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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2581-2587
Synergism Between Mycophenolate Mofetil and Cyclosporine
in Preventing Graft-Versus-Host Disease Among Lethally Irradiated
Dogs Given DLA-Nonidentical Unrelated Marrow Grafts
By
Cong Yu,
Kristy Seidel,
Richard A. Nash,
H. Joachim Deeg,
Brenda
M. Sandmaier,
Alexander Barsoukov,
Erlinda Santos, and
Rainer Storb
From the Clinical and Public Health Sciences Divisions of the Fred
Hutchinson Cancer Research Center, Seattle, WA; and the University of
Washington, Seattle, WA.
 |
ABSTRACT |
Mycophenolate mofetil (MMF) was evaluated either alone or combined
with cyclosporine (CSP) for preventing graft-versus-host disease (GVHD)
in dogs given 9.2 Gy total body irradiation and DLA-nonidentical
unrelated marrow grafts. Marrow autograft studies showed gut toxicity
as limiting MMF side effects. Four groups were studied for GVHD
prevention: six dogs in group 1 received MMF 10 mg/kg twice
daily subcutaneously (SC) on days 0 to 27. They died
between 8 to 28 days from infection or GVHD; survival was better than
that of 72 controls given no immunosuppression (P = .04), but
not different from 19 dogs given CSP. Four dogs in group 2 received MMF
as described, along with CSP at 10 to 15 mg/kg twice daily on days 0 to
27. They died at 6 to 98 days from CSP-associated toxicity, weight
loss, or infection. Nine dogs in group 3 received MMF SC twice daily 6 mg/kg/d for 3 days, followed by 10 mg/kg twice daily until day 27, along with CSP as described; four died between 7 to 106 days with
intussusception, infection, or GVHD, and five became long-term
survivors. Six dogs in group 4 received shortened MMF (21 days) and
reduced doses of CSP given through day 100. Three died with GVHD or
infection between days 38 to 119, and three became long-term survivors. Results support the notion of synergism between MMF and CSP, as evidenced by stable graft-host tolerance in greater than 50% of dogs.
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INTRODUCTION |
THE PURPOSE of immunosuppressive drugs
administered after allogeneic marrow grafts is to prevent
graft-versus-host disease (GVHD) and to facilitate the induction of
graft-host tolerance that will persist even after immunosuppression has
been discontinued. We have evaluated immunosuppressive drugs for GVHD
prevention in a model of marrow allografting from dog leukocyte antigen
(DLA)-nonidentical unrelated donor dogs. Without immunosuppression,
hyperacute and fatal GVHD occurred uniformly. Significant delay of GVHD
and prolongation of survival were seen when methotrexate (MTX),
cyclosporine (CSP), FK506, azathioprine, deoxyspergualin,
6-mercaptopurine, or succinylacetone were used as single agents, while
other drugs were ineffective.1-3 GVHD prevention was most
impressive when MTX was combined with either CSP2 or
FK506,3 but other drug combinations were not
effective.1 The advantages of MTX/CSP and MTX/FK506 were
confirmed clinically first in Phase I/II studies and then in randomized
trials.4-6 Nevertheless, while the drug combinations worked
well in transplants from HLA-identical sibling donors, results were
less impressive with marrow grafts from alternative donors, either
phenotypically HLA-identical unrelated individuals or
HLA-haploidentical relatives. Therefore, the search for alternative
immunosuppressive drugs, which are more effective in GVH prevention,
has continued.
Mycophenolic acid (MPA) is an antimetabolite with a broad range of
activities that include antitumor, antifungal, antibacterial, antiviral activities, and immunosuppression.7-9 MPA
selectively inhibits inosine 5 -monophosphate dehydrogenase
(IMPDH), the key enzyme that controls the de novo synthesis
of guanosine monophosphate (GMP).9,10 Lymphocyte activation
and proliferation depends on the supply of guanine nucleotides (GTP)
for protein and nucleic acid synthesis through the de novo pathway
while other cells have a salvage biosynthesis pathway.10,11
MPA blocks T- and B-cell proliferation in response to antigens and
mitogens, thereby suppressing cellular and humoral immune
responses.12,13 In addition, MPA inhibits lymphocyte
binding and downregulates expression of adhesion molecules, and that
way reduces lymphocyte migration and interaction with
macrophages.12,13 Mycophenolate mofetil (MMF), a
semisynthetic derivative of MPA, has been studied in solid organ
transplants and has been shown to prolong allografts of skin, lung,
small intestine, heart, liver, and kidney in animals and in
humans.14
The present study evaluated the usefulness of MMF for GVHD prevention
in dogs given marrow grafts from DLA-nonidentical unrelated donors. MMF
alone delayed the onset of acute GVHD and resulted in a modest
prolongation of survival. When MMF was combined with CSP, approximately
half of the dogs studied became long-term survivors.
