| Part I The clinical study of intestinal microbiota and the emergence of acute graft-versus-host disease in patients undergoing allogeneic hematopoietic stem cell transplantationObjective: We carried out a clinical study to investigate the alterations of intestinal microbiota during the procedure of allogeneic hematopoietic stem cell transplantation(alloHSCT)and their correlation with the emergence of acute graft-versus-host disease(a GVHD).Methods: 47 patients who experienced allo-HSCT and suffered from a GVHD at the Department of Hematology,First Affiliated Hospital of Soochow University were enrolled in this study.18 patients were female,and 29 patients were male.The median age at the time of transplantation was 36 years.The donor of the transplantation included HLA-identical siblings(n=16),matched unrelated donors(n=15)and haplo-identical related donors(n=16).Fecal specimens were collected from these patients at three different time-points during transplantation period: prior to the administration of conditioning regimen,during the first week after day 0 and after engraftment.The bacterial genomic DNA was extracted.Then it was amplified for the V3 hypervariable regions of the 16 S r RNA gene.Polymerase chain reaction(PCR)products were sequenced on a 454 GS FLX platform.Then,the changes of intestinal microbiota and the clinical manifestations of these patients were evaluated.Results: All patients achieved hematopoietic reconstitution and maintained donor engraftment.During the follow-up period,16 patients(34%)had no a GVHD,while 31 patients(66%)developed a GVHD.Among those patients with a GVHD,19 had grade I-II a GVHD and 12 had grade III-IV a GVHD.There were 19 patients suffered from gastrointestinal(GI)a GVHD,and the other 12 a GVHD patients had skin and/or liver a GVHD without intestinal involvement.Data analysis was based on the sequencing results of those 141 samples.Firmicutes,Proteobacteria,Bacteroidetes and Actinobacteria were the most abundant phyla in these patients’ intestine.Their abundances all changed during the course of transplantation.After engraftment,the composition of intestinal microbiota had many differences between patients without a GVHD and patients with a GVHD.At genus level,Bacteroides and Escherichia-Shigella had higher abundance in patients without a GVHD compared with patients with a GVHD,while Enterococcus notably increased in patients with a GVHD.These changes were more obvious between patients without a GVHD and patients with GI a GVHD.Conclusion: These results demonstrated that intestinal microbiota shifted during the transplantation course.Some alterations of intestinal microbiota correlated with the emergence of a GVHD,especially GI a GVHD.Part II Treatment of steroid refractory gastrointestinal acute graft-versus-host disease with fecal microbiota transplantationObjective: We conducted a clinical study to assess safety and efficacy of fecal microbiota transplantation(FMT)in treating steroid refractory GIa GVHD.Methods: 8 patients who had steroid refractory GI a GVHD after allo-HSCT at the Department of Hematology,First Affiliated Hospital of Soochow University were enrolled in this study.5 patients were female,and 3 patients were male.The median age was 35.6 years.The donor of the transplantation included HLA-identical siblings(n=2),matched unrelated donors(n=1),and haplo-identical related donors(n=5).Once diagnosed as steroid refractory GI a GVHD,all patients were treated with one or more second-line therapy,but had no responses.Then,FMT was applied.(1)The safety and efficacy of FMT in treating steroid refractory GI a GVHD were observed.(2)Fecal samples from 2 healthy donors and 6 patients(before FMT and a week after the first FMT)were collected.16 S r RNA sequencing was used to show the composition and the diversity of the intestinal microbiota of these patients.Results:(1)Clostridium difficile infection(CDI)was not observed among all these 8 patients.The mean time of the onset of GI a GVHD(days after allo-HSCT)was +79.7 days.After diagnosed as a GVHD,the patients received standard first-line treatment with corticosteroids,and then one or more second-line therapy,but had no responses.Before FMT,the mean stool volume of all patients was 1,777 mL/day,with the mean stool frequency 11.5 times/day,and 4 patients suffered from abdominal pain.(2)FMT was applied in four patients once and four patients twice.After the administration of FMT,3 patients had nausea and vomiting,while 2 patients had bloating.1 patient had cytomegalovirus(CMV)activation in 30 days after FMT.All these patients acquired clinical symptomatic remission after the first FMT.Their stool volumes and frequencies were reduced.Among those 4 patients with abdominal pain,3 had alleviation.4 patients’ diarrhea disappeared in the 4 weeks after the first FMT and they were alive at 90 days.2 patients’ a GVHD recurred during the treatment and died because of severe complications.1 patient died of cerebral hemorrhage caused by thrombocytopenia.Another patient gave up the treatment due to economic reasons and died.