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This is to certify that the

thesis entitled

TRANSFER FACTOR DERIVED FROM WHOLE BLOOD AND ITS USE

IN THE TREATMENT OF MULTIPLE SCLEROSIS

presented by

Nancy L. Threadgill

has been accepted towards fulfillment
of the requirements for

mug degree in (@104, AJZTMH? Silo/11:749.

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Major professor

 

Date fligL/Jj/?7d)

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TRANSFER FACTOR DERIVED FROM WHOLE BLOOD AND ITS USE

IN THE TREATMENT OF MULTIPLE SCLEROSIS

BY

Nancy L. Threadgill

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1978

ABSTRACT

TRANSFER FACTOR DERIVED FROM WHOLE BLOOD AND ITS USE
IN THE TREATMENT OF MULTIPLE SCLEROSIS

By

Nancy L. Threadgill

Transfer factor (TF) derived from whole blood appears to have
some properties similar to that of TF derived from leukocytes. It is
a non-protein, non-antigenic substance which will stimulate lympho-
cytes in vitro. This was demonstrated by apparently nonspecific
responses by lymphocytes in active rosette and blastogenesis studies.
Chemical data suggest a low molecular weight polynucleotide.

Transfer factor specificity for immunoprophylaxis for multiple
sclerosis (MS) cannot be determined until a specific antigen related

to the disease is identified and purified.

ACKNOWLEDGEMENTS

My sincere appreciation and gratitude is expressed to:

J. R. Kateley, Ph.D., Director of Immunology, Edward W. Sparrow
Hospital, for his assistance in the selection of a research project,
his help in securing patient specimens and reagents, and for allowing
me to use his department's space and equipment to complete my project.

W. E. Maldonado, M.D., Director of Laboratories, Edward W.
Sparrow Hospital, for allowing me freedom in my work schedule to
complete the requirements for this degree; for his support; and for
allowing me to use reagents and equipment.

M. Thomas, M.S., M.T.(ASCP), my advisor, for her guidance,
patience, and support of my achieving this degree, and for allowing
me to complete several requirements outside Michigan State University.

T. Bell, D.V.M., Ph.D., J. Dyke, Ph.D., and J. R. Kateley, Ph.D.,
my advisory committee.

F. K. Neumann, Administrator of Edward W. Sparrow Hospital, for
the financial support received during the graduate program.

J. K. Gahan, M.S., M.T.(ASCP), SBB, my supervisor and friend,
for continuing guidance, patience, and support.

All the patients with multiple sclerosis and my friends and

colleagues who supplied blood specimens for me to complete my project.

ii

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . . .
LITERATURE REVIEW . . . . . . . .
Transfer Factor. . . . . .
Multiple Sclerosis . . . .
Transfer Factor - Multiple
MATERIALS AND METHODS . . . . . .
Transfer Factor. . . . . .
Biochemical Studies. . . .
Immunoglobulin Levels. . .
Lymphocyte Studies . . . .
RESULTS 0 O O O O O O O O O O O 0
DISCUSSION. 0 O O O O O O O O O 0
SUMMARY AND CONCLUSIONS . . . . .
APPENDIX. 0 O O O O O O O O O O O

BIBLIOGRAPHY. . . . . . . . . . .

VITA O O O O O O O O I O O O O O O

Sclerosis

iii

Page

16
16
18
21
22
26
33
40
41
44

48

LIST OF TABLES
Table Page

1 Percentage comparison of 16 major histocompatibility
antigens between 44 multiple sclerosis patients and
a normal population. . . . . . . . . . . . . . . . . . . 26

2 Chemical evaluation of whole blood transfer factor
using the Sequential Multiple Analyzer-Computerized
(SMAC) o o o o o o o o o o o o o o o o o o o o o o o o o 27

3 Spectrophotometric ratio, osmolality, and pH of
whole blood transfer factor. . . . . . . . . . . . . . . 28

4 Evaluation of whole blood transfer factor for iso-
agglutinins and alloagglutinins. . . . . . . . . . . . . 28

5 Immunoglobulin levels of 9 MS patients before and
after TF therapy . . . . . . . . . . . . . . . . . . . . 29

6 Lymphocyte populations (percent) in peripheral blood
of controls and MS patients following TF therapy . . . . 29

7 In vitro effect of TF on active T-rosettes of con-
trols and MS patients (percent rosettes) . . . . . . . . 29

8 Results of Limulus Amebocyte Lysate (LAL) tests on
20 TF samples plus the pH and osmolality of each . . . . 3O

9 Lymphocyte blastogenesis stimulation indices of MS
patients and controls (Mitogen - Leukoagglutinin). . . . 31

lO Blastogenesis stimulation indices of lymphocytes
incubated with TF thirty minutes, washed three times,
and then cultured with Leukoagglutinin (0.5 ug/
culture) . . . . . . . . . . . . . . . . . . . . . . . . 31

ll Blastogenesis stimulation indices of lymphocytes

incubated with TF twenty-four hours, washed three
times, and then cultured with Leukoagglutinin. . . . . . 32

iv

INT RODUCT ION

Transfer factor (TF) is one of many substances or lymphokines
that are produced by lymphocytes as part of the cellular immune
response to foreign antigen. In 1949 Lawrence (29) discovered a
method to recover this factor from leukocytes. Its mechanism of
action and biochemical structure is yet unknown. In the past ten
years researchers have tried using TF to treat some cancerous, auto-
immune, and infectious diseases in which impaired immunity may be
involved. Many of the clinical trials have had promising results
while others have been disappointing.

Recently transfer factor therapy has been tried in the treatment
of multiple sclerosis (MS), a disabling neurologic disease which
afflicts young people. The etiology of the disease is unknown and
no cure is available, although many treatments have been tried with
only limited success.

The Human Immunology Foundation in Red Bank, New Jersey (J.
Angers, M.D., Director) has tried treating MS patients with a transfer
factor derived from whole blood rather than leukocytes. They report
a favorable response in 62 out of 80 patients, or over 80%.

Edward W. Sparrow Hospital in Lansing, Michigan, has begun a
similar program for treating multiple sclerosis under the direction
of John Kateley, PhJD., Director of Immunology. The transfer factor

is obtained from whole blood donated by household contacts of the

2
MS patients following the protocol of Angers et a1. (5). This TF
is then administered to the patients by themselves or their own
private physicians.
The purpose of this study was to attempt to identify some of the
chemical properties of the transfer factor derived from whole blood
and to determine its effect on lymphocytes using in vitro techniques

such as blastogenesis and rosette formation.

LITERATURE REVIEW

Transfer Factor

 

In humans the immune response to foreign antigens such as bac-
teria and viruses is a complex system involving lymphocytes. There
are at least two types of lymphocytes: B-cells and T-cells. B-cells
produce the specific humoral antibodies which are responsible for
antibody mediated immunity; T-cells initiate the events of cell-
mediated immune reactions either through 1) direct cell-to-cell
interactions or 2) production of lymphokines (48). Some of these
lymphokines include: macrophage inhibition factor (MIF), chemotactic
factor (CF), lymphotoxin (LT), blastogenic factor (BF), interferon,
and transfer factor (TF). All of these lymphokines are currently
being evaluated for use in controlling disease. Within the last ten
years one of these, TF, has been used as therapy for diseases which
are either caused by or associated with defects in the cell mediated
immune (CMI) system. Transfer factor is a dialyzable substance which
can initiate, as well as increase, delayed hypersensitivity (DH) and,
therefore, CMI.