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MATERIALS AND METHODS |
Dogs and DLA typing.
Litters of beagles, harriers, Walker hounds, and crossbred dogs were
used in this study. Dogs were either bred at the Fred Hutchinson Cancer
Research Center or purchased from Department of Agriculture licensed
vendors located in the states of Washington and Michigan. Dogs were
immunized against leptospirosis, distemper, hepatitis, and parvovirus,
dewormed, and observed for disease for at least 2 months before being
entered on study. Dogs weighed from 5.8 to 18.6 (median, 10) kg and
were 7 to 36 (median, 10) months old. The experimental protocols and
the facilities used were approved by the Fred Hutchinson Cancer
Research Center's (FHCRC) Internal Animal Care and Use Committee per
guidelines stipulated in the Experimental Animal Welfare Act of 1985 administrated through the National Institutes of Health.
Donors and recipients were unrelated and were obtained from different
breeding colonies or were of different pedigrees for at least five
generations. They were determined to be DLA-nonidentical on the basis
of mismatching for DLA-DR BB1 antigens identified by direct gene
sequencing.15,16
MMF.
MMF was provided by Roche Bioscience (formerly Syntex Discovery
Research, Palo Alto, CA) as lyophilized powder (intravenous [IV] formula without TWEEN 80). The drug was prepared as
a fresh suspension in 5% Dextoses at a concentration of 25 mg/mL and
stored at room temperature until use.
Total body irradiation (TBI), marrow transplantation, and supportive
care.
For both autologous and allogenic grafts, marrow cells were aspirated
under general anesthesia through needles inserted into humeri and
femora and stored in heparinized tissue culture medium at 4°C for
no more than 6 hours.17 Recipients were administered TBI at
a single dose of 920 cGy delivered at 7 cGy/minute from two opposing
cobalt-60 sources.17 Within 4 hours of TBI, harvested marrow cells were infused IV into recipients at doses of 1.3 to 4.1 (mean, 3.2) × 108 nucleated cells/kg. The day of
marrow grafting was designated as day 0. All allogeneic recipients were
given, in addition, IV infusions of peripheral blood buffy coat cells
obtained by leukapheresis from the marrow donor on days 1 and 2, at
doses of 6.3 to 19.6 (mean, 12.7) × 108 nucleated
cells/kg to assure consistent hematopoietic engraftment.18
Supportive care included oral and systemic antibiotics, subcutaneous
fluids with electrolytes, and platelet transfusions.17 Complete blood counts were obtained daily before and for the first 3 weeks after transplant and twice weekly thereafter. Serum chemistry levels were obtained once weekly to monitor kidney and liver functions. Dogs were checked and weighed daily, and the percentage of weight loss
calculated as the weight at day 0 (W0) minus the least
weight after transplant (Wl) divided by the weight at day 0 (W0): [W0  Wl] /
W0 × 100%.
Hematopoietic engraftment was assessed by sustained increases in
granulocyte and platelet counts after the postirradiation nadir, by
documentation of donor originated cells with microsatellite marker
studies in specimens from peripheral blood and marrow,19 by
histologic features of the marrow from biopsy or autopsy specimens, and
by clinical and histopathologic findings of GVHD in allogeneic recipients.
Clinical signs of GVHD included severe diarrhea due to gut involvement
(G), conjunctival or skin erythema (S), and elevations of liver enzymes
and bilirubin (L). Acute GVHD was defined as disease manifested before
day 100, and chronic GVHD was defined as disease present after day 100. Dogs either died of or were euthanized because of complications of the
study, or they were euthanized after completion of the study. The study
was considered complete once dogs lived more than 200 days after
transplant with or without clinical evidence of GVHD.
