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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3267-3275
Influence of Intestinal Bacterial Decontamination Using
Metronidazole and Ciprofloxacin or Ciprofloxacin Alone on the
Development of Acute Graft-Versus-Host Disease After Marrow
Transplantation in Patients With Hematologic Malignancies: Final
Results and Long-Term Follow-Up of an Open-Label Prospective
Randomized Trial
By
Dietrich W. Beelen,
Ahmet Elmaagacli,
Karl-Dieter Müller,
Herbert Hirche, and
Ulrich W. Schaefer
From the Departments of Bone Marrow Transplantation, Medical
Microbiology, and Medical Informatics, Biometry and Epidemiology,
University Hospital of Essen, Essen, Germany.
 |
ABSTRACT |
In a single-center open-label prospective study, a total of 134 marrow transplant recipients with hematologic malignancies were
randomly assigned to a bacterial decontamination medication using
metronidazole and ciprofloxacin (n = 68) or ciprofloxacin alone (n
= 66) during 5 weeks posttransplant. The development of grades II to
IV acute graft-versus-host disease (GVHD) was defined as the primary
study endpoint. According to the intention-to-treat, 17 patients (25%)
randomized to the combined decontamination medication and 33 patients
(50%) randomized to ciprofloxacin alone developed grades II to IV GVHD
(P < .002). The higher frequency of grades II to IV acute
GVHD in patients randomized to ciprofloxacin alone resulted from a more
than twofold increased number of patients developing liver or
intestinal involvement with acute GVHD compared with patients
randomized to the combined decontamination medication (P < .003). The influence of the study medication on grades II to IV acute
GVHD was significant only in recipients of transplants from
genotypically HLA-identical sibling donors (n = 80), whereas in
recipients of transplants from donors other than HLA-identical siblings
(n = 54), grades II to IV acute GVHD frequencies between the study
arms were not significantly different. The combined decontamination was
associated with a significant reduction of culture growth of intestinal
anaerobic bacteria during 5 weeks posttransplant (P < .00001). In addition, the number of cultures with growth of anaerobic
bacteria (P < .005) as well as the median concentrations of
anaerobic bacteria in the posttransplant period (P < .0001)
were higher in patients contracting grades II to IV acute GVHD. Neither
chronic GVHD nor overall survival was significantly different between
the two study arms. In patients with HLA-identical sibling donors who
were treated in early disease stages, the 5-year survival estimate was
slightly, but not significant, higher after the combined
decontamination medication (60% ± 11%) compared with ciprofloxacin
alone (46% ± 9%). In conclusion, the present study provides
evidence that antimicrobial chemotherapy targeted to intestinal
anaerobic bacteria in marrow transplant recipients significantly
reduces the severity of acute GVHD and supports the theory that the
intestinal anaerobic bacterial microflora plays a role in the
pathogenesis of acute GVHD after human marrow transplantation.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE GRAFT-VERSUS-HOST disease (GVHD)
after allogeneic bone marrow transplantation (BMT) primarily results
from an inflammatory process of recipient tissues that is induced by
alloreactive donor T-cell clones recognizing differences between the
marrow donor's and recipient's gene products encoded by the human
major histocompatibility complex (MHC) or by polymorphic tissue
antigens outside the MHC. Besides the outstanding significance of
histoincompatibility, the development of acute GVHD is influenced and
modulated by other factors, such as the type of immune prophylaxis,
patient and donor age, underlying disease, and the state of
alloimmunization of female donors donating marrow for male
recipients.1-4
Current pathophysiologic models of acute GVHD emphasize the role of
host tissue activation and damage caused by the myeloablative conditioning preceding BMT. This leads to a dysregulated release of
proinflammatory cytokines and enhanced expression of tissue antigens
that, in turn, may augment recognition of unshared recipient tissue
antigens by alloreactive donor T cells and may amplify clonal donor
T-cell expansion.5
Early studies in germ-free or completely decontaminated rodents
demonstrated that the resident intestinal bacterial microflora contributes to the pathogenesis of experimental acute GVHD, because the
absence or complete growth suppression of intestinal bacteria prevented
the development of acute GVHD even in recipient animals of