Historically, in 1906, Pirquet (20) described a patient who
demonstrated skin sensitivity to tuberculin after that patient
recovered from a tuberculous infection. This reaction (red welt at

injection site), elicited by Very small quantities of tuberculin,

4
remained positive long after the infection. Pirquet suspected this
response was related to immunity.

In the early 1940's Landsteiner and Chase (20,31) discovered
that tuberculin hypersensitivity in guinea pigs was transmitted by
leukocytes. They also found that this reactivity could be transferred
from one animal to another by injecting intact leukocytes from the
donor into the recipient. After injection of the leukocytes the
recipient also had a positive response where previously there had
been no response. Investigators have observed that, in humans, a
smaller dose of cellular material can elicit a similar response and
that the sensitivity lasts longer.

Landsteiner and Chase realized that this transfer of reactivity
could have great significance in the treatment of immune deficiency
diseases. However, there was a serious problem in the use of intact
leukocytes, particularly in the immune suppressed patient who could
develop graft-versus-host (GVH) disease.

In 1949 H. Sherwood Lawrence (20,28,36) reported that cell—free
leukocyte extracts could transfer the same reactivity from donor to
recipient as intact leukocytes. He was the first to name the respon-
sible agent "transfer factor", based on the fact that he could transfer
immunity from a sensitized donor to an unsensitized patient, and that
patient in turn became sensitized in a short time. For about 15 years
Lawrence's discovery was largely ignored; however, increasing clinical
use in the 1970's has brought TF back into immunological research.

Lawrence's method for producing TF utilized frozen-thawed leuko-
cytes from a sensitized donor. The cells were treated with DNAse and

dialyzed against distilled water to remove serum proteins. Transfer

5
factor remains in the cell-free dialysate. Many modifications of
Lawrence's method have been described for preparation of TF. These
modifications make comparisons between different preparations diffi-
cult to interpret.

Molecular weight estimates indicate TF to be approximately
10,000 daltons. Transfer factor is a non-antigenic, polynucleotide-
polypeptide complex which is resistant to DNAse, pancreatic RNAse,
and trypsin but not to pronase (35). It is not an immunoglobulin
nor does it effect changes in the immunoglobulin levels of the
recipient.

The presence of TF in a leukocyte extract is usually demonstrated
indirectly by the use of an in vivo or in vitro immunoassay. If TF
is made from a donor who is known to be sensitized to a specific
antigen such as tuberculin (PPD) and that TF is given to a recipient
who is PPD negative, that recipient will exhibit a positive PPD skin
test within hours. This method of testing has also proved that
transfer factor is immunologically specific. When TF is derived
from a donor sensitized to tuberculin but not diphtheria toxoid and
is then given to a recipient who is not sensitized to either substance,
that recipient will become sensitized to tuberculin only.

Recently, other methods for testing the response to TF therapy
in vitro have been reported. They include a) an increase in the
number of active T-lymphocyte rosettes, b) an increase in macrophage
inhibition factor (MIF), and c) an increase in lymphocyte blasto-
genesis. Lawrence and colleagues have utilized the latter procedure
extensively. For example, if T? prepared from tuberculin sensitive

cells is incubated with nonsensitive lymphocytes, no immunologically

6
specific events are observed. However, if tuberculin is then added
to the culture, a small number of lymphocytes will undergo transfor-
mation to lymphoblasts. These cells act as if they are now sensitized
to the antigen (31). All the in vitro testing methods provide easier
ways of testing patient responses and the reactivity of individual
TF preparations.

The mechanism by which TF works in vivo is a question as yet
unanswered, although there are many reasonable hypotheses available.
Kirkpatrick (36) suggests that TF allows cells which previously could
not respond to a specific antigen to produce lymphokines such as MIF
or chemotactic factor. Fudenberg (36) projects that TF increases
the number of specifically sensitized lymphocytes by recruiting
previously uncommitted cells. Possibly TF induces macrophages to
act on lymphocytes to uncover new antigen receptor sites or it causes
precursor cells to become immunocompetent T-cells. Lawrence (28)
suggests that TF may be an informational molecule acting as an
antigen specific receptor on T-lymphocytes or that TF may act as a
derepressor of normal lymphocytes. In any case, small populations
of normal circulating lymphocytes become antigen responsive, transform
and undergo clonal proliferation (29,35). Further research in this
area is required and more will be possible when animal models are
found who respond in a similar fashion as humans. At this time
results in this area are inconclusive (7).

Therapeutically, transfer factor has been used in the treatment
of various diseases. Many bacterial, parasitic and fungal diseases
such as disseminated coccidioidomycosis, tuberculosis and schistoso-

miasis have been treated with TF therapy, usually in conjunction with

7
antibiotics, with favorable responses in most patients. One disease,
disseminated mucocutaneous candidiasis, appears to react the best.
Many of these patients whose disease has been resistant to protracted
amphotericin therapy have responded with clinical improvement or even
total remission. In any infectious disease TF appears to reverse the
course of an overwhelming infection, reduces the need for toxic anti-
biotics, and gives the immune deficient patient capability to respond
to pathogens in his environment (26).

Specific immunodeficiency diseases have been treated with transfer
factor also. The Wiscott-Aldrich syndrome, a sex-linked (x) recessive
deficiency state in children characterized by thrombocytopenia,
eczema, and recurrent infections, has been successfully treated with
TF. Following TF therapy many children with this disease can lead
amost normal lives without continual illnesses.

Treatment of cancer is another area of TF research. Malignant
melanomas in some patients have reduced in size following injection
of TF. In any cancer, however, immunotherapy alone cannot destroy
large tumor nodules but, if used as an adjunct to surgery, chemotherapy
and radiation, TF could increase the chances of long term survival (32).

For the studies reported above, the donors for TF can often be
screened for specific cell mediated immunity. Frequently, the donors
have had the disease themselves.

There are essentially no side effects in patients given TF. It
causes no inflammatory response (except perhaps a small localized
reaction at the site of injection). No hematological, biochemical,
or enzymatic abnormalities have been detected even when large doses

are given (30). One possible side effect could be the transfer of

8
unwanted hypersensitivities from donor to recipient (for example,
to poison ivy or bee stings). However, taking careful donor histories
should make this possibility negligible.

Clearly, the use of transfer factor therapy shows promising
results, although many problems and inconsistencies have been
reported. Patients and their diseases are heterogeneous. Other
variables result from the many different methods of TF production
and the variations in dosages used. Clinicians agree that the time
has come for more controlled trials where uniform TF is given in
uniform doses under uniform protocols. Hopefully, some of these

trials will begin soon.

Multiple Sclerosis

 

Multiple sclerosis (MS) is a disabling neurological disease
which usually affects young adults between the ages of 20 and 40.
Its symptoms are caused by a localized inflammatory reaction in
which the myelin surrounding the nerve fibers disintegrates and is
replaced by scar tissue which forms plaques. This occurs primarily
in the white matter of the central nervous system (CNS), particularly
the periventricular areas, optic nerves, and the cerebellum. The
plaques distort or block nerve impulses which control such functions
as gait, sight, speech, and memory. The disease is characterized by
alternating periods of attacks (exacerbations) and remissions. Some
patients may have only one or two attacks and can lead relatively
symptom-free lives. Others will suffer from progressive exacerbations
leading to total disability in only a few years from onset. No two
patients follow the same pattern and, although most patients will show

some improvement after each attack, none will ever recovery completely.

