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【Objective】 To investigate our experience in the diagnosis and treatment of acute graft-versus-host disease (aGVHD) after liver transplantation in surgical ICU. 【Methods】 We retrospectively analyzed the general data, clinical manifestation, diagnosis, and treatment strategies of five patients with aGVHD after liver transplantation in The First Affiliated Hospital of Xi’an Jiaotong University from January 2000 to December 2019. 【Results】 The incidence rate of aGVHD was 5/850 (5.88 ‰), and all the five patients were male and aged 40-64 years (mean age 56 years). Diabetes, hepatocellular carcinoma, liver transplantation, transcatheter arterial chemoembolization (TACE), and high concentration of immune agents were the main risk factors associated with the development of aGVHD. The average time from surgery until clinical symptom of aGVHD was 15 to 32 days. In our patients with aGVHD, the most common symptom was fever (5/5), followed by skin rash (5/5), pancytopenia (5/5), diarrhea (3/5), and secondary pulmonary infection (3/5). However, liver functions were not remarkable affected. Diagnostic criteria for aGVHD in our center include acute onset, risk factors, typical clinical manifestation, and histopathology after exclusion of differential diseases. Our treatment strategies include high-dose methylprednisolone, stopping/reducing current immunosuppressive protocol, and antilymphocytic agents as second-line treatment. Empirical antibiotics and antifungal agents play a vital part in infections after transplantation. Hematopoietic cytokine was administered to treat pancytopenia. Patients also received supportive therapy, such as isolation and nutritional support, with the goal of benefiting the entire condition. Despite intensive treatment, two of five patients (40%) with aGVHD died due to sepsis and multiorgan failure. One case (20%) died of intracranial hemorrhage and one case (20%) died of tuberculosis. Only one case (20%) stayed alive after 1-year follow-up without complications. 【Conclusion】 The diagnosis of aGVHD relies on clinical suspicion and is confirmed by skin pathology. The patients with aGVHD had early onset (38.5 ℃), large rash range (>50%), complication of sepsis, and poor response to hematopoietic cytokine therapy indicate poor prognosis. Intensive treatment should be started immediately after aGVHD diagnosis. In conclusion, we strongly suggest an early identification, diagnosis, and vigorous treatment strategy, which is the key to improving the prognosis of aGVHD.
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【Objective】 To investigate the preventive effects of early apoptotic splenic mononuclear cells induced by extracorporeal photopheresis (ECP) on acute graft versus host disease (aGVHD) in mice and explore the underlying mechanisms. 【Methods】 1) Splenic mononuclear cells were extracted from C57BL/6 mice and treated with different concentrations of 8-MOP (50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 600 ng/mL). After treatment, irradiate the cells with 2 J/cm2 of ultraviolet light. Then, use the Annexin V-FITC/PI apoptosis detection kit to assess the early apoptosis rate of the cells and determine the optimal concentration of 8-MOP for the experiment.2) There were 35 SPF-grade female BALB/C mice (H-2Kd) aged 6-8 weeks. After whole-body irradiation with 8Gy X-rays, the mice were divided into five groups: sham irradiation group received intravenous infusion of 0.2 mL of normal saline, the syngeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of BALB/C mouse bone marrow nucleated cell suspension (including a cell count of 1×107), the allogeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of C57BL/6 mouse bone marrow nucleated cell suspension (including a cell count of 1×107), the aGVHD group received intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells (including a cell count of 1×107) and splenic mononuclear cells (including a cell count of 1×107) in 0.2 mL, the ECP prevention group received pre-transplant intravenous infusion of 0.2 mL of ECP-treated splenic mononuclear cells of C57BL/6 mice (including a cell count of 1×107 ) 48 hours before transplantation, and on the day of transplantation, intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells (including a cell count of 1×107) and splenic mononuclear cells (including a cell count of 1×107 ) in 0.2 mL.The preventive effects of ECP on aGVHD were observed, and the concentrations of IFN-γ, IL-2, TNF, IL-4 and IL-6 in mouse serum were measured using CBA. Th1 cell counts were determined by flow cytometry. 【Results】 Different concentrations of 8-MOP (50 ng/mL,100 ng/mL, 200 ng/mL, 300 ng/mL, 600 ng/mL) were used to treat mouse splenic mononuclear cells. The early apoptosis rates (%), observed after treatment were as follows: (14.18±0.865) vs (16.76±0.407) vs (18.83±0.404) vs (19.27±0.404) vs (14.5±0.529). The appropriate concentration of 8-MOP was determined to be 200 ng/mL.In vivo experiment, the results showed that the aGVHD group had decreased survival rate, reduced body weight, and increased clinical scores compared to the syngeneic and allogeneic bone marrow transplantation groups (P<0.01), and the chimerism of bone marrow cells in mice after transplantation was over 90%. ECP significantly improved the survival rate of mice after transplantation, reduced clinical scores (P<0.05), and decreased the concentrations of Th1 cell cytokines in serum (P<0.05) and the counts of Th1 cells in the spleen (P<0.05). 【Conclusion】 ECP-induced early apoptotic single nuclear cells from the spleen can prevent the occurrence of aGVHD by reducing the Th1 response in mouse.
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OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.