MHC-mismatched transplants.6-8 In rhesus monkeys, complete bacterial decontamination was similarly effective in preventing acute
GVHD after MHC partially matched, 1 log10 T-cell depleted transplants.9 The mechanisms by which intestinal bacteria
may modulate experimental acute GVHD are currently unclear. It has been
hypothesized that increased cytokine release of macrophages stimulated
by bacterial breakdown products, such as lipopolysaccharide (LPS) and
lipoteichoic acids, or cross-reactions of donor T cells with bacterial
antigens may promote the allorecognition process underlying acute
GVHD.10,11
Whether the intestinal bacterial microflora is involved in the
pathogenesis of acute GVHD after human marrow transplantation is
currently controversial. Single small studies in leukemia patients did
not find an association between intestinal bacterial growth suppression
and the development of acute GVHD, whereas one randomized study in
aplastic anemia patients as well as two uncontrolled trials in patients
with malignant and nonmalignant hematologic diseases suggested that
decontamination of intestinal bacteria reduces the incidence of grades
II to IV acute GVHD.12-16
The most comprehensive analysis including 194 consecutive patients from
our institution demonstrated that ineffective growth suppression of
intestinal anaerobic bacteria, which represent more than 99% of the
entire intestinal bacterial flora, was independently associated with a
1.7-fold higher risk of grades II to IV acute GVHD compared with
sustained anaerobic bacterial decontamination during 5 weeks of the
posttransplant course.16 However, only 21% of patients in
this analysis achieved a decontaminated state with regard to anaerobic
bacteria. We therefore asked whether the addition of oral
metronidazole, which has specific bactericidal activity against
anaerobic bacteria, to the broadly applied quinolone antibacterial
prophylaxis in marrow transplant patients may improve anaerobic
decontamination efficacy and, in turn, may reduce the incidence of
grades II to IV acute GVHD. The present work describes the results
obtained in an open-label prospective randomized trial that compared
the influence of oral metronidazole in combination with ciprofloxacin
to oral ciprofloxacin alone on the development of grades II to IV acute
GVHD and on intestinal anaerobic bacterial decontamination efficacy in
recipients of marrow transplants from genotypically HLA-identical
sibling donors, partially HLA-matched extended family donors, or
matched unrelated donors.
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PATIENTS AND METHODS |
Study protocol.
Adult patients (15 to 57 years of age) with hematologic malignancies
for whom a genotypically HLA-identical sibling donor, a partially
HLA-matched extended family donor, or a matched unrelated donor
according to the German consensus criteria had been identified were
eligible for this trial.17 Patients were enrolled after the
scientific background, details of the study protocol, and the potential
benefits and hazards of treatment in each of the two study arms had
been explained and written informed consent to participate in this
trial had been obtained. The study protocol had been approved by the
Ethics Committee at the Medical Faculty of the University Hospital of
Essen (Essen, Germany). Patient accrual was initiated in September 1993 and terminated in August 1995.
The study was performed as a single-center open-label prospective
randomized trial, which compared two different strategies of intestinal
bacterial decontamination. Patients randomly assigned to the combined
intestinal decontamination medication were treated with oral
metronidazole at 400 mg three times daily (tid) and oral ciprofloxacin
at 750 mg twice daily (bid; study arm A), whereas patients assigned to ciprofloxacin alone received oral ciprofloxacin 750 mg bid (study arm B). Treatment according to study arm was initiated on day  14 (day 0 designates the day of marrow
infusion) and was maintained until day 35 posttransplant, the diagnosis of acute GVHD, or death, whichever came first. Demographic and treatment characteristics of patients according to study arm are summarized in Table 1.
The development of a maximum clinical grade of severity of acute GVHD
greater than grade I was defined as the primary analytical end- point
of the study. The final evaluation of this endpoint was performed at
100 days posttransplant or at the time of death, if this occurred
earlier. The secondary analytical study aim was a quantitative
comparison of intestinal anaerobic bacterial growth suppression between
the two study arms and the evaluation of the association between
bacterial growth suppression and the development of grades II to IV
acute GVHD.
