pH was then adjusted to 7J3and the solution was brought to‘a final volume of 100 ml. 7. EDTA Phosphate Buffered Saline (EPS) The EPS was prepared by solubilizing 9.0 g of NaCl, 3.58 g of (N3)EDTA, and 1.08 g of KH2P04 (monobasic) in approximately 0.99 liter of distilled water. The pH was L __ 73 then adjusted to 7.3 and the solution was brought to a final volume of 1.0 liter. 8. 2 x EPS (double strength EPS) The 2 x EPS was prepared by solubilizing 9.0 g of NaCl, 3.58 g of (N3)EDTA, and 1.08 g of KH2P04 (monobasic) in approximately 0.49 liter of distilled water. The pH was then adjusted to 7.3 and the solution was brought to a final volume of 0.5 liter. 9. Histopaque 1077 (Ficoll) from Sigma Chemical Company 10. Unopette WBC/Platelet dilution chambers C. Tissue Sampling and Extraction 1. Total leukocyte studies The mink were anesthetized by ether inhalation and whole blood was extracted via jugular venipuncture. The hair on the ventral aspect of the neck over the jugular veins was clipped. A 12 ml syringe containing 1.0 ml of the 0.18 M (K3)EDTA was used to extract 9.0 ml of blood, achieving an approximate volume of 10.0 ml. Three mink were bled each time and this procedure was repeated for each. White blood cell and platelet counts, as well as smears for performing differentials, were prepared for each sample. Unopette WBC/Platelet dilution systems were used and the counts were completed manually as time permitted. The smears were stained with Wright's stain and the differentials were also completed as time permitted. 74 While centrifuging the whole blood, the tubes for the white blood cell wash were prepared by pipetting 4.0 m1 of distilled water into 1 tube and 4.0 ml of 2 x EPS into another tube. This was duplicated for each sample and the tubes were placed in a 37°C. The blood samples were centrifuged at 3000 G for 15 min. and the plasma portion was discarded. The buffy coat was carefully removed and transferred to another labelled 15 ml centrifuge tube. In a 2 step procedure, the red blood cells in the buffy coat were lysed and the white blood cells were washed. The RBC lysis was accomplished by hypotonic shock by adding the 4.0 ml of distilled water and vortexing immediately at a low setting. After approximately 15 sec., the 2 x EPS was added and the sample was vortexed again, thus restoring the cell suspension medium to an isotonic state. The sample was then returned to the water bath until each buffy coat was similarly handled. These steps were repeated for each buffy coat. The samples were recentrifuged at‘900(;for 8 minutes and the supernatants were removed and discarded. The lysing steps were repeated, the samples were again centrifuged at 900 G for 8 minutes and the supernatants were again discarded. Approximately 1.0 m1 of the EPS was then added to each pellet and the samples were vortexed. The 3 samples were then combined in a polycarbonate centrifuge tube and each tube was washed with approximately'l.0'ml of EPS, which was also added to the combined sample. Using EPS the sample was brought to a 75 final volume of 10.0 ml and the cells and platelets were counted. The counts were done manually in duplicate using Unopette WBC/Platelet dilution chambers and were completed within an hour as time permitted. Exactly 100 microliters of the cell sample was added to an equal volume of 15% Bovine Serum Albumin and direct smears were made for completing differentials. The sample was then centrifuged at 900(3for 81ninutes and the supernatant was removed and discarded. Precisely 250 microliters of ice cold 7% perchloric acid was added to the pellet and it was then vortexed and immediately placed in an ice-water bath. The sample was centrifuged at 12,000 G - 1.0°C. for 20 minutes and the supernatant was collected. It was brought to a final volume of 0.5 ml and maintained in an ice-water bath until measured. In order to increase the final nucleotide concentrations in the samples, the pH of the samples was not altered. This allowed for much smaller sample volumes but the samples could not be stored in the acid and therefore had to be assayed the same day. 2. Granulocyte and non-granulocyte studies The procedure for tissue sampling for this part of the experiments was basically the same as in the total leukocyte studies with a few differences. A centrifuge tube containing 3 ml of Ficoll was prepared for each sample and placed in a 37°C. water bath. Following the second red blood cell lysis step, the samples were carefully layered on the Ficoll before centrifugation. The samples 76 were centrifuged at 1,800 G for 10 minutes and the supernatants were removed and discarded. The non- granulocytes from each sample were harvested from the upper aspect of the Ficoll and pooled in a polycarbonate centrifuge tube. The central portion of the Ficoll gradient was discarded and the