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Mice , Female , Animals , Mice, Inbred C57BL , Bone Marrow Transplantation , Graft vs Host Disease , Stem Cells , OrganoidsABSTRACT
Objective:To investigate the effect and underlying mechanism of BRCC3 knockout on acute GVHD(aGVHD) of mice.Methods:A total of 12 recipient C57BL/6J mice were divided into two groups, including 6 wild type(WT) and BRCC3 -/-(KO). The recipients were exposed to 4.5 Gy + 4.5 Gy 60Co γ-rays in total body irradiation (TBI) at 30 min intervals. At 6 h post-irradiation, 1×10 7bone marrow cells and 8×10 6 splenocytes from BALB/c mice were infused into C57BL/6J mouse via tail vein to develop aGVHD mouse model. BRCC3 was specifically knocked out in aGVHD mouse model. The organ damage was examined through histopathology. The levels of serum cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cytometric bead array (CBA), respectively. Spleen, liver and small intestine lymphocytes were isolated at 9 d post-transplantation, and the infiltration and activation of T cells in the target organs were assayed using flow cytometry. Results:The absence of BRCC3 in recipient mice significantly shortened survival ( P<0.05) with increased liver injury of aGVHD mice. In BRCC3 -/-recipient mice, the proportions of CD8+ T cells and CD8+ CD25+ T cells were significantly higher than those in the spleen( t=6.53, 5.52, P<0.05), and the proportions of CD8+ T cells and CD8+ CD25+ T cells were significantly increased in the liver ( t=3.74, 3.19, P<0.05). Similarly, the proportions of CD8+ T cells, CD8+ CD25+ T cells and CD8+ CD69+ T cells were significantly elevated in the small intestine ( t=3.52, 4.06, 3.29, P<0.05). Conclusions:BRCC3 deletion increased the proliferation and activation of donor CD8+ T cells and aggravated aGVHD, which might provide a new prevention and treatment target for aGVHD.
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Allogeneic hematopoietic stem cell transplantation ( allo-HSCT) is an effective therapy for the treatment of various malignant and non-malignant hematological diseases. Acute graft-verse-host-dis-ease ( aGVHD) , a major complication following allo-HSCT, is one of the predominant causes of GVHD-re-lated mortality. The development of aGVHD is a typical pathologic process with the release of inflammatory cytokines in great quantities, resulting in the occurrence ofcytokine storm and causing specific pathologi-cal damages by attacking the recipient organ. Therefore, identification of biomarkers specific for aGVHD of-fers promise to the treatment of aGVHD. In this review, we summarizes the functions of several typical in-flammatory cytokines, including IL-1β, IL-6, IL-17A and IL-10, and their mechanisms in the development of aGVHD in order to provide references for the prevention and treatment of aGVHD.
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Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the major curative treatment for patients with malignant hematologic diseases. However, a number of serious complications limits successful outcomes. Acute graft-versus-host disease(GVHD) is a major cause of death following allo-HSCT.The mortality rate is 50 % in Ⅱ -Ⅳ aGVHD. At present, the major prevention methods conclude inhibiting T cell activation and proliferation, depleting of T cell in graft, and decreasing the dose of preconditioning. And only one quarter of these patients will have a complete response to first-line corticosteroid therapy. Patients with steroid resistant acute GVHD require second-line therapy to which the response rate is only 30 %-50 %.Second-line therapies including monoclone antibody, chemotherapy and cytokine inhibitor were used. Secondline options can impair immune reconstitution and increase the risk for infection. Therefore, novel approaches to prevent and treat GVHD are critically needed. This work will review novel immuno-modulatory therapy currently being employed both for the prevention and treatment of GVHD.
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Objective The study was aimed to investigate the effects of transforming growth factor beta1(TGF-?1)on acute graft-versus-host disease(aGVHD)after allogeneic bone marrow transplantation(allo-BMT).Methods The recipient was male BABL/C.The donor was male C57BL/6.The murine model of aGVHD had been established by allo-BMT with donor derived T cells.There were four groups in this study:control group,radiation group,transplantation control group and TGF-?1 group.The mice in TGF-?1 group were administered TGF-?1[1 ?g/(kg?d)]subcutaneously two days before transplantation until seven days after it.Results The study showed that the survival time of TGF-?1 group was significantly longer than the transplantation control group,and the aGVHD pathological changes were milder in TGF-?1 group than in transplantation control group.Seven days after transplantation,the level of IL-2 of TGF-?1 group was higher than control group,but significantly lower than transplantation control group.The level of IL-10 of TGF-?1 group was significantly higher than transplantation control group.Conclusion TGF-?1 can prevent the lethal aGVHD and raise the survival rate after allo-BMT in murine model.It may prevent the lethal aGVHD by accommodating the Th1 and Th2 cytokine level in vivo.
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Objective:To investigate the expression profile of Ly49A in a mouse GVHD model and the ex vivo expression alteration of it in T cells exposal to CsA, IL 4, and IL 15, respectively.Methods:Based on mouse aGVHD model, two color fluorescence flow cytometry was used to assay the expression of Ly49A on splenic and bone marrow CD3 +, CD4 + and CD8 + T cell subsets, before and after engraftment in different time interval. Cell culture was used to analyse in vitro Ly49A expression on splenic T cells treated with IL 4 and IL 15.Results:①In normal B6 and F1 strain, the frequencies of Ly49A + cells in BMCs , splenocytes and PMNCs remained at a low level and almost no, if any, such cells were detected in thymus; ②The proportions of CD3 +/Ly49A + andCD8 +/Ly49A + cells rose in early stage(7th,day) , tended to decline until 5 w post engraftment, and thereafter arose and exceed that pretreatment, which was down regulated in the presence of CsA; ③With or without ConA, the CD3 +/Ly49A + proportion were up modulated in purified CD3 +splenic cells, by IL 4 and IL 15 with no different effect demonstrated between those cytokines.Conclusion:KIR +T lymphocytes were most belong to peripheral lymphocytes. There exist a immunological tolerant tendency between grafts and host in semimatched transplant, and some cytokines and immunosuppressants can enhance the tolerance.