Biometric study plan.
On the basis of our previous published analysis,16 the
hypothetical difference between the two study arms with regard to the
primary study endpoint was estimated to be in the range of 20%. To
demonstrate a difference of this magnitude between the two study arms
with a one-sided  -error of .05 and a power of 80%, a total number
of 140 study patients was prospectively calculated. To allow interim
analyses after 50 and 100 patients had been enrolled in the study, a
group-sequential study plan was used. For rejection of the 0-hypothesis
in these two interim analyses, adjusted -errors of .0015 for the
first 50 patients and of .018 for the first 100 patients were
prospectively determined. These interim analyses were performed after
the 50th and the 100th study patient had been observed for more than
100 days posttransplant or until death.
To ensure an equal distribution of potential influencial factors for
acute GVHD between the study arms, a stratified randomization design
with the following three stratification criteria was chosen: partially
HLA-matched extended family donors or matched unrelated donors versus
genotypically HLA-identical sibling donors, chronic myelogenous
leukemia (CML) as underlying disease versus other hematologic
malignancies, and female donor to male recipient transplants versus
other donor and recipient gender combinations.
Statistical analysis.
Time-to-event estimates (±standard errors) were calculated by the
product-limit method with right-censoring of subjects at the last time
point at which they were at risk for a given event.18 For
the primary study endpoint, those patients who developed graft failure
or died without grades II to IV acute GVHD were censored at the
respective time points of these events. Accordingly, patients surviving
without grades II to IV acute GVHD were censored at the time of last
follow-up.
All evaluations with regard to the primary study endpoint were based on
the intent-to-treat with no exclusions due to protocol violations or
drop-outs. The log-rank test was used to test the homogeneity of
time-to-event distribution functions across strata.19 To
evaluate the influence of different explanatory variables on the times
to achieve the respective analytical endpoints, stepwise proportional
hazards general linear model analyses were performed.20,21 Differences between frequencies were compared using the two-tailed Fisher's exact test (2 × 2 frequency tables) or by the
Mantel-Haenszel  2 test (2 × n frequency tables).
Comparisons of continuous variables were analyzed using the Wilcoxon
rank-sum test. Exponential measures were compared using the Savage
test. For the comparison of timely repeated continuous measures between
strata, repeated measures analysis of variance was
applied.22 With the exception of the proportional hazards
general linear model analyses, all indicated P values are given
without adjustment for multiple testing.
Microbiologic analyses.
Fecal samples of study patients were monitored twice weekly for the
growth of aerobic and anaerobic bacteria as well as fungi using
modified microbiologic culture techniques as previously published.16,23 Quantitative bacterial cultures were
performed using conventional plate counting technique. Standardized
dilutions of stool samples were cultured on several aerobic and
anaerobic culture agars (CLED agar, Clostridium difficile selective
agar with D-cycloserine and cefoxitin, Mannitol Salt agar, Rose-Bangal Chloramphenicol agar, Yeast-extraxt Cystein blood agar with gentamicin and nalidixic acid, and Drigalski agar). Bacteria were categorized according to culture growth conditions as either aerobic or anaerobic. Single colonies were identified by conventional biochemical tests. Quantification of bacterial culture growth was expressed as the log10 of colony-forming units (CFU) with a detection
threshold below 103 CFU per gram of sample. For the purpose
of analyses on bacterial culture growth suppression, cultures with no
CFU growth were calculated as 100 CFU. In patients who
developed grades II to IV acute GVHD in the posttransplant course, only
samples taken before the diagnosis of this condition were considered in
analyses on the association of intestinal bacterial growth suppression
and acute GVHD. In case of diarrhea, fecal samples were additionaly
analyzed with regard to enterotoxines, enteropathic viruses, and parasites.
Supportive care.