9
The diagnosis of MS is difficult. There is no symptom or clinical
test which clearly defines the disease. Currently, the only way to
prove the existence of MS in a patient is to study the nervous system
lesions at autopsy; however, some reasonable standards have been
developed (37,44). Multiple sclerosis is classified as clinically
definitive if:

1) There is a history of remitting and relapsing symptoms
with two or more episodes.

2) There are documented neurological lesions at two or
more separate sites in the CNS white matter.

3) The age of onset of symptoms is between ten and fifty.
4) The history of signs or symptoms is six months or longer.

5) There is no better neurologic explanation for the
observed symptoms.

Other classifications of probable or possible MS are limited varia-
tions of the above criteria.

One laboratory test which is helpful in supporting the diagnosis
of MS is the identification of immunoglobulin G present in the cerebro-
spinal fluid (CSF). Sixty to eighty percent of MS patients exhibit
an oligoclonal pattern of IgG which may be present even when the amount
of immunoglobulin in the CSF is normal. This pattern is found more
often in patients with evidence of a currently active disease process
and may signify immunological activity in the brain (16). The finding
of oligoclonal bands in the CSF is not diagnostic itself, but it does
provide strong supporting evidence when syphilis, sarcoidosis, chronic
infections of the CNS and certain other rare diseases are excluded
(37,44).

Racial, genetic, and environmental factors appear to play a major

role in determining susceptibility to multiple sclerosis. Approximately

10
ten percent of MS patients will have a closely related member of
the family affected. Identical twins may have both or only one twin
affected. Women have MS slightly more often than men (ratio 1.4 to l)
and it is particularly prevalent in caucasians of European descent.
Wbrldwide distribution shows the disease to be more common in the
higher latitudes on both sides of the equator. Multiple sclerosis
is rare in the Japanese (and probably other orientals) and in
African Blacks. Migration from a high risk area to a low risk area
during childhood will confer low risk to the individual. Migration
at a later age will modify the risk to a lesser degree. Migrants
from a low risk area to a high risk area will acquire the risk
factor of the new area, especially if they are susceptible (geneti-
cally predisposed) to the disease (1).

Increasing evidence shows that histocompatibility antigens
(HLA) may be involved in the susceptibility to MS, particularly
HLA-A3, HLA-B7, and HLA-DwZ (formerly LD—7a). These antigens
are significantly more common in MS patients than in control popu-
lations (13). They are not diagnostic for MS, but possibly allow
patients to be predisposed to the agents which cause the disease.

It is also possible that another gene closely linked to an HLA gene
may be responsible.

Histocompatibility genes may also influence the progress of MS.
Jersild et a1. (22) has reported that a rapid progression of the
demyelinating disease is more prevalent in patients with HLA-Dwz.

The etiology of MS is not known, but several areas of investiga-
tion are open. The primary consideration now is that MS may be due

to a viral infection. Three features of the disease, 1) the

ll
demyelination, 2) the intermittent course, and 3) the lengthy dura-
tion, are each characteristic of some known virus diseases and each
can be associated with virus infections of the central nervous system
(CNS) (39). The virus most often implicated is the measles virus,
although other paramyxoviruses may be involved.

In 1976, Alter pointed out that measles occurs later in childhood
in industrialized than in primitive societies and suggested that MS
may be a rare response to measles in late childhood. Multiple
sclerosis patients tend to have had measles later in life than
controls (2).

In 1962, Adams and Imagawa (16) first demonstrated slightly
increased titers of measles antibody in patients with MS when com-
pared to normal populations. Antibodies to other viruses have been
found by some workers, but the measles antibodies are the most
consistent. In those patients who have the abnormal band of IgG in
the CSF, only a small part of this immunoglobulin is measles specific.
By contrast, in subacute sclerosing panencephalitis (SSPE), a disease
known to be caused by the measles virus, the CSF IgG is almost all
measles specific (16). In approximately one-third of MS patients,
there is an IgM which is specific for an antigen on the surface of
measles infected test cells. Clearly, more testing needs to be done,
particularly with purified antigens, to determine the specificity of
the antibodies present in the serum and CSF of MS patients.

Additional evidence that a virus is involved was first reported
by Carp et al. (10) in 1972 and confirmed by Koldovsky and Henle (23)
in 1975. When materials from the brain, serum, Spleen, or CSF of

MS patients were injected into mice, there was a decrease in circulating

12
polymorphonuclear neutrophils (PMN's) within 16 to 48 hours. This
PMN depression was serially transmitted to normal mice by inoculation
of sera from the infected mice. Multiple sclerosis material added
to tissue cultures of transformed mouse fibroblasts (PAM cells) which
contain an RNA tumor virus resulted in decreased cell yields. Cell-
free lysates from these cultures also produced PMN reduction in
mice (38). The effect of the transmissible agent was neutralized
by sera from MS patients and their relatives who did not have the
disease (16,38). Although these studies were promising, they have
not been confirmed by others, nor can Carp and Henle repeat their
results (45).

Immunologically, there is evidence that cell-mediated immune
mechanisms may be involved in multiple sclerosis. Lymphocytes are
present at lesion sites. In the peripheral blood there are changes
in lymphocyte subpopulations and their functions, and there are simi-
larities between MS and some known autoimmune diseases in humans.
Unfortunately, cell-mediated immune functions in MS patients are
difficult to assess due to many technical variables in the testing,
particularly the complexity of reactions and the impurity of test
antigens.

It has been demonstrated that lymphocytes are present around the
CNS lesions of MS patients indicating an immune response. Whether
this response causes the original damage or develops as a result of
damage induced by viruses or other causes is unknown.

Using cell-surface markers, T- and B-cells in the peripheral
blood are generally found to be in different proportions when compared

to normal populations. Many patients have shown a decrease in T-cells,

13
particularly during acute exacerbations (Oger et al.; Lisak et a1.)
(25). In others, however, the number may be unchanged. In addition,
some investigators have shown reduced T-cell blast transformation
with plant lectins, although others cannot confirm this finding (25).
Most workers (25) agree that the number of B-cells is slightly
increased, which may explain the increase in viral antibody titers
in some patients. These same results are also found in autoimmune
diseases such as rheumatoid arthritis and systemic lupus erythematosus
(25).

At present there are no animal models available to study multiple
sclerosis. It is possible to induce experimental allergic encephalo-
myelitis (EAE) in animals which is characterized by demyelination,
one of the most prominent pathologic features of MS. Antibodies
specific to the basic protein of myelin can be demonstrated in animals
with EAE; however, these antibodies cannot be demonstrated in humans,
which suggests some other antigen is involved in MS.

Current treatment of multiple sclerosis lies in relief of symptoms
through the use of drugs to control spasticity and physical therapy.
All have only modest results (6). Steroids may shorten the disability
resulting from an exacerbation, but chronic steroid therapy does not
significantly alter the outcome of the disease (42).

Because MS is of such long duration and is subject to spontaneous
exacerbations and remissions, and because there is a wide variation in
symptoms between individuals, the effectiveness of any single form of

treatment is difficult to evaluate in a controlled fashion.