The immune function in long-term survivors was assessed by established
techniques. These included in vitro testing of lymphocyte responsiveness to alloantigens in a standard mixed lymphocyte culture
and to concanavalin A,20 natural killer (NK) cell
function,21 cytotoxic T-lymphocyte function, and flow
cytometry for lymphocyte phenotyping.22 Also, antibody
responses to sheep red blood cells (SRBC) in vivo were determined. For
that purpose, dogs were given an IV injection of 1 mL of a 10%
suspension of SRBC in 0.15 mol/L NaCl. Antibody titers were determined
as described.20
Statistical methods.
Survival times and times to granulocyte or platelet recoveries were
compared between treatment groups using log-rank statistics. Granulocyte and platelet recoveries were defined as the first days
after the postirradiation decline when granulocyte counts rose above
500/µL and platelet counts above 20,000/µL for two consecutive measurements. Pearson's  2 test was used to
compare proportions experiencing graft failure in different groups.
Historical and concurrent controls.
Results in current recipients were compared with those in previous and
concurrent DLA-nonidentical recipients receiving either no
immunosuppression (n = 72), a long course of CSP for 100 days (n = 19),
or a short course of MTX along with 45 days of CSP (n = 6)
(unpublished), and 100 days of CSP (n = 10).2
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RESULTS |
Dose finding and toxicity studies in dogs with marrow autografts.
Five dogs given 9.2 Gy TBI and autologous marrow grafts were scheduled
to receive MMF from day 0 to day 27 after transplant delivered either
as a continuing IV infusion or SC. All three dogs given 40 mg MMF/kg/d
developed severe diarrhea along with dehydration and loss of 20% to
29% of body weight. They had poor hematopoietic engraftment and were
euthanized on days 11, 12, and 13 because of poor general condition.
Marrow cellularity was <5%, <5%, and 20% of normal,
respectively. The fourth dog was given MMF 20 mg/kg/d as a continuous
IV infusion, and the fifth dog was given MMF 10 mg/kg SC three times a
day on days 0 to 8 and twice a day on days 9 to 27. Both dogs developed
transient diarrhea and 14% to 19% of body weight loss, and both had
prompt and complete hematopoietic recovery. No significant toxicities in central nervous system, kidney, and liver were observed in the five
autologous transplant recipients.
DLA-nonidentical, unrelated recipients.
Given the findings in dogs with autografts, a dose of MMF of 10 mg/kg
twice daily (20 mg/kg/d) SC was thought to be safe for the GVHD
prevention studies. Because of the pronounced weight loss seen with
MMF, we administered the drug for no more than 4 weeks. Owing to the
considerable cost of the drug, we elected to discontinue CSP on the
same day we stopped MMF. The CSP doses used were arrived at
empirically, and they were shown in previous studies to enhance
hematopoietic engraftment in dogs given suboptimal conditioning
programs.23,24
Results are summarized in Table 1. Six dogs
in group 1 were scheduled to receive MMF alone at a dose of 10 mg/kg SC
twice daily on days 0 to 27 after transplant. Two of the six dogs had persistent severe diarrhea from day 0. One of two was euthanized on day
8 because of poor clinical condition, while the other died on day 12 of
pneumonia and intestinal bleeding. Both dogs had a severely hypoplastic
marrow at autopsy and no evidence of GVHD. The remaining four dogs had
successful allografts as indicated by rapidly rising granulocyte and
platelet counts and by microsatellite marker studies. They developed
clinical and histologic evidence of severe GVHD and died between days
18 and 28. All six dogs had reduced food intake and lost between 17%
and 36% of their body weight. Serum chemistries showed normal levels
of creatinine, urea nitrogen, bilirubin, and slightly increased levels
of aspartate aminotransferase and/or alanine aminotransferase (data not
shown).
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Table 1.