All patients were protectively isolated in reverse isolation rooms
equipped with high efficiency particular air filtration systems. These
conditions were initiated on day 7 and usually maintained until
day +35 posttransplant. Aseptic techniques were used during patient
contacts throughout this time period. In addition to the study
medication, all patients received oral or parenteral fluconazole at 200 mg bid for the prevention of candida infections. Blood component
substitution, oral and parenteral nutrition, and treatment of suspected
or documented bacterial or mycotic infections followed the published
guidelines.16 Aciclovir treatment of suspected or
documented herpes simplex virus infections was performed in 54 patients
(79%) of study arm A and in 50 patients (76%) of study arm B over a
median time period of 11 days (range, 3 to 20 days) and of 12 days
(range, 5 to 35 days), respectively. Starting at 2 to 3 weeks
posttransplant, patient peripheral blood cells were weekly monitored
for replicative cytomegalovirus infections by a qualitative
pp65-antigenemia assay. Based on a positive pp65-antigenemia assay,
preemptive ganciclovir treatment was instituted in 27 patients (40%)
of study arm A and in 27 patients (41%) of study arm B for a median
treatment duration of 13 days (range, 6 to 21 days) and of 13 days
(range, 7 to 21 days), respectively.
Acute adverse effects.
Acute adverse effects and organ toxicities were daily recorded and
graded according to the WHO toxicity scale.24
GVHD.
Prophylaxis of acute GVHD consisted of short-course methotrexate (sMTX;
15 mg/m2 body surface area day +1, 10 mg/m2
days +3, +6, and +11) and cyclosporine A (CyA; 3 mg/kg of body weight
per day, continuous intravenous [IV] infusions between day 1
and day +35, oral CyA bid after day 35) in patients with partially
HLA-matched family donors or matched unrelated donors, in patients with
CML as underlying disease, and in male recipients of female donor
transplants.1 Patients fulfilling none of these criteria
exclusively received continuous CyA infusions. All but 2 patients
assigned to sMTX+CyA prophylaxis received the full prescribed
methotrexate dosages. Steady-state full blood levels of CyA and its
major metabolites were routinely monitored twice weekly using a
polyclonal antibody-based detection system (Tdx Cyclosporine and
metabolites serum assay; Abbott Laboratories, Abbott Park, IL).
Adjustments of CyA doses were allowed to keep blood levels within the
therapeutic range of 400 to 800 ng/mL. Overall median CyA and
metabolite blood levels during the first 5 weeks posttransplant were
564 ng/mL (range, 17 to 2,844 ng/mL) in 579 samples of study arm A and
530 ng/mL (range, 28 to 2,079 ng/mL) in 576 samples of study arm B (not
significant [NS]).
The assessment and grading of acute and chronic GVHD was primarily
based on clinical findings and followed the commonly accepted diagnostic criteria.25,26 In case of uncertain organ
involvement with acute GVHD of stages 2 to 4, histologic evaluations of
target organs were additionally performed. Thereby, the clinical
diagnosis of acute GVHD was histologically confirmed in 28 of 89 patients (31%) with cutaneous disease (study arm A: 13 of 42 patients
[31%]; study arm B: 15 of 47 patients [32%]), in 18 of 26 patients (69%) with liver disease (study arm A: 7 of 10 patients
[70%]; study arm B: 11 of 16 patients [69%]), and in 17 of 26 patients (65%) with gastrointestinal disease (study arm A: 4 of 8 patients [50%]; study arm B: 13 of 18 patients [72%]).
Initial treatment of acute GVHD consisted of 2 mg/kg of body weight IV
prednisolone with dose tapering according to the clinical response of
acute GVHD symptoms. In patients not responding to escalated (5 to 10 mg/kg) prednisolone doses, antithymocyte globuline was used as
second-line treatment.
| |
RESULTS |
Acute adverse effects.
In 63 patients (93%) of study arm A and in 59 patients (89%) of study
arm B, nausea and vomiting (NAV) of grades 2 to 4 was documented (NS).