14

Transfer Factor - Multiple Sclerosis

 

One of the diseases for which transfer factor therapy has been
tried is multiple sclerosis, but only a few of the clinical trials
have been reported. Because the etiologic agent of MS is unknown, an
adequate method of determining the immunity of a donor to MS is
unavailable. Therefore, the reactivity of the TF to MS when given
to the patient is unknown and can only be assessed by patient response.

Behen et al. (8) published a study in which 30 MS patients
received TF prepared from household contacts. It was felt that rela-
tives would have more chances of producing an immunity to MS than
random donors. No substantial improvement was noted in these patients
who received two injections over a six-month interval.

Platz et al. (20) reported improvement of three out of eleven
patients who received high doses of TF over a two-year period. Fog
and Zabriskie reported no improvement in their studies (15,50).

The most promising report was published by Angers et a1. (4) in
which TF prepared from household contacts and given at weekly intervals
induced improved clinical response in greater than 50% of MS patients.
Clinical signs which were improved included bladder function, mobility,
coordination, stamina, and balance.

The wide variation in the results of the studies reported are
probably due to donor selection and the variable status of the disease
itself. In addition, most studies use dialysates from frozen-thawed
leukocytes (TFD), while Angers used frozen-thawed whole blood (TFWB).
Moreover, the dosage schedules differed in each study. Angers' proto-

col used TFWB at weekly intervals, while other investigators adminis-

tered TF less frequently.

15
Until the etiologic agent of MS is known, adequate donor selec-
tion is impossible to control. However, continued efforts can be
made to determine the structure and the action of TF and to continue

to monitor effects of TF therapy by patient response.

MATERIALS AND METHODS

Transfer Factor

 

Donors

The majority of the donors of the whole blood required to pre-
pare the TF were relatives of MS patients. Some were friends and
co-workers. Every donor was required to meet the age, health, and
physical requirements set forth in the Standards of the American
Association of Blood Banks (1976) for routine blood donation as well
as closer scrutiny relative to viral diseases and allergies. In
addition, the following laboratory tests were performed to insure the
donor's health: complete blood count (CBC), erythrocyte sedimentation
rate (ESR), VDRL, HBsAg (for hepatitis) and a screening test for
infectious mononucleosis. Any abnormal results were reviewed by a
pathologist to determine acceptability of using the donated blood.

After meeting these requirements, one unit of blood (450 ml) was
drawn into a plastic bag containing heparin as the anticoagulant.a
The blood was immediately frozen (without addition of cryopreserva-

tives) at -70°C and maintained at that temperature until used.

 

a
Fenwal Corporation, Chicago, IL.

16

17

Transfer Factor Preparation

 

Whole blood was thawed at 4°C and placed in a sterile beaker.
A 1/200 volume of Tween 80b (25% solution) was added and the mixture
was stirred for 5-10 minutes at room temperature (25°C). During this
time dialysis tubingC (6 ft. length) which had been soaking for 3-5
days in distilled water was prepared. Immediately before use it was
rinsed in reagent grade denatured alcohol (95%)d and then rinsed 5-6
times in sterile distilled water. A double knot was made in one end
of the tubing. The blood was poured into the other end, the tubing
tied and placed in a 3800 ml wide-mouthed Erlenmeyer flask filled
with cold pyrogen-free distilled water. Both ends of the tubing were
placed outside the flask and secured with masking tape. The flask
was then placed at 4°C for 24 hours while the water was gently agi-
tated using a stir bar and magnetic stir rod. After 24 hours the
dialysate was concentrated using an Amicon Ultrafiltration apparatus
with filters having a 1000 M.W. cutoff (High Output UF Cell #2000).e
The blood was redialyzed with cold, sterile water for another 24
hours and the dialysate concentrated. The concentration of both
dialysates was achieved by positive pressure using an inert nitrogen
propellant at 30-60 psi. The ultrafiltration was performed in the
cold and a flow rate of 60-80 ml/hr is optimal. The ultrafiltration

units and filters were sterilized by rinsing with 10% alcohol in

 

b I I I I I I

Tween 80 - #T-l64, Fisher SCientific Co., Livonia, MI.
c I I I I I

DialySis Membrane Size 3OFO, Union Carbide Corp., Chicago, IL.
d I C l I

Mallnnckrodt #7008, SCientlfic Products, Allen Park, MI.

e . .
Amicon Corp., LeXington, MA.

18

sterile distilled water. The units are periodically screened for
bacterial contamination. All of the above work was carried out in
the laboratory walk-in refrigerator, monitored at 4°C :_2°.

The 20-25 ml of TF recovered after filtration was diluted to a
50 ml volume with 1.5% NaCl and was then filtered through a 0.22 u
Milex filterf and dispensed in 5 ml volumes (10 vials) and stored
at -20°C until needed for use. Transfer factor was released to the
patient's physician only when the sterility screen had been com-

pleted and the results were negative.

Dosage Schedule

 

Each dose of 5 ml was administered subcutaneously. Each patient
received a dose three times during the first week of therapy. There-
after, only one injection per week was given. This dosage may have

been modified later depending upon patient response.

Biochemical Studies

 

The following tests were done to determine some of the chemical

characteristics of TFWB.

PE
The pH of 19 samples was determined using the Beckmang Model 3560

Digital pH Meter which was first standardized with buffers of pH 7.40

and 6.86.

 

fMillipore Corp., Bedford, MA.

gBeckman, Fullerton, CA.

l9

Osmolality

 

Five microliters of TFWB was placed onto filter paper which
then fit inside the vapor chamber of the #5100 Vapor Pressure

Osmometer.h The osmolality results were read from a digital readout.

SMAC
Samples of TFWB identical to that used by the patients were
analyzed on the SMAC (Sequential Multiple Analyzer Computerized).l

The following parameters were determined:

Albumin CO 2

Alkaline phosphatase CPK

ALT (SGP-T) Creatinine

AST (SGO-T) Glucose
Bilirubin, total LDH

Bilirubin, direct Phosphorus

BUN Potassium
Calcium Protein, total
Cholesterol Sodium
Chloride Uric acid

260/280 Ratios

 

A 1:20 dilution of TFWB was made with distilled water. These

diluted samples were then scanned on the Beckman DB Spectrophotometer:l
over a range of 240nm—300nm. The optical densities at 260 nm and
280 nm were determined and a ratio obtained. It was previously

determined that diluting the sample would not alter the ratio.

 

hWescor, Inc., Logan, UT.
1Technicon Corp., Tarrytown, NY.

JBeckman, Fullerton, CA.

20

Limulus Amebocyte Lysate (LAL)k

 

This test was developed to detect endotoxins of gram-negative
bacteria.

Glass vials were provided which already contained lyophilized
lysate (0.1 ml/vial). This lysate was previously standardized to
detect at least 1.0 ng/ml E. coli 0111:B4 endotoxin. Using a fresh
pipette for each sample, 200 ul of transfer factor was added to each
vial of lysate. Pyrogen free distilled water served as the negative
control, while E. coli 0111:B4 endotoxin was the positive control.
Each vial was mixed by tilting and gentle rolling until the contents
were in solution. All the vials were then left undisturbed in a
37°C water bath for one hour. After incubation each vial was care-
fully placed in an inverted position on a flat surface. A positive
test was characterized by the formation of a solid gel. A negative
test was characterized by the absence of a gel or clot.