Dogs Administered 9.2 Gy TBI, Hematopoietic Grafts From
DLA-Nonidentical, Unrelated Donors, Followed by MMF With or Without CSP Treatment
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Four dogs in group 2 were given MMF at 10 mg/kg twice daily SC from
days 0 to 27 and CSP IV at 10 mg/kg twice daily from days 0 to 9, followed by CSP by mouth at 15 mg/kg twice daily from days
10 to 27. Two dogs were euthanized because of MMF and CSP-associated toxicities at days 6 and 8, which included intussusception and massive
diarrhea. The two remaining dogs had prompt engraftment. One of the two
had clinical signs of gut GVHD and was euthanized at day 28 due to
severe weight loss (over 40%), and the other died of myocarditis of
unknown etiology at day 98 without clinical signs of GVHD.
Because of the observed drug toxicities in dogs of group 2, the dose of
MMF in dogs of group 3 was reduced from 10 to 6 mg/kg twice daily SC
for the first 3 days followed by 10 mg/kg twice daily SC until day 27. The CSP dose was that described for dogs in group 2. All 9 dogs had
prompt engraftment. Four of the 9 died: 1 died of pneumonia and
meningitis on day 72, 1 was euthanized on day 7 because of septicemia
secondary to intussusception (a CSP-associated toxicity in dogs), 1 because of acute GVHD with 38% weight loss on day 48, and 1 because of
severe chronic skin GVHD on day 106. The remaining 5 dogs became
long-term survivors without clinical manifestation of GVHD, even though
all had histopathologic evidence of GVHD on autopsy at the completion
of study (n = 5). All 9 dogs in this group had weight loss ranging from
17% to 38% while on MMF. In dog E071, MMF was discontinued
prematurely on day 23 because of weight loss. Dogs recovered their body
weight after immunosuppression was stopped.
To further reduce MMF- and CSP-related toxicities, the six dogs in
group 4 were given the same regimen of MMF as was used in dogs of group
3 except that MMF was stopped after 21 instead of 27 days, and the IV
CSP dose was reduced to 5 mg/kg twice daily for the first 6 days,
followed by CSP by mouth at 15 mg/kg twice daily until day 20. Given
that GVHD was observed between days 27 and 100 in dogs of groups 2 and
3 in which CSP was stopped on day 27, we elected to continue CSP until
day 100 using a gradual taper schedule as outlined in the footnote of
Table 1. All six dogs had prompt engraftment. Three died. One died on
day 95 of GVHD, systemic infection, and renal failure. CSP was
discontinued early (day 66) in this dog because of extensive
papillomata on all extremities, which were related to prolonged
administration of CSP. The second dog developed severe skin GVHD and
was euthanized on day 119. In this dog, CSP was also stopped
prematurely (day 79) because of papillomata. The third dog was
euthanized on day 38 because of continuous seizures caused by a
purulent meningitis. The three remaining dogs all had a complete course
of posttransplant immunosuppression and they became long-term
survivors. Skin GVHD developed in one of the long-term survivors on day
140 and resolved after 2 weeks of treatment with CSP at 10 mg/kg/d by
mouth.
Peripheral blood leukocytes.
Table 2 shows the flow cytometric analyses
of leukocytes after hematopoietic reconstitution in four allografted
dogs from group 3 tested between 80 to 120 days after transplant. The
proportions of B and T cells were in the normal range, and the CD4/CD8
ratios were inverted in all four dogs.
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Table 2.
Flow Cytometric Analysis of Peripheral Blood Leukocytes
From Normal Dogs and Dogs With Marrow Allografts
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Antibodies to SRBC.
Figure 1 illustrates the eight long-term
survivors' primary humoral antibody titers in response to SRBC.
Results were compared with those from five concurrently injected normal
dogs. All but one of the marrow graft recipients showed lower than
normal hemagglutinin titers on day 5 after injection. Subsequently, the
majority of dogs reached antibody titers in the normal range, while at
least three of the animals continued to show subnormal antibodies
during the 25-day period of serum collection.
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| Fig 1.
Hemagglutinin titers after a single injection of SRBC in
five normal control dogs and eight long-term survivors after marrow transplantation. Shown for controls are mean titers ± 1 standard deviation (SD) (shaded area). Transplant recipients were administered SRBC between days 80 and 120 after transplant.
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NK and T-cell function.