In the majority of affected patients, NAV developed during the time
period of myelablative conditioning. In association with the ingestion
of the study medication, 21 episodes of NAV were noted in 8 patients
(12%) of study arm A compared with 8 episodes in 3 patients (5%) of
study arm B (NS). Oral mucositis of grades 2 to 4 developed in 56 patients (82%) of study arm A and in 61 patients (92%) of study arm B
(NS). A more than 2.5-fold increase of baseline serum creatinine levels
was observed in 9 patients (13%) of study arm A as opposed to 7 patients (11%) of study arm B (NS). Hepatic veno-occlusive disease (2 patients [3%] of study arm A, 3 patients [6%] of study arm B),
grades 2 to 4 acute cardiac toxicity (6 patients [9%] of study arm
A, 4 patients [6%] of study arm B), noninfectious interstitial
pneumonia syndrome (3 patients [4%] of study arm A, 5 patients
[8%] of study arm B), and multiorgan toxicity (4 patients [6%] of
study arm A, 3 patients [5%] of study arm B) occurred with
comparable frequencies in the two study arms. Duration of neutropenia
less than 500/µL was 18 days (range, 6 to 31 days) in study arm A and
18 days (range, 10 to 30 days) in study arm B (NS). Furthermore, no
differences were detectable between both study arms in the number of
red blood cell and platelet transfusions, in the number of days with
fever greater than 38°C and of days with IV antibiotics or
antimycotics, as well as in the frequencies of documented invasive
bacterial and mycotic infections (data not shown).
Primary study endpoint.
Table 2 summarizes information on the
number of subjects treated according to the study protocol as well as
on protocol violations and on drop-out patients in the two study arms.
At the time of the first interim analysis, which included the first 50 patients, the cumulative estimates of grades II to IV acute GVHD were
18% ± 8% for study arm A and 52% ± 11% for study arm B
(P < .011). Because the prospectively adjusted -error for
a premature completion of the study had not been reached by this
analysis, the next 50 subjects were enrolled. The consecutive interim
analysis showed cumulative estimates of grades II to IV acute GVHD of
22% ± 6% for study arm A and 54% ± 7% for study arm B
(P < .0014). Because of the decrease of the -error below
.018, this analysis led to completion of the study protocol. At the
time of the second analysis, an additional 34 subjects had entered the
study protocol, thus resulting in a total of 134 patients included in
the final evaluation (Fig 1).
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| Fig 1.
Cumulative probabilities of grades II to IV acute GVHD in
134 patients randomly assigned to intestinal bacterial decontamination
using ciprofloxacin (750 mg PO bid) and metronidazole (500 mg PO tid)
or ciprofloxacin (750 mg PO bid) alone.
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The influence of the study arm on grades II to IV acute GVHD was
significant only in recipients of transplants from HLA-identical sibling donors, with estimates of 18% ± 6% for study arm A
compared with 54% ± 8% for study arm B (P < .0005). In
contrast, recipients of transplants from partially HLA-matched family
donors or matched unrelated donors had comparable estimates of grades
II to IV acute GVHD, ranging between 36% ± 9% in study arm A and
46% ± 10% in study arm B (NS).
Organ involvement with acute GVHD.
The stages of organ involvement with acute GVHD in the two study arms
are included in Table 3. The most striking
differences were documented in the stages of liver and intestinal
involvement, but the differences between the stages of acute cutaneous
GVHD also reached significance. Consequently, the number of patients with involvement of two or three organs was more than twofold higher in
study arm B (P < .02). As for the primary study endpoint, differences between the study arms resulted primarily from the significantly lower frequencies of organ involvement with acute GVHD in
patients with HLA-identical sibling donors
(Table 4).
Intestinal bacterial decontamination and acute GVHD.