granulocytes from each sample were harvested from the pellet and pooled in a polycarbonate centrifuge tube. Both the granulocytes and the non- granulocytes were diluted to 10 ml with FPS and cell counts and differentials were completed. The samples were then centrifuged at 900 G for 8 minutes and the supernatants were discarded. Approximateley 250 microliters of the 7% perchloric acid was added to each sample and the sample was placed in an ice-water bath. From this point the procedure was the same as described in the total leukocyte studies. D. Measurement In this study, the nucleotides were measured with the same HPLC procedure as described in the mouse renal studies, except that a 50 microliter injection loop was used instead of the 10 microliter loop employed in the mouse renal studies. III. Formula for Calculating the AEC 1/2 [ATP] + [ADP] AEC = [ATP] + [ADP] + [AMP] STATISTICAL ANALYSIS OF DATA The data sets with even sample numbers were evaluated statistically with a two tailed T—test for paired samples with even sample numbers. The data sets with uneven sample numbers were evaluated statistically with atnuztailed T- test for data with uneven sample numbers. 77 RESULTS I. Mouse Renal Protein Content The renal protein content was compared in CHS and normal mice. The mean total protein content of kidneys was 0.254;:(L025 mg-protein / mg-kidney in affected mice and was 0.263 1 0.031 mg-protein / mg-kidney in the normal controls. These values are not significantly different. The results are depicted graphically in Figure 1. II. Mouse Renal ATPase Activity ATPase activities were measured in kidney extracts of CHS and normal mice. The mean total ATPase activities were 97.30 1 19.77 uM-activity / mg—protein / hour in the affected mice and 95.20 -_l-_ 6.90 uM-activity / mg-protein / hour in the controls. The mean Na+-K+ ATPase activities were 26.05 i 7.57 uM-activity / mg-protein / hour in affected mice and 32.17 1 10.66 uM-activity / mg-protein / hour in controls. The mean Mg++ ATPase activities were 68.47 : 18.50 uM—activity / mg-protein / hour in beige mice and 61.25 i 7.52 uM-activity/ mg-protein / hour in control mice. The renal ATPase activities of the control and the affected mice were not significantly different. These results are depicted graphically in Figure l. 78 79 FIGURE 1. THE TOTAL PROTEIN CONTENT AND THE ATPase ACTIVITIES OF KIDNEY TISSUE FROM CHS AND NORMAL MICE - CONTROL E222 - AFFECTED asthma" 1.. i / , M32333? 0.3 / / T + I 80 QL/ Sodium-Potassium é]. // T / I ATPase % .0 ¢ 1 3i” .0 ,1 0.0 /// é // /// * The units for the total protein content are mg-protein / mg—kidney. * The units for the ATPase activities are uM-activity / mg-protein / hour. III. Adenine Nucleotide Concentrations and AEC Values of Kidneys from CHS and Normal Mice Adenine nucleotide concentrations were measured in extracts from kidneys of CHS and normal mice. Initially, the nucleotides were extracted from kidneys which were dissected from the mice following euthanasia by cervical 8O dislocation. Because of exceptionally low AEC values, the procedure was modified and the experiments were repeated. In the modified technique, the kidneys were surgically removed using ether anesthesia. In the dissected samples, the mean ATP concentrations were 2.302;:1"066 uM / g-dry weight in the affected group and 2.019 : 1.078 uM / g-dry weight in the control group. The mean ADP concentrations were 2.679 1 1.241 uM / g-dry weight in the affected group and 2.536 1 0.956 in the control group. The mean AMP concentrations were 3.095 : 0.803 in the affected group and 3.548 : 1.315 in the control group. The AEC values were calculated using the formula described in the Materials and Methods, and the mean AEC values were 0.437 1 0.072 for the affected samples and 0.401 : 0.069 for the control samples. Neither the nucleotide cencentrations nor the AEC values were significantly different in the control and the affected mice. In the experiment using the surgical technique, the mean ATP concentrations were 3.411;:lu856 in the affected mice and 3.140 : 2.414 in the control mice. The mean ADP concentrations were 2.436;:(L720 in the affected mice and 2.714 1 0.907 in the control group. The mean AMP concentrations were 1.485;:(L926 in the affected mice and 2.013 : 1.464 in the control mice. Again the AEC values were calculated; the mean AEC values were 0.625 1_0.094 for the affected samples and 0.579 1 0.099 for the control samples. Neither the nucleotide concentrations nor the AEC 81 values were significantly different in the control and the affected mice. Some improvement in recovery of the nucleotides was attained by using the surgical technique for removal of the kidneys. This was most obvious in the increased AEC values in those samples. The results of both of these experiments are depicted graphically in Figure 2. FIGURE 2. THE PHOSPHOADENYLATE CONCENTRATIONS AND THE ADENYLATE ENERGY CHARGES OF KIDNEY TISSUE FROM BEIGE AND NORMAL MICE J - CONTROL / - AFFECTED DISSECTED SURGICALLY REMOVED 8.0 8.0 AEC x 10 ABC x 10 I mo 64) ATP «.0 ”P I .I, {—1, 4,0 . ADP . AMP 24) ll[ 5!! 