Several vials containing 0.1 ml lysate already treated with
endotoxin also had TF added to them using the testing procedures
already listed. These vials were used to determine whether TF was
inhibitory to the reaction. A positive reaction was expected. If
a negative reaction was observed, the entire test was considered

invalid.

Agglutinins

 

Two drops of TF and one drop of appropriate cells were com-

WB
bined in a 10 x 75 mm glass tube.

 

kMicrobial Associates, Bethesda, MD.

21

 

Agglutinin Test Cell

Isoagglutinins l
Anti—A a cells
Anti-B b cells

Alloagglutinins m
i.e., Anti-D 0 cells

Two drOps of 22% bovine albuminn were added to the tubes containing
the 0 cells. All the tubes were centrifuged and then observed for
hemolysis and/or agglutination. The tubes testing for alloagglutinins
were then incubated for 30 minutes at 37°C. Again they were centri-
fuged and read. The cells were washed three times in 0.85% NaCl.
After the third wash the supernatant was decanted and two drops of
anti-human globulin (Coombs reagent)n were added to the cells
remaining. The tubes were again centrifuged and then observed for

hemolysis and/or agglutination.

Immungglobulin Levels
Levels of IgG, IgM and IgA were determined in the sera of MS
patients both before and after receiving TF. Five microliters of
patient serum, standards and controls were placed in the wells of
specific Tri-Partigen radial diffusion plates.0 The plates were
allowed to stand at room temperature for a minimum of 50 hours before

the diameters of the precipitin rings were measured using a calibrated

 

l . . . .
Affirmagen, Ortho Diagnostics, Raritan, NJ.
m . . .
Selectogen, Ortho Diagnostics, Raritan, NJ.
nOrtho Diagnostics, Raritan, NJ.

0 . . . .
Behring Diagnostics, SomerVille, NJ.

22
magnifier. The immunoglobulin level was then determined from a

standard curve.

Lymphocyte Studies

 

Lymphocyte Preparation

 

Peripheral blood was collected in heparinized tubes and 0.1 ml
dextran coated carbonyl iron (DCCI) was added to every 2.0 ml of
the anticoagulated blood. The tubes were placed in a 37°C water
bath for 20 minutes and the contents mixed every five minutes. The
blood was then transferred to a Ficoll-Hypaque (F-H) gradient (1.0
ml of blood to 1.0 ml of gradient). The cell suspensions were centri—
fuged on the gradient at 1800 rpm for 25 minutes after which the
upper layer of plasma was removed and discarded. The lymphocytes at
the interface of the F-H gradient were collected with a Pasteur
pipette and transferred to another centrifuge tube. To remove residual
gradient, the lymphocyte suspension was washed with Hanks' Balanced
Salt Solution (HBSS)-Incomplete by centrifuging at 1200 rpm for 10
minutes. Two washes were generally sufficient to remove all gradient
components. Platelets were removed from the lymphocyte suspensions
by a third wash at 500 rpm for five minutes. The supernatants were
discarded following each centrifugation step. The lymphocytes were
resuspended in Roswell Park Memorial Institute 1640 (RPMI 1640)
supplemented with 2% human AB plasma for blastogenesis studies or
Hanks' Balanced Salt Solution (HBSS) if rosette analyses or histo-
compatibility typing tests were required. The lymphocyte suspensions

were adjusted to 2 x 106 cells per ml with the appropriate media.

23

Histocompatibility Testing

 

The standard National Institutes of Health (NIH) technique of
microlymphocytotoxicity was used for determining the histocompati-
bility antigens on the lymphocytes of patients with multiple sclerosis.
The antisera were supplied by NIH. Please see the Appendix for the

complete procedure.

Rosette Analysis

 

Preparation of neuraminidase-treated erythrocytes (EN cells)
for identification of thymus-derived (T) lymphocytes: Sheep erythro-
cytes (SRBC) were washed with Hanks' Balanced Salt Solution (HBSS)
three times. The SRBC's were adjusted to a 5% suspension with HBSS.
Four-tenths milliliter vibriocholera neuraminidase (VCN) was added
to 2.0 ml of the 5% SRBC's and incubated at 37°C for 60 minutes.
After incubation, the VCN treated SRBC's were washed three times
with HBSS and then readjusted to a 5% suspension. The working sus-
pension for the assay was 0.5% SRBC‘s.

Preparation of human erythrocytes with added complement (HEAC
cells) for bone-marrow derived (B) lymphocytes: Group 0 human
erythrocytes (RBC) were washed five times with HBSS. The cells were
adjusted to a 2.5% suspension and then 2.0 ml of the human RBC's
were incubated with 2.0 ml of a 1:20 dilution of rabbit anti-human
RBC serum for 40 minutes at 37°C. Then the cells were centrifuged
and the supernatant removed. Two-tenths milliliter of mouse complement
was added and the cells were incubated for an additional 30 minutes.
After being washed four times with HBSS the cells were adjusted to

a 5% suspension. The working suspension for the assay was 0.5% RBC's.

24

Rosette assay: One-tenth milliliter lymphocyte suspension
(2 x 106/m1) was mixed with 0.1 ml of EN or HEAC cells in a 10 x 75
mm glass tube. The tube was centrifuged at 600 rpm for eight
minutes. Approximately 0.1 ml of supernatant was removed from the
tube and replaced with 0.1 m1 of a 0.01% gentian violet solution.
The pellet was gently resuspended with a Pasteur pipette and one
drop was transferred to a hemacytometer. All the lymphocytes in the
four large corner squares were counted, both rosettes and lymphocytes
with no RBC's attached. Lymphocytes with three or more EN or HEAC
cells attached were considered rosettes. The percentage of rosette
forming cells (RFC) was determined by dividing the number of rosettes
by the total number of lymphocytes counted.

Active T-Rosettes: One—tenth milliliter of lymphocyte
suspension (2 x 106/md) was mixed with 0.2 ml inactivated fetal calf
serum (FCS) and 0.2 m1 of 0.5% suspension of SRBC's which had been
washed four times in HBSS. This mixture was incubated in a 10 x 75 mm
glass tube for 60 minutes at 37°C and then centrifuged at 400 rpm
for 7 minutes. Approximately 0.3 m1 of supernatant was removed from
the tube and replaced with 0.1 ml of 0.01% gentian violet suspension.
The pellet was gently resuspended and one drop was transferred to a
hemacytometer. All the lymphocytes in the four large corner squares
were counted and the percentage of rosette forming cells was determined.

Active T-Rosettes with TF: Lymphocytes (5 x 106 ml) from
both MS patients and controls were incubated with 0.5 ml TF at 37°C
for 30 minutes. The cells were then washed three times with Medium

199 and resuspended to a 2 x lOG/ml suspension. Then the percentage

of active T-rosettes was determined as above.