A total of 866 fecal samples were evaluable for bacterial culture
growth during the posttransplant course. The proportion of fecal
samples with no detectable anaerobic bacteria was more than twofold
higher in study arm A (236 of 446 [53%]) compared with study arm B
(96 of 420 [23%]; P < .00001). Concentrations of anaerobic
CFU in the two study arms during the posttransplant course are depicted
in Fig 2. In patients who contracted grades II to IV acute GVHD, the proportion of samples successfully
decontaminated of anaerobic bacteria was significantly lower (80 of 257 [31%]) compared with patients with grades 0 to I acute GVHD (252 of
609 [41%]; P < .005). Whereas the concentrations of
anaerobic CFU in patients who developed grades II to IV acute GVHD
reached a plateau in the range of 104 to 105
posttransplant, these concentrations declined in patients with grades 0 to I acute GVHD (Fig 3). Median overall
concentrations of anaerobic CFU in the posttransplant course were
100-fold higher in patients contracting grades II to IV acute GVHD
compared with patients with grades 0 to I acute GVHD (P < .0001).
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| Fig 2.
Median concentrations (±standard errors of the median)
of anaerobic bacterial CFU per gram of fecal sample in patients treated
with 500 mg metronidazole PO tid and 750 mg ciprofloxacin PO bid ( ;
n = 68) or with 750 mg ciprofloxacin PO bid alone ( ; n = 66) in
the week before and during 5 weeks after allogeneic marrow
transplantation. The CFU detection threshold is 103 per
gram of sample. Median concentrations of anaerobic bacterial CFU in
patients treated with metronidazole and ciprofloxacin were below the
detection threshold between 3 and 5 weeks posttransplant. Differences
between both patient groups were compared using repeated measures
analysis of variance.22
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| Fig 3.
Median concentrations (±standard errors of the median)
of anaerobic bacterial CFU per gram of fecal sample in patients with
grades 0 to I (; n = 84) or grades II to IV (; n = 50) acute
GVHD in the week before and during 5 weeks after allogeneic marrow
transplantation. Median concentrations of anaerobic bacterial CFU in
patients with grades 0 to I acute GVHD were below the detection
threshold (103 per gram of sample) in week 5 posttransplant. Differences between both patient groups were compared
using repeated measures analysis of variance.22
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Chronic GVHD.
Fifty-three patients in each study arm survived longer than 100 days
posttransplant and were thus evaluable for chronic GVHD. The cumulative
estimate of chronic GVHD was 64% ± 8% in study arm A and 70% ± 7% in study arm B (NS). Two independent predictors of chronic
GVHD were identified by multivariate analysis. The sMTX+CyA regimen
reduced the relative risk of chronic GVHD to 0.38 (95% confidence
limits, 0.19 to 0.76) compared with CyA alone (P < .006),
whereas preceding grades II to IV acute GVHD increased the relative
risk to 1.80 (95% confidence limits, 1.06 to 3.06) compared with
grades 0 to I acute GVHD (P < .03).
Transplant outcome.
With a median follow-up period of 51 months (range, 40 to 63 months) in
both study arms, 33 of 68 (49%) patients in study arm A and 28 of 66 (42%) patients in study arm B are currently alive (NS). The 5-year
survival estimates of different patient subsets are outlined in
Table 5. Multivariate analysis identified two independent predictors of survival in this study. In early disease
stages (ie, first complete remissions or first chronic phase), the
relative survival probability was 2.02-fold (95% confidence limits,
1.27 to 3.20) higher compared with more advanced stages (P < .003). Furthermore, grades II to IV acute GVHD decreased the relative
survival probability by a factor of 2.75 (95% confidence limits, 1.72 to 4.39) compared with grades 0 to I acute GVHD (P < .0001).
Causes of death in the two study arms are summarized in
Table 6.
| |
DISCUSSION |
Based on our previous retrospective observation that sustained growth
suppression of intestinal anaerobic bacteria after allogeneic sibling
donor marrow transplantation independently reduces the risk of grades
II to IV acute GVHD, an open-label prospective randomized trial was
conducted that compared the influence of intestinal bacterial
decontamination using metronidazole combined with ciprofloxacin to
ciprofloxacin alone on the development of grades II to IV acute GVHD as
the primary study endpoint.16 Because of the specific
bactericidal activity of metronidazole against anaerobic bacteria, it
appeared justified to anticipate that the decontamination efficacy
against intestinal anaerobic bacteria is much higher in patients
receiving the combined medication. In consequence of our previous
observation, this should result in different frequencies of grades II
to IV acute GVHD in the two study arms.