3;: 2h A. 'l/{/ 1 f * The units for the phosphoadenylate concentrations are uM / g-dry weight. The adenylate energy charges were calculated using the formula described in Materials and Meth“ds. .— \\ .— \\\‘ \\ \\\i ———4 \ fi \\‘ \ WES—T * 82 IV. Adenine Nucleotide Concentrations and AEC Values in Peripheral Blood Leukocytes of CHS and Normal Mink Phosphoadenylate concentrations were measured in extracts from peripheral blood leukocytes in CH8 and normal mink. The mean ATP concentrations were 6.610;:lu554 nM / 107 cells in cells from affected mink and 6.667 r 2.002 nM / 107 cells in cells from control mink. The mean ADP concentrations were 1.40 i 0.85 nM / 107 cells in cells from affected mink and 2.05 i 1.27 nM / 107 cells in cells from control mink. The mean AMP concentrations were 0.78 i 0.86 nM / 107 cells in cells from affected mink and 0.89;:(L85 nM / 107 cells in cells from control mink. The mean cAMP concentrations were 0.37 i 0.32 nM / 107 cells in cells from affected mink and 0.83;:(L76 nM / 107 cells in cells from control mink. Again the AEC values were calculated and the mean AEC was 0.83 i 0.07 in the affected samples and 0.80 i 0.09 in the control samples. Neither the adenine nucleotide concentrations nor the AECs were significantly different in the affected and the control groups. The results of these studies are depicted graphically in Figure 3. V. Adenine Nucleotide Concentrations and AEC Values in Granulocytes from CHS and Normal Mink Phosphoadenylate concentrations were also measured in extracts of granulocytes from CHS and normal mink. The mean ATP concentrations were 7.30 : lu07 rut / 107 cells in cells from affected mink and 7.25 i 1.81 nM / 107 cells in 83 FIGURE 3. THE PHOSPHOADENYLATE CONCENTRATIONS AND THE ADENYLATE ENERGY CHARGES OF LEUKOCYTES FROM CHS AND NORMAL MINK I - CONTROL // - AFFECTED __% 16 firm * The units for the phosphoadenylate concentrations are nM / 107 cells. 7% 7/ 2 6 cells from control mink. The mean ADP concentrations were 2.09 i 2.09 nM / 107 cells in cells from affected mink and 2.95 + 2.35 nM / 107 cells in cells from control mink. The mean AMP concentrations were 0.55 : 0.45 nM / 107 cells in cells from affected mink and 0.94 + 0.18 nM / 107 cells in cells from control mink. The mean cAMP concentrations were 0.52 i 0.45 nM / 107 cells in cells from affected mink and 84 1.07 i 0.38 in cells from control mink. The AEC values were 0.83 1 0.07 in the affected samples and 0.78 i 0.07 in the control samples. Neither the adenine nucleotide concentrations nor the AECs were significantly different in the control and the affected mink. The results of these experiments are depicted graphically in Figure 4. FIGURE 4. THE PHOSPHOADENYLATE CONCENTRATIONS AND THE ADENYLATE ENERGY CHARGES OF GRANULOCYTES FROM CHS AND NORMAL MINK - CONTROL [/4/ - AFFECTED ATP AEC x 10 8.0 ‘C\ l—-——-l \\D F— 6.0 ADP AMP cAMP Ebb—£— \\\\\\\ e\\\\\\\\\\\‘*\-~ §\\ * The units for the phosphoadenylate concentrations are nM / 107 cells. * The adenylate energy charges were calculated using the formula described in Materials and Methods. 85 VI. Adenine Nucleotide Concentrations and AEC Values in Non-granulocytes from CH8 and Normal Mink Adenine nucleotide concentrations were measured in non-granulocytes of CHS and normal mink. The mean ATP concentrations were 6.41 :_l.34 nM / 107 cells in cells from affected mink and 7.47 i 1.67 nM / 107 cells in cells from control mink. The mean ADP concentrations were 0.89 i 0.46 nM / 107 cells in cells from affected mink and 1.17;:(L51 nM / 107 cells in cells from control mink. The mean AMP concentrations were 0.94 i 1.13 nM / 107 cells in cells from affected mink and 0.13 i 0.16 nM / 107 cells in cells from control mink. The mean cAMP concentrations were 0.12 i 0.09 nM / 107 cells in cells from affected mink and 0.23 i 0.25 nM / 107 cells in cells from control mink. The mean AEC values were 0.84 i 0.12 in the affected samples and 0.91 1 0.05 in the«control samples. Neither the phosphoadenylate concentrations nor the AECs were significantly different in the control and the.affected groups“ The results Of these experiments are depicted graphically in Figure 5. 