25

Lymphocyte Blastogenesis

 

The lymphocytes were prepared and adjusted to a suspension of
2 x 106 cells/ml with RPMI 1640 medium with 2% FCS. Mitogen concen-
trations were adjusted to give a wide dose range (Leucoagglutinin -
20 ug/ml to 0.5 ug/ml). Control cultures (no mitogen) and 2 or 3
mitogen doses were usually performed for each leukocyte culture.
One-tenth milliliter cells and 0.1 ml mitogen were added to micro-
liter plates with an Eppendorf pipette. Cultures were set up in
duplicate and triplicate if enough cells were available. The cultures
were incubated at 37°C in a 5% CO2 humidified atmosphere and the
blastogenic response was determined approximately 72-80 hours after
culture initiation. Fifty microliters of tritiated thymidine (3H-Tdr)
was added about 18 hours prior to harvesting the cultures using a
MASH-II. The filter paper disks were placed in scintillation vials
and 10 ml of Scintiverse was added. The cultures were dark adapted
in the scintillation counter for two hours and were then counted for
two minutes. The stimulation index (SI) was then calculated:

(cpm cultures with mitogen) 4 (cpm background cultures)
(cpm background cultures)

Lymphocyte Blastogenesis with TF

Two milliliters of prepared lymphocytes (5 x 106/ml) from both
MS patients and controls were incubated with 1.0 ml TF at 37°C for
different time intervals. The cells were then washed three times
with RPMI 1640 and resuspended to a 2 x 106/m1 suspension. The blasto-

genesis cultures were then set up as outlined above.

RESULTS

The results obtained are listed in the following tables.

Taflel.

Percentage comparison of 16 major histocompatibility
antigens between 44 multiple sclerosis patients and a

normal population (49)

 

HLA Antigen

% MS Patients

% Normal Cauca-
sian Population

 

HLA-Al
HLA-AZ
HLA-A3
HLA-A9
HLA-AlO
HLA-All
HLA-A28
HLA-A29

HLA-BS
HLA-B7
HLA-BB
HLA-BlZ
HLA-B13
HLA-Bl4
HLA-BlB
HLA-B27

27
36
3O
20
14

ll
48
18
14

11
ll

28
50
26
20
12
11

ll
24
21
29

O‘mmb

 

26

27

Table 2. Chemical evaluation of whole blood transfer factor using
the Sequential Multiple Analyzer-Computerized (SMAC)

 

 

Standard Normal Serum
Parameter Mean Range Deviation Values
Albumin 0.0 3.5-5.0 g/dl
Alkaline phosphatase 0.0 30-105 U/L
ALT (SGPT) 0.0 2-45 U/L
AST (SGOT) 0.0 10-40 U/L
Bilirubin - total 0.0 0.2-1.0 mg/dl
Bilirubin - direct 0.0 0.0-0.3 mg/dl
BUN 0.0 6-23 mg/dl
Calcium 8.4 2.1-15 3.2 8.5-10.5 mg/dl
Cholesterol 8.5 4-22 3.9 150-250 mg/dl
Chloride >130 96-107 mEq/L
CO2 0.0 24-33 mM/L
CPK 0.0 25-145 U/L
Creatinine 0.6 0.1-1.3 0. 0.6-1.1 mg/dl
Glucose 5.7 0-15 4.9 65-110 mg/dl
LDH 0.0 100-225 U/L
Phosphorus 4.9 0.6-9.9 3.2 2.5-4.5 mg/dl
Potassium 7.1 1.7-10.0 2.9 3.3-4.6 mEq/L
Protein - total 0.0 6-8 g/dl
Sodium >160 135-145 mEq/L
Uric acid 0.0 2.5-8.0 mg/dl

 

28

Table 3. Spectrophotometric ratio, osmolality, and pH of whole
blood transfer factor

 

 

Parameter Mean Range Standard Deviation
260/280 ratio 6.7 1.2-12.9 1.8
Osmolality 320 213-469 46

pH 7.39 6.73-7.68 1.67

 

Table 4. Evaluation of whole blood transfer factor for isoagglutinins
and alloagglutinins

 

No isoagglutinins (anti-A, anti-B) or alloagglutinins were demonstra-
ted in 20 samples of transfer factor.

In 2 cases in which anti-D was known to exist in the donor blood, the
antibody was not demonstrated in the transfer factor prepared from
that blood.

No unexpected antibodies were demonstrated in 20 multiple sclerosis
patients up to 6 months following transfer factor therapy.

 

29

Table 5. Immunoglobulin levels of 9 MS patients before and after

 

 

TF therapy
T-test
Ig Pre (mean) Post (mean) Values Normal Values
G 958 996 0.40 548-1768 mg/dl
M 153 151 0.08 45-153 mg/dl
A 152 158 0.20 78-322 mg/dl

 

Using the Student's t-test, there is no significant difference between
the pre and post values at P=0.05.

Table 6. Lymphocyte populations (percent) in peripheral blood of
controls and MS patients following TF therapy

 

 

Group Number B-cells T-cells Active T-cells
Controls 20 18.1il.9 69.4:3.7 20.6:3.8

MS patients 25 17.0i3.6 67.8:4.7 20.1:5.l
T-test values 1.31 1.19 0.32

 

Using the Student's t-test, there is no significant difference between
these two populations at P=0.05.

Table 7. In vitro effect of TF on active T-rosettes of controls and
MS patients (percent rosettes)

 

 

Group No TF With TF T-test Values
Controls l9.7il.7 24.1:3.4 2.28
MS patients 18.2i3.0 23.4i3.0 2.20

 

Using the Student's t-test, there is a significant difference between
the number of active T-rosettes with and without TF at P=0.05.

30

Table 8. Results of Limulus Amebocyte Lysate (LAL) tests on 20 TF
samples plus the pH and osmolality of each

 

 

Sample No. LAL Results pH Osmolality
l — 6.73 413
2 + 7.38 375
3 + 6.95 226
4 - 7.01 346
5 + 7.55 261
6 + 7.48 256
7 + 7.41 325
8 - 7.43 469
9 + 7.53 403

10 + - 289
11 + 7.62 302
12 + 7.52 367
13 + 7.49 443
14 - 7.45 365
15 + 7.15 320
16 + 7.68 275
17 - - 345
18 - 7.35 313
19 - - -

20 + 7.60 234

 

Positive control = +
Negative control = -
All inhibition controls = +

31

Table 9. Lymphocyte blastogenesis stimulation indices of MS patients

and controls (Mitogen - Leukoagglutinin)

 

Leukoagglutinin (pg/culture)

 

 

2.0 1.0 0.5 0.1 0.05

MS Patient #1 8.11 8.23 8.89 6.63 5.56
#2 9.73 10.30 11.44 15.81 9.52

#3 9.81 9.74 10.92 9.07 5.30

#4 7.60 7.80 7.78 8.98 5.99

#5 43.55 52.30 47.66 3.23 9.00

Controls #1 41.51 41.99 45.78 28.78 13.55
#2 26.17 26.89 29.30 7.85 2.99

#3 26.00 27.26 28.67 14.85 7.33

#4 77.47 86.56 93.18 25.05 4.44

 

Table 10. Blastogenesis stimulation indices of lymphocytes incubated

with TF thirty minutes, washed three times, and then cul-
tured with Leukoagglutinin (0.5 ug/culture)

 

fi—

 

With TF Without TF
MS patient #1 101.72 102.13
#2 111.55 103.28
#3 127.81 121.14
Controls #1 118.82 101.11
#2 107.31 113.36
#3 100.98 131.88

 

Table 11. Blastogenesis stimulation indices of lymphocytes incubated

with TF twenty-four hours, washed three times, and then
cultured with Leukoagglutinin

 

Leukoagglutinin (Hg/culture)

 

 

 

 

 

 

2.0 1.0 0.5 0.1 0.05
MS patient #1
with TF 393.48 432.02 440.77 175.83 96.85
without TF 118.21 115.05 111.19 4.67 2.33
MS patient #2
with TF 291.52 326.19 333.12 131.15 74.44
without TF 96.94 103.28 98.43 28.06 3.52
Control #1
with TB 329.38 312.35 266.98 68.39 14.91
without TF 160.37 146.74 121.19 6.75 1.08
Control #2
with TF 247.84 257.12 247.49 72.99 24.08
without TF 89.13 156.34 167.28 37.82

72.20

 

DISCUSSION

Studies of immune competence in patients with multiple sclerosis
are numerous and many give conflicting results (25). Dysfunctions in
humoral as well as cell mediated immunity have been noted.