In accordance with the first hypothesis, a pronounced reduction of
intestinal anaerobic bacterial growth posttransplant could be
demonstrated for the combined decontamination medication in comparison
to ciprofloxacin alone, with differences in median concentrations of
anaerobic bacteria in the range of 104 to 108.
Most importantly, both study arms differed significantly with regard to
the primary study endpoint, and a significant association between
anaerobic bacterial growth suppression and the development of grades II
to IV acute GVHD could be ascertained. It is of note that the reduction
of grades II to IV acute GVHD resulted primarily from a more than
twofold lower number of metronidazole-treated patients who developed
liver or intestinal involvement with acute GVHD.
The biological basis by which the intestinal anaerobic bacterial
microflora may modulate the development of acute GVHD after human
marrow transplantation is currently unknown. Dysregulated and increased
secretion of proinflammatory cytokines during the induction phase of
acute GVHD is regarded as a major promoting factor of the allospecific
donor T-cell activation process.5 A recent murine study
supports previous clinical observations that the severity of acute GVHD
increases with an intensified radiation dose of total body irradiation
(TBI).27 This was associated with a systemic increase in
tumor necrosis factor- (TNF-) levels, induced both by greater
sensitivity of macrophages to LPS and by an increased translocation of
LPS into the circulation of allogeneic recipient animals conditioned
with the higher TBI dose. Thus, irradiation synergized with LPS
stimulation of macrophages and potentiated TNF- production in this
murine model. It is therefore tempting to speculate that a substantial
reduction of LPS and other soluble bacterial breakdown products by
decontamination of the preponderant resident anaerobic bacterial
microflora in the intestinal lumen substantially decreases entry of
bacterial products into the portal and systemic circulation, which are
able to induce an alloantigen-independent inflammatory process. This, in turn, would diminish or even abolish TNF- production by monocytes and macrophages, because it is known that, in nonmalignant cells, radiation only induces TNF- mRNA without protein
production.28,29 Because TNF- is thought to be a
particularly important mediator of liver and intestinal cell damage,
this hypothesis is consistent with the finding of the present study
that the lower frequency of grades II to IV acute GVHD in patients
treated with the combined decontamination medication is primarily a
consequence of a reduced liver and intestinal involvement with acute
GVHD.30-33
A further potential mechanism by which decontamination of intestinal
anaerobic bacteria may modulate liver and intestinal acute GVHD is the
downregulation of MHC and adhesion molecule expression on professional
antigen-presenting cells (APCs) in the liver and the intestinal wall
due to a reduced activation by bacterial products penetrating the
intestinal mucosa.34,35 In consequence, the cellular
interactions between APCs and donor T cells leading to allospecific
clonal T-cell activation and expansion would not take effect.
Similarly, a downregulated expression of these antigens on liver and
intestinal epithelial cells would render these cells less susceptible
to the cytolytic and apoptotic actions mediated by the allospecific
effector cells of acute GVHD. Direct evidence for the clinical
significance of these mechanisms is difficult to attain, because liver
and intestinal tissue cannot be serially investigated during the early
posttransplant course, and cell injury induced by TNF- and other
proinflammatory cytokines may largely depend on paracrine cytokine
effects in these tissues, which are not reflected by systemic cytokine
levels.36 It would therefore be interesting to study
whether systemic application of neutralizing soluble receptors for
bacterial LPS may be similarly effective in preventing acute GVHD after
experimental and human marrow transplantation such as decontamination
of the intestinal anaerobic bacterial microflora.