(3)Before FMT,the diversity of intestinal microbiota of the patients decreased compared with that of the donors(P<0.01).After FMT,the diversity had a significant increase compared with that before FMT(P<0.01),but was still lower than that of the donors(P<0.01).The ratio of Firmicutes to Proteobacteria got imbalanced in patients with steroid refractory GI a GVHD and the ratio of Bacteroidetes significantly decreased.1 week after FMT,the diversity of intestinal microbiota improved,and the composition was reconstructed,showing a trend back to normal.Conclusion: The implication of the treatment for steroid refractory a GVHD with FMT was found to be safe and efficient by improving the diversity and composition of the intestinal microbiota.But further evaluations are still needed.Part III The role of probiotics in murine acute graft-versus-host disease modelObjective: Our lab isolated a lactic acid bacteria strain of probiotics named Pro15.We studied the role of Lactobacillus rhamnosus GG(LGG)and Pro15 strain in murine a GVHD model,and explored their role in a GVHD.Methods:(1)BALB/c(H-2d)mice were divided into three groups,including PBS control group,LGG strain group and Pro15 strain group.On day-7,-5,-3 and-1,mice were given with PBS,LGG or Pro15 strain by gavage administration.BALB/c mice were given lethally 7.5Gy total body irradiation,followed by the infusion of 1×107 bone marrow cells and 5×106 spleen cells from C57BL/6 mice.(2)The body weight and clinical manifestations of a GVHD of the recipient mice were monitored every two days,while survival were observed daily after transplantation.(3)Histopathology of a GVHD target organ,such as lung,liver,small intestine and colon,was detected in recipient mice after 4 days post transplantation.(4)The immune cell types of spleen,lung,liver and mesenteric lymph nodes(MLN)were analyzed by flow cytometry after 4 days post transplantation in recipient mice.(5)The levels of IFN-γ,IL-2,IL-4,IL-6,TNF-α,IL-10 and IL-17 A in recipient mice were assessed by Cytometric Beads Array.(6)We also used heat-killed LGG or Pro15 strain,culture supernatant of LGG or Pro15 strain to gavage the mice.And then we performed the transplantation procedure as mentioned above.Survivals of these groups were observed.(7)At 4 days post transplantation,the permeability of intestinal tract of recipient mice were evaluated by FITC-dextran examination.The levels of lipopolysaccharide(LPS)in recipient mice were also measured at the same time.Then,the expressions of TLR2 and TLR4 of intestinal epithelial cells of these three recipient mice were tested.(8)The changes of intestinal microbiota of these a GVHD mice were monitored.Microbial diversity was estimated by calculating shannon index.We also observed the differences of intestinal microbiota between PBS group and Pro15 strain group.Results:(1)The results showed that mice treated with LGG or Pro15 strain can reduce the severity of clinical a GVHD.LGG strain group and Pro15 strain group had better survival rate compared with PBS group.Pro15 strain group had the best survival.4 days after transplantation,the organs of recipient mice were harvested and pathology analyses conducted.(2)Histological examination showed a significant attenuation of inflammation reaction in Pro15 strain group.(3)We found that Pro15 strain administration significantly reduced the proportions of activated CD4+T cells in the spleen and lung of recipient mice compared with PBS treatment(P<0.01).Meanwhile,the percentage of activated CD8+T cells in the spleen,lung and MLN of recipient mice receiving Pro15 was also obviously decreased relative to that of PBS-treated control mice(P<0.01).(4)The level of IFN-γ were decreased in LGG strain group and Pro15 strain group,but had no significant differences compared with PBS group.(5)Both heat-killed LGG or Pro15 strain and culture supernatant of LGG or Pro15 strain can’t prolong survival of murine a GVHD model.(6)The serum level of FITC-dextran in Pro15 strain group was lower than PBS group(P<0.05)at 4 days post transplantation.Pro15 administration also significantly reduced the level of serum LPS compared with PBS group(P<0.001).Pro15 treatment can downregulate TLR4 expression of intestinal epithelial cell compared with PBS treatment(P<0.05).(7)Microbial diversity had no differences among PBS group,LGG strain group and Pro15 strain group at 1 day before and 2 days post transplantation.But at 5 days,shannon index of intestinal microbiota was much higher in Pro15 strain group than in PBS group(P<0.05).(8)At 5 days post transplantation,the composition of intestinal microbiota had many differences between PBS group and Pro15 strain group.Conclusion: Our studies demonstrated that Pro15 strain can attenuate the severity of clinical GVHD in murine model,and prolong their survival.Pro15 strain was benefit for the integrity of intestinal barrier,and inhibited TLR4/LPS pathway.Also,Pro15 strain preserved the overall diversity and modulated the composition of intestinal microbiota during a GVHD. |