Sixty to eighty percent of MS patients will have an abnormal
oligoclonal IgG in their CSF while serum immunoglobulin levels remain
within normal ranges. Reduced mixed lymphocyte reactions, blasto-
genic responses to phytolectins and impaired delayed hypersensitivity
reactions to microbial and fungal skin test antigens have been
reported. Other studies, however, have failed to duplicate these
results (25).

Virus-specific immune responses in MS patients have also been
variable. Sever and Kurtzke (46) reported delayed hypersensitivity
in skin reactions to measles and mumps antigen preparations in MS
patients. A reduced blastogenic response of these patients to
measles viral antigens was observed by Dan and Peterson (11); however,
Knowles and Sanders (26) found no significant differences. Utermohlen
and Zabriskie and Ciongoli et al. (25) demonstrated a diminished
capacity of MS peripheral blood lymphocytes (PBL) to synthesize
migration inhibition factor (MIF) when stimulated in vitro with
measles antigen. In general, there appears to be a tendency toward
a reduced activity of CMI responses against measles and paramyxoviruses,

as compared with other antigens.

33

34

Because of the demyelinating nature of MS the response to
neural antigens has also been studied. Lisak et a1. (25) observed
no delayed hypersensitivity to CNS antigens in MS patients and also
were unable to show the production of MIF with MS cells exposed to
myelin basic protein. Other workers, however, have reported other-
wise (25). Both positive and negative results for lymphocyte
transformation in response to basic protein have been reported (25).

It has been proposed that altered cellular immunity in MS
patients could result from changes induced by viruses or by responses
to viral antigens cross-reacting with neural antigens.

This study confirms many of the observations already reported.

Of the histocompatibility antigens generally associated with MS,
HLA-A3 and HLA-B7, only one, HLA-B7, was Significantly increased.
It was found in twice as many of the MS patients as would normally
be expected. There was only a very slight increase in the HLA-A3
antigen. (The HLA-Dwz antigen was not included in this testing.)
Two antigens, HLA-A2 and HLA-B12, were expressed less often than in
the normal caucasian population. (All the patients tested were
caucasian.) (Table l)

The lymphocyte populations (T- and B-cells) in the peripheral
blood of the MS patients occurred in proportions similar to those
of normal controls tested at the same times. Other workers have
reported a decrease in T—cells with a relative increase in B-cells
(25). However, the MS patients used in this study were already using
TF for therapy. It is possible that the TF brought their T-cells
back up to normal levels but pre and post TF studies need to be done

to prove this (Table 6).

35

It has also been reported that blastogenic response is reduced
in MS patients and this study tends to confirm this report (Table
9). Four out of five of the patients studied had stimulation
indices significantly lower than normal controls when their lympho-
cytes were cultured with varying amounts of Leukoagglutinin.

The major objective of this study was to evaluate the influence
of TFWB on lymphocytic activities and several observations can be
made.

Antibody production, either active or passive, is not stimulated

by TF . No irregular antibodies were found in twenty MS patients

WB
up to six months following the onset of TF therapy (Table 5). Neither
were there any changes in the serum immunoglobulin levels in a group
of patients studied before and after TF therapy. This suggests that
TF does not stimulate the humoral immune system, a fact which has

WB
also been demonstrated with TFD (29).

Isoantibodies (anti-A and anti-B) and alloantibodies (anti-Rho[D])
known to exist in donor blood were not found in TFWB (Table 4).
Because antibodies are immunoglobulins they are molecules too large
to pass the dialysis membrane.

One of the more interesting results of this study was the
influence of TFWB on the active-rosette population in MS and control
volunteers (Table 7). Transfer factor derived from whole blood
stimulated both patient and normal control active T-lymphocytes.
This suggests a non-specific response of the cells to the TF. The
active rosettes are generally considered to be T-cells which are

actively engaged in the immune response at the time of testing. Yu

(21) concluded that the active cells are a subpopulation with

36

different numbers of membrane sulfhydryl and sialic acid groups
which lead to a different surface distribution of SRBC receptors.
Holtzman and Lawrence (21) have demonstrated that TFD will increase
the number of active rosettes in vitro in the normal population
following a three hour incubation. They concluded that this was a
non-specific response. This study shows that TFWB also will increase
the number of active rosettes following only a thirty minute
incubation.

It should be noted here that inconsistencies arose in the
active rosette testing procedure when RBC's from different sheep were
used from day to day. Results became reproducible when the red cells
from the same sheep (#20 from Microbiological Associates) were used
throughout the experiment.

Blastogenic procedures were tried using Leukoagglutinin as the

mitogen and various combinations of TF The results were variable.

WB'
The TFWB was not mitogenic, nor was it cytotoxic. The percentage of
viable control cells and TF treated cells was 90% at 24 hours, 85%

at 48 hours, and 75% at 72 hours incubation at 37°C.

Lymphocytes from both MS patients and normal controls were incu-
bated with TFWB for thirty minutes, washed three times, and then
cultured with Leukoagglutinin (0.5 ug/culture). A sub-optimal dose
of the mitogen was used so that elevation in the stimulation index
would be possible. In all cases there was essentially no change in
lymphocyte stimulation by incubating for a short time with TFWE
(Table 10).

However, when lymphocytes from patients and controls were incu-

bated with TFWB for 24 hours before culture, there was a marked change

37
in the blastogenic response. The two MS patients had a 3- to 4—fold
increase in stimulation and the normal controls had a 2- to 3-fold
increase.
As with the active rosettes, there appears to be a non-specific

response by the cells to the TF since both the patient and normal

WB

control cells responded to TFWB incubation. This only points out the
need for further characterization of the active molecules of transfer
factor and their separation into adjuvant and antigen-specific
activities.

The chemical studies show that transfer factor derived from
whole blood appears to have properties similar to those reported for
TF derived from leukocytes. It is a non-immunogenic substance that
contains no protein (Table 2). The sodium and chloride levels are
high due to the addition of normal saline in the preparation of the
product. The other molecules present in significant amounts are ions
(calcium, potassium, and phosphorus) which have molecular weights
less than 10,000.