The influence of the combined decontamination medication on acute GVHD
demonstrated for the entire study population resulted predominantly
from a significant reduction of grades II to IV acute GVHD in patients
with genotypically HLA-identical sibling donors, whereas in patients
with donors other than HLA-identical siblings this influence was much
less pronounced despite identical anaerobic bacterial decontamination
efficacies (data not shown). The latter patient subset is presumed to
have a higher degree of minor and major histocompatibility antigen
disparity with their donors compared with recipients of HLA-identical
sibling donor transplants. It can therefore be postulated that the
proposed mechanisms by which anaerobic bacterial decontamination
reduces acute GVHD are less effective, with an increasing degree of
histoincompatibility. Possible pathophysiologic explanations for the
observed differences include, among others, activation-independent
expression and presentation of MHC and minor antigens by APCs in target
tissues as well as enhanced allospecific donor T-cell activation due to
multiple histocompatibility antigen disparities. Regardless of the
quality of anaerobic bacterial decontamination, this would promote
acute GVHD more strongly in recipients of transplants from partially HLA-matched family donors or matched unrelated donors compared with
HLA-identical sibling donor transplant recipients.
Chronic GVHD developed with similar frequencies in the two study arms.
This is not surprising, because a previous randomized trial, which
compared sMTX+CyA to CyA alone for prophylaxis of acute GVHD after
HLA-identical sibling marrow transplants, has demonstrated that an
improvement of acute GVHD prophylaxis does not inevitably result in a
reduction of chronic GVHD.1,2 The development of chronic
GVHD in the present study was significantly associated with grades II
to IV acute GVHD, which is in accordance with most analyses on risk
factors for chronic GVHD.37 Contrary to the results of the
mentioned randomized trial, the probability of chronic GVHD was
significantly lower in patients treated with the combined prophylactic
regimen compared with patients exclusively receiving CyA. However, in
the present study, patients with donors other than HLA-identical
siblings were also included and the prophylactic regimen was chosen
according to risk features of acute GVHD. This heterogeneity in patient
populations and treatment strategies therefore precludes a meaningful
comparison of the influence of the immunoprophylactic regimen on
chronic GVHD between the two studies. The documented effect of the
combined decontamination medication on grades II to IV acute GVHD
together with the independent influence of the sMTX+CyA regimen on
chronic GVHD suggest that a combination of both strategies will
synergize in reducing chronic GVHD.
Despite the significant reduction of grades II to IV acute GVHD in
patients treated with the combined decontamination medication, overall
survival was nearly identical in the two study arms. This analytical
endpoint was adversely influenced by an advanced disease stage at the
time of transplant and by the development of grades II to IV acute
GVHD, both of which are generally accepted as the strongest predictors
of transplant outcome. Because the effect of anaerobic bacterial
decontamination on acute GVHD was largely restricted to patients with
HLA-identical sibling donors, it was interesting to analyze whether
this patient subset had a survival benefit associated with the study
medication, if only patients in early disease stages were considered.
This comparison showed a 14% superior 5-year survival estimate for
patients treated with the combined decontamination medication, but this
difference did not reach statistical significance due to the limited
number of comparable patients in both study arms. Nevertheless, this
difference provides a basis for a future study in this patient subset
investigating the influence of anaerobic bacterial decontamination on
survival as the primary study endpoint.
In conclusion, this open-label prospective randomized study provides
evidence that antimicrobial chemotherapy targeted to intestinal
anaerobic bacteria in marrow transplant recipients significantly
reduces the severity of acute GVHD. The results of this study further
support that the intestinal anaerobic bacterial microflora plays a role
in the pathogenesis of acute GVHD after human marrow transplantation.
| |
ACKNOWLEDGMENT |
The authors are indebted to the nursing staff of the Department of Bone
Marrow Transplantation at the University Hospital of Essen for their
excellent patient care. The authors thank Angelika Hussel for her
support in data aquisition and documentation. This work was made
possible by the outstanding efforts of the technicians of the
Department of Microbiology at the University of Essen.
| |
FOOTNOTES |
Submitted September 14, 1998; accepted January 14, 1999.
This work is dedicated to Prof. Ulrich W. Schaefer at the occasion of
his 60th birthday.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dietrich W. Beelen, MD, Department of Bone
Marrow Transplantation, University Hospital of Essen, Hufelandstr. 55, 45122 Essen, Germany; e-mail: dietrich.beelen{at}uni-essen.de.
| |
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ABSTRACT
INTRODUCTION