Data on NK function in four of the long-term surviving dogs and four
normal dogs are summarized in Table 3. NK function was within the normal range in all but one dog (E072) in which low NK
function was seen at effector:target cell ratios of 80:1 and 40:1,
respectively. Cytotoxic T-cell function as determined by cell-mediated
lympholysis assays was within the normal range in the same four dogs as
were the lymphocyte responses to alloantigens in mixed leukocyte
culture and to concanavalin A (data not shown).
Granulocyte and platelet recoveries in MMF/CSP-treated dogs.
Figure 2 compares granulocyte and platelet recoveries
in dogs given MMF/CSP (n = 19) to those in 16 dogs treated with another antimetabolite, MTX, combined with CSP. No significant differences between the two groups of dogs were found, within regard to the tempo
of platelet (P = .27) or granulocyte recoveries (P = .21).
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| Fig 2.
Granulocyte and platelet recovery in dogs administered
hematopoietic grafts from DLA-nonidentical, unrelated donors followed by either MMF/CSP (n = 19; solid lines) or MTX/CSP (n
= 16; interrupted lines).
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Comparison of current results to those in concurrent and historical
controls given either no immunosuppression, CSP alone, or MTX/CSP
combined.
Table 4 and Fig 3 summarize the
results. Dogs in group 1 given MMF alone survived significantly longer
than dogs not given immunosuppression (P = .04).1,25 Dogs in group 2 were not used for statistical
comparisons given that the study was prematurely stopped after four
dogs were entered owing to the drug toxicities encountered.
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Table 4.
Comparison of Survivals in Dogs Administered 9.2 Gy TBI
and Hematopoietic Grafts From DLA-Nonidentical Unrelated Donors
With or Without Posttransplant Immunosuppression
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| Fig 3.
Survival of dogs administered 9.2 Gy TBI followed by
hematopoietic grafts from DLA-nonidentical donors. Dogs were
administered either no immunosuppression, MMF alone, CSP alone,
MTX+CSP, or MMF+CSP. For details on the immunosuppressive regimens,
see Tables 1 and 4.
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Dogs in groups 3 and 4 given the combination of MMF/CSP survived
significantly better than dogs not given immunosuppression (P < .001). Also, there was a suggestion that their survival was better
than that of dogs given MTX/CSP (P = .078). Of note, the addition of MMF to CSP reduced the risk of graft failure in dogs given
CSP alone, a finding that was comparable to that previously made with
CSP after addition of MTX.2
| |
DISCUSSION |
The model used to test the efficacy of immunosuppressive agents in
controlling GVHD involves dogs given hematopoietic grafts from
DLA-nonidentical unrelated donors. By combining marrow and peripheral
blood buffy cells for the transplant, engraftment, and subsequent
development of hyperacute and fatal GVHD are the rule,18
thus making this a stringent model for studies of GVHD prevention.1
Many single immunosuppressive agents have been tested in the model, and
seven drugs MTX, CSP, FK506, azathioprine, deoxyspergualin, succinylacetone, and corticotropin releasing factorhave led to significant prolongations of recipient survival.1-3,26
However, long-term survival after discontinuation of immunosuppression was not seen with any of the drugs except for MTX (<10% of dogs). As
seen with most other single agents, dogs treated with MMF alone lived
significantly longer than controls, but there were no long-term survivors. Previously, best survivals were seen when MTX was combined with either CSP or FK506.25 We interpreted this to
represent synergisms between these agents in regard to controlling
GVHD, an assumption that was also supported by subsequent clinical
trials.4,5 We failed to observe synergism when combining
another antimetabolite, azathioprine, with
CSP.27
The current results with a combination of MMF and CSP are encouraging.
Eight of 15 dogs studied in groups 3 and 4 that received MMF for 21 to
27 days on a reduced schedule and CSP either for 27 days or for 100 days became long-term survivors, a result that may have been better
than that seen with MTX/CSP. Even though all immunosuppression was
discontinued, most dogs survived either with no clinical evidence of
chronic GVHD or only mild skin disease. However, when dogs were
euthanized at the completion of the studies, autopsy findings showed
subtle histologic signs of GVHD in various organs. It is unknown
whether chronic GVHD would have become clinically manifested had the
animals been permitted to live. While no data on synergism between MMF
and CSP have been reported for the marrow transplant setting, there is
at least one publication showing synergism between the two drugs in a
rat hind limb transplant model.28
Two of six dogs given MMF alone experienced graft failure, a figure
which was not statistically different from the 53% seen previously in
dogs given CSP alone. Apparently, both MMF and CSP when administered
alone block the graft enhancing effect of the donor T cells, while not
abrogating the host-versus-graft reaction which, in this model, is
thought to be effected by large granular lymphocytes with NK
function.29,30 Of interest, when the two drugs were
combined (groups 2 to 4), no graft failure was seen among 19 dogs
studied.