Spectrophotometrically, TF exhibited a significant absorption

WB
peak at 254 nm. This observation suggests that nucleotides are
present in TFWB preparation since absorbance in the range of 260 nm
is usually due to nucleotides while absorbance at or near 280 nm is
usually due to protein. Little, if any, protein is contained in
TFWB which was determined by the SMAC analysis. All the TFWB prepa-
rations tested by this method had a peak absorbance near 260 nm
which gave a 260/280 ratio greater than 1.0 (Table 3). Groust et
al. (18) reported 260/280 ratio values from 1.03 to 1.35 on TF

derived from leukocytes (TFD). The mean value in this study using

38
TFWB was 6.7 with a range of 1.2 to 12.9. It therefore is possible

that there are more nucleotides in TFWB than in TFD as a direct
result of either the erythrocytes or something in the plasma. It is
also possible that something other than nucleotides is causing the
absorbance at that wavelength. Further studies using different
separation techniques would be helpful in the determination. This
also might indicate where the active moiety of TF lies - whatever
that may be.

Another test for the existence of nucleotides in TFWB is the
Limulus Amebocyte Lysate (LAL) test for endotoxin of gram-negative
bacteria. Elin and Wolff (14) and Levin et a1. (33) have demonstrated
that some compounds such as polynucleotides, heavy metals, blood
products, or high osmolality may give false positive or false nega-
tive results and that the pH range of the samples must be 6.8 to
7.5 for maximum efficiency of the reaction. It is highly unlikely
due to aseptic techniques and cold temperatures throughout all pro-
cedures that endotoxin is present in TF. Moreover, each preparation
exhibited no bacterial growth on culture. However, as noted in Table
8, thirteen out of twenty samples of TFWB tested demonstrated a
positive reaction to LAL. The pH and osmolality of each sample is
within range for appropriate reaction and all controls gave expected
results. If no endotoxin is present, something else is responsible
for the non-specific positive reaction. Again, separation techniques
or even dilution studies will give more answers to this question.

It is difficult to fully assess TFWB produced for multiple -

sclerosis because a specific antigen for the disease has not yet been

found. Donor selection is purely hypothetical and patient response

39
is inherently variable. However, more specific testing needs to
be done. Possibilities may include blastogenesis using other mitogens
or more extensive macrophage inhibition studies, etc. There is so

much more yet to be learned.

SUMMARY AND CONCLUSIONS

Transfer factor derived from whole blood appears to have some
preperties similar to that of TF derived from leukocytes. It is a
non-protein, non-antigenic substance which will stimulate lymphocytes
in vitro. Its specificity for immunoprophylaxis for multiple sclerosis
cannot be determined until a specific antigen related to the disease
is identified and purified. Its clinical effects on MS patients is
very difficult to evaluate due to the highly variable nature of the
disease. Long-term exacerbation-free intervals in several patients
at risk for relapses would provide positive information on its
clinical usefulness.

Clearly, further studies are necessary to purify and characterize
the active moieties of TF, but these studies must wait until more
specific information is known about the etiologic agent in multiple

sclerosis.

4O

APPENDIX

Histocompatibility Testing

 

Preparation of antisera trays - All histocompatibility typing
sera (supplied by NIH) were dispensed into polystyrene micro-
histocompatibility typing trays. Liquid petroleum (0.004 ml) was
added to each well with a 250 lambda Terisaki dispenser. Typing
serum (0.001 ml) was added to each wall under the oil with a 50
lambda Terisaki dispenser. Known positive and negative sera were
added as controls and the trays were stored at -70°C until needed.

Test procedure - Lymphocytes were prepared as on page and
adjusted to a suspension of 2 x 106 cells/ml in HBSS. The mixed
lymphocytes (0.001 ml) were added to each of the tray wells with a
50 lambda Terisaki dispenser making sure the antisera and cells
mixed thoroughly. The tray was then incubated at room temperature
(22-26°C) for thirty minutes. Rabbit complement (0.005 ml) was
added to each well and the tray was incubated at room temperature
for sixty minutes. Five percent eosin solution (0.003 ml) followed
by 0.005 ml buffered formaldehyde were added to each well. A 50 x
75 mm microscope slide was lowered onto the wells in order to
flatten the tops of the droplets. The wells were then observed with
an inverted phase contrast microscope for cell viability. Living

lymphocytes were small and refractile while dead reactive lymphocytes

41

42

were larger and stained red. A positive reaction was noted when a

majority of the cells were killed.

Dextran-Coated Carbonyl Iron (DDCI)

Dextran-T-70 (70,000 M.W.) - Pharmacia, Piscataway, NJ
Carbonyl iron (3 u particles) - GAF Corp., NY

One gram carbonyl iron is mixed with 12 ml of 5% dextran in
0.85% NaCl and placed in a 37°C water bath for 10 minutes.
Mix occasionally. Store at 4°C.

Ficoll—Hypaque Gradient (F-H)

Ficoll (400,000 M.W.) - Pharmacia, Piscataway, NJ (9% solution)
Hypaque (sodium diatrizoate) - Winthrop Laboratories, NY
(33% solution)

For the gradient combine 24 parts Ficoll to 10 parts Hypaque.
The specific gravity should be 1.08.

The following may be obtained from GIBCO - Grand Island Biological

Corporation, Grand Island, NY

RPMI 1640 with 25 mM Hepes Buffer (with glutamine -
without antibiotics)

HBSS - Hanks' Balanced Salt Solution (1X)

Hanks' BSS (10X) - Hanks' Incomplete
without calcium, magnesium, sodium bicarbonate

Medium 199

Vibrio cholera neuraminidase (VCN)

1.0 m1 VCN is mixed with 1.0 ml HBSS and then the pH is
adjusted to 7.0-7.4 with 0.1 N NaOH. VCN can be stored at
4°C.

Leukoagglutinin - Pharmacia, Piscataway, NJ

Fetal Calf Serum (FCS) - Reheis #50-204; absorbed on ice with sheep

and human erythrocytes

43
Tritiated Thymidine (3H-Tdr) - New England Nuclear

6.7 Ci/mM, #NET-027 Thymidine: 1.0 mCi, 0.036 mg stock
solution
0.1 m1 H-Tdr + 9.9 m1 RPMI 1640

Scintillation Cocktail - Scintiverse - Fisher Scientific (#SO--X-1)
MASH - Multiple Automated Sample Harvester

Liquid Petroleum - Paraffin oil (mineral oil) - White, Light
Fisher Scientific Co., Silver Spring, MD

Terisaki Dispenser — Hamilton Co., Inc., Whittier, CA

BI BLI OGRAPHY

10.

11.

12.

BI BLI OGRAPHY

Acheson, E. D.: Epidemiology of Multiple Sclerosis. British
Medical Bulletin, 33:9-14, January 1977.

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VITA

VITA

The author was born in Honolulu, Hawaii, on February 23, 1950.
She moved with her family to Pennsylvania in 1954, Louisiana in 1963,
and Ohio in 1966. She graduated from Indian Hill High School in
Cincinnati in 1968 and later that year entered Michigan State Uni-
versity. In 1972 she graduated with a B.S. Degree in Medical
Technology. Following a twelve-month clinical internship at E. W.
Sparrow Hospital in Lansing, Michigan, she became certified with the
Registry of Medical Technologists of the American Society of Clinical
Pathologists.

She has continued at Sparrow Hospital, where she is currently
working full time in the blood bank while completing an M.S. Degree
in the Clinical Laboratory Science program, Department of Pathology,
Michigan State University.

She is currently a member of the Michigan and American Societies
for Medical Technologists and the Michigan and American Associations

of Blood Banks.

48

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