86 FIGURE 5. THE PHOSPHOADENYLATE CONCENTRATIONS AND THE ADENYLATE ENERGY CHARGES OF NON-GRANULOCYTES FROM CHS AND NORMAL MINK _,f - CONTROL - AFFECTED ATP 8.0 W l 161/ / / 4.0 7/ j??? AMP 2 0 //// ADP “ cAMP / IE}; // . +571 ghee. * are nM / 107 cells. * ‘ 3’ Fl IIIO X H G R\\\\\\\\\\\\>§ra The units for the phosphoadenylate concentrations The adenylate energy charges were calculated using the formula described in Materials and Methods. DISCUSSION The hypothesis for this study was developed based on the proven defect in the storage of ATP, ADP, serotonin, and other secretable compounds in CHS platelets. Furthermore, our initial studies showed that leukocytes from CHS mink and cattle had significantly lower levels of ATP and ADP than normal. These experiments were designed to test the hypothesis that the CHS defect was characterized by defective storage of nucleotides in other tissues as well. To strengthen the initial findings, it was necessary to test the hypothesis in another affected species and in another affected tissue. Renal tissue of beige mice was chosen. In the experiments on renal tissues from beige and control mice, no differences were noted in the concentrations of ATP or ADP. These results conflicted with those of the initial leukocyte studies. There were also no differences in the AMP or cAMP concentrations. In the present studies, the AEC was calculated using the formula described in the Materials and Methods. The AEC is a measurement of the extent the ATP-ADP-AMP system is filled with high energy phosphate bonds and is probably the most powerful indicator of the phosphoadenylate energy 87 88 status of a cell. Again there was no difference between the control and the affected values. In measuring the nucleotides, considerable variation was experienced. Several factors were considered as the source of this variation. First of all, the variation may have been due to loss of nucleotides during the dissection procedure. Considering the extremely short half life of nucleotides in tissues, this possibility was very likely. Theoretically, variations of as little as l or 2 seconds in the period between removal and freezing of the kidney could create marked differences in the results. A second possibility was that nucleotides were somehow being lost during the extraction. This possibility was tested by spiking several of the samples at the beginning of the extraction procedure and by processing standards through the extraction procedure. In fact, nearly 98% of the nucleotides were consistently recovered. This essentially eliminated the possibility that the extraction procedure was a major contributing cause of the variation. A third possibility was that there was variation in that part of the procedure used for correcting the measured values. In the mouse renal studies, the values were corrected based on the dry weight of the pellet from the extracted tissue. The pellets were very small and weighing them accurately proved to be difficult. Interestingly enough, much of the variation was not evident in the adenylate energy charges. Because calculating the AEC 89 utilizes a ratio which includes the correcting value in both the numerator and the denominator, this variable is mathematically eliminated. This would suggest that much of the variation was in fact due to inaccuracies in weighing the dry pellets. Initially, the kidneys were dissected following cervical dislocation. In those samples the ATP concentrations were consistently low and the AMP concentrations were consistently high. Because of this, the AECs for those samples were also very low. We started removing the kidneys surgically, using ether anesthesia, and there was a marked improvement in the nucleotide retentions as indicated by higher ATP concentrations, lower AMP concentrations, and higher AECs. Unfortunately, this did not generate a measurable improvement in the consistency of the results. The renal phosphoadenylate concentrations described in these studies compare favorably with those in other studies 1J1 which adenine nucleotide concentrations have been reported24o. Renal ATPase activities were also evaluated in these experiments. The total ATPase activity as well as the sodilun-potassium ATPase and the magnesium ATPase activities were examined in kidney homogenates from control and beigearnice. There were no differences in the ATPase activities between the control and affected mice. 90 The levels of ATPase activity reported in these studies are similar to those in other reports in the literature in whhfllrenal ATPase activities were measured241'242. Because of the observed similarity between the control and the affected nucleotide concentrations in the mouse renal studies, the leukocyte findings were reexamined. Peripheral blood leukocytes were harvested from the buffy coats of whole blood samples as described in the Materials and Methods. Adenine nucleotides were extracted and measured and the ABC was calculated. There were no differences in the concentrations of ATP, ADP, AMP, or cAMP nor in the AEC between affected and control samples. The leukocytes were also separated into granulocytic and non- granulocytic fractions and phosphoadenylates were measured in both fractions. Again there were no