MMF given for 21 to 27 days in the doses applied to dogs in groups 3 and 4 along with CSP did not result in a delay of hematopoietic recovery after transplant over that seen in dogs given a short course
of MTX (days 1, 3, 6, 11) along with CSP. Thus, no limiting hematopoietic toxicity of MMF in this setting was seen. The major limiting toxicity of MMF in this model has been gastrointestinal, although it is not clear how much of this toxicity was contributed by
CSP. All animals developed some degree of diarrhea after transplant, and three allografted dogs died with intussusception or severe gut
hemorrhage. All dogs experienced significant weight loss which, however, corrected itself after discontinuation of the agents. Another
problem related to the profound postgrafting immunosuppression has been
papilloma virus infection, usually manifested as extensive warts on the
dog's paws. This finding has resulted in a papilloma virus vaccination
program at FHCRC.
In previous studies, we administered immunosuppressive therapy for
approximately 3 months after transplant. This was based on observations
in MTX-treated dogs, which showed superior immunosuppression when the
drug was administered for an extended period of time. In the current
study, we reduced the immunosuppressive therapy to approximately 4 to 5 weeks after transplant in an effort of cost containment, except for
dogs in group 4. Comparing the results in dogs of group 3 given MMF and
CSP for 28 days with those in group 4 given MMF and CSP for 22 and 101 days, respectively, there was no statistically significant difference
in survival. It appears that effective GVHD prevention in this model
can be accomplished with MMF and CSP administered for only 4 weeks.
Nevertheless, a number of dogs exhibited signs of mild chronic skin
GVHD, as well as histologic evidence of chronic GVHD in skin, gut, and liver on autopsy. Presumably, these findings are related to the severe
degree of histoincompatibility between current unrelated pairs. Despite
the persisting subclinical chronic GVHD, immune functions in long-term
survivors were either in the near normal or normal range. As in
previous studies in dogs and humans, the T4/T8 ratio was inverted.
In conclusion, MMF was effective in delaying the onset of or completely
preventing acute GVHD and prolonging survival in dogs given
DLA-nonidentical unrelated hematopoietic grafts. When combined with
CSP, slightly more than half of the dogs so treated became long-term
survivors, even though both MMF and CSP were discontinued as early as
day 27. The limiting toxicities of this dose regimen in dogs were
gastrointestinal. The significant synergism between MMF and CSP
observed in the current study and confirmed in an independent study on
hematopoietic engraftment23 warrants exploration of this
drug combination in clinical marrow transplantation.
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FOOTNOTES |
Submitted September 2, 1997;
accepted November 7, 1997.
Supported in part by Grants No. CA15704, CA31787, HL36444, and DK42716
from the National Institutes of Health, Department of Health and Human
Services (DHHS), Bethesda, MD. The laboratory was also
supported through a prize from the Josef Steiner Krebsstiftung, Bern,
Switzerland, awarded to R.S.
Presented in part at the 37th Annual Meeting of the American Society of
Hematology, Seattle, WA, December 1-5, 1995.
Address reprint requests to Cong Yu, MD, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave N, D1-100, PO Box 19024, Seattle, WA
98109-1024.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Dr George Sale for providing pathology
reports, Jim Works and Dr John Wagner for their expertise in DLA
typing. We would also like to thank Lori Ausburn, Doug Jones, Eric
Bell, Alix Smith, Bonnie Larson, Harriet Childs, Dr Barbara Johnston,
and the technicians of the hematology/pathology and bacteriology
laboratories for their excellent assistance. We also would like to
thank Dr Tom Matthews at Roche Bioscience for providing MMF.
| |
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Abstract
Introduction
Abstract