Human Amniotic Membrane-Derived Mesenchymal Stem Cells Prevent Acute Graft-Versus-Host Disease in an Intestinal Microbiome-Dependent Manner.

Acute graft-versus-host disease (aGVHD) represents a fatal severe complication after allogeneic hematopoietic stem cell transplantation. As a promising cell therapeutic strategy of aGVHD, the mechanism of mesenchymal stem cells (MSC) to ameliorate aGVHD has not been fully clarified, especially in the field of intestinal homeostasis including the intestinal microbiome involved in the pathogenesis of aGVHD. The present study aimed to explore the effect of MSC on intestinal homeostasis including the intestinal barrier and intestinal microbiome and its metabolites, as well as the role of intestinal microbiome in the preventive process of hAMSCs ameliorating aGVHD. The preventive effects of human amniotic membrane-derived MSC (hAMSCs) was assessed in humanized aGVHD mouse models. Immunohistochemistry and RT-qPCR were used to evaluate intestinal barrier function. The 16S rRNA sequencing and targeted metabolomics assay were performed to observe the alternation of intestinal microbiome and the amounts of medium-chain fatty acids (MCFAs) and short-chain fatty acids (SCFAs), respectively. Flow cytometry was performed to analyze the frequencies of T immune cells. Through animal experiments, we found that hAMSCs had the potential to prevent aGVHD. HAMSCs could repair the damage of intestinal barrier structure and function, as well as improve the dysbiosis of intestinal microbiome induced by aGVHD, and meanwhile, upregulate the concentration of metabolites SCFAs, so as to reshape intestinal homeostasis. Gut microbiota depletion and fecal microbial transplantation confirmed the involvement of intestinal microbiome in the preventive process of hAMSCs on aGVHD. Our findings showed that hAMSCs prevented aGVHD in an intestinal microbiome-dependent manner, which might shed light on a new mechanism of hAMSCs inhibiting aGVHD and promote the development of new prophylaxis regimes for aGVHD prevention.

Therapeutic Effects of HLA-G5 Overexpressing hAMSCs on aGVHD After Allo-HSCT: Involving in the Gut Microbiota at the Intestinal Barrier.

Background: Acute graft-versus-host disease (aGVHD) initiated by intestinal barrier dysfunction and gut microbiota dysbiosis, remains one of the main obstacles for patients undergoing allogenic hematopoietic stem cell transplantation (allo-HSCT) to achieve good prognosis. Studies have suggested that mesenchymal stem cells (MSCs) can suppress immune responses and reduce inflammation, and human leukocyte antigen-G5 (HLA-G5) plays an important role in the immunomodulatory effects of MSCs, but very little is known about the potential mechanisms in aGVHD. Thus, we explored the effect of HLA-G5 on the immunosuppressive properties of human amnion MSCs (hAMSCs) and demonstrated its mechanism related to the gut microbiota at the intestinal barrier in aGVHD. Methods: Patients undergoing allo-HSCT were enrolled to detect the levels of plasma-soluble HLA-G (sHLA-G) and regulatory T cells (Tregs). Humanized aGVHD mouse models were established and treated with hAMSCs or HLA-G5 overexpressing hAMSCs (ov-HLA-G5-hAMSCs) to explore the mechanism of HLA-G5 mediated immunosuppressive properties of hAMSCs and the effect of ov-HLA-G5-hAMSCs on the gut microbiota at the intestinal barrier in aGVHD. Results: The plasma levels of sHLA-G on day +30 after allo-HSCT in aGVHD patients were lower than those in patients without aGVHD, and the sHLA-G levels were positively correlated with Tregs percentages. ov-HLA-G5-hAMSCs had the potential to inhibit the expansion of CD3+CD4+ T and CD3+CD8+ T cells and promote Tregs differentiation, suppress proinflammatory cytokine secretion but promote anti-inflammatory cytokines release. Besides, ov-HLA-G5-hAMSCs also could reverse the intestinal barrier dysfunction and gut microbiota dysbiosis in aGVHD. Conclusion: We demonstrated that HLA-G might work with Tregs to create a regulatory network together to reduce the occurrence of aGVHD. HLA-G5 mediated hAMSCs to exert higher immunosuppressive properties in vivo and reverse the immune imbalance caused by T lymphocytes and cytokines. Furthermore, HLA-G5 overexpressing hAMSCs could restore gut microbiota and intestinal barriers, thereby ameliorating aGVHD.

Human Amniotic Mesenchymal Stem Cells Alleviate aGVHD after allo-HSCT by Regulating Interactions between Gut Microbiota and Intestinal Immunity.

Acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation poses one of the most vexing challenges. Gut microbiota dysbiosis can proceed aGVHD and mesenchymal stem cells (MSCs) have promising therapeutic potential for aGVHD. However, whether hAMSCs affect the gut microbiota during aGVHD mitigation remains unknown. Accordingly, we sought to define the effects and underlying mechanisms of human amniotic membrane-derived MSCs (hAMSCs) regulating the gut microbiota and intestinal immunity in aGVHD. By establishing humanized aGVHD mouse models and hAMSCs treatment, we found that hAMSCs significantly ameliorated aGVHD symptoms, reversed the immune imbalance of T cell subsets and cytokines, and restored intestinal barrier. Moreover, the diversity and composition of gut microbiota were improved upon treatment with hAMSCs. Spearman's correlation analysis showed that there was a correlation between the gut microbiota and tight junction proteins, immune cells as well as cytokines. Our research suggested that hAMSCs alleviated aGVHD by promoting gut microbiota normalization and regulating the interactions between the gut microbiota and intestinal barrier, immunity.

Establishment of Prognosis Model in Acute Myeloid Leukemia Based on Hypoxia Microenvironment, and Exploration of Hypoxia-Related Mechanisms.

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic neoplasm with poor survival outcomes. However, the routine clinical features are not sufficient to accurately predict the prognosis of AML. The expression of hypoxia-related genes was associated with survival outcomes of a variety of hematologic and lymphoid neoplasms. We established an 18-gene signature-based hypoxia-related prognosis model (HPM) and a complex model that consisted of the HPM and clinical risk factors using machine learning methods. Both two models were able to effectively predict the survival of AML patients, which might contribute to improving risk classification. Differentially expressed genes analysis, Gene Ontology (GO) categories, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to reveal the underlying functions and pathways implicated in AML development. To explore hypoxia-related changes in the bone marrow immune microenvironment, we used CIBERSORT to calculate and compare the proportion of 22 immune cells between the two groups with high and low hypoxia-risk scores. Enrichment analysis and immune cell composition analysis indicated that the biological processes and molecular functions of drug metabolism, angiogenesis, and immune cell infiltration of bone marrow play a role in the occurrence and development of AML, which might help us to evaluate several hypoxia-related metabolic and immune targets for AML therapy.

Human Amniotic Mesenchymal Stem Cells Inhibit aGVHD by Regulating Balance of Treg and T Effector Cells.

BACKGROUND: Acute graft versus host disease (aGVHD) remains a leading cause of transplant-related mortality following allogeneic haematopoietic cell transplantation (allo-HCT). Human amniotic mesenchymal stem cells (hAMSCs) are a novel mesenchymal stem cells (MSCs), which have stronger proliferation and immunomodulatory ability compared with bone marrow mesenchymal stem cells (BM-MSCs). Besides, as the amniotic membrane is often treated as medical waste after delivery, hAMSCs can be obtained conveniently and noninvasively. The aim of this study was to explore the therapeutic efficacy and underlying mechanisms of hAMSCs transplantation for the humanized aGVHD mouse model. METHODS: We established a humanized aGVHD mouse model by transplanting human peripheral blood mononuclear cells (PBMCs) into NOD-PrkdcscidIL2rγnull (NPG) mice, human amniotic membrane collected from discarded placenta of healthy pregnant women after delivery and hAMSCs were extracted from amniotic membrane and expanded in vitro. Mice were divided into untreated group (Control), aGVHD group (aGVHD), and hAMSCs treatment group (aGVHD+hAMSCs), the hAMSCs labeled with GFP were administered to aGVHD mice to explore the homing ability of hAMSCs. T effector and regulatory T cells (Tregs) levels and cytokines of each group in target organs were detected by flow cytometry and cytometric bead array (CBA), respectively. RESULTS: We successfully established a humanized aGVHD mouse model using NPG mice. The hAMSCs have the ability to inhibit aGVHD in this mouse model through reduced villous blunting and lymphocyte infiltration of the gut while reducing inflammatory edema, tissue destruction and lymphocyte infiltration into the parenchyma of the liver and lung. hAMSCs suppressed CD3+CD4+ T and CD3+CD8+ T cell expression and increased the proportion of Tregs, and besides, hAMSCs can reduce the levels of IL-17A, INF-γ, and TNF in aGVHD target organs. CONCLUSION: The NPG murine environment was capable of activating human T cells to produce aGVHD pathology to mimic aGVHD as in humans. The hAMSCs controlled aGVHD by decreasing inflammatory cytokine secretion within target organs by modulating the balance of Tregs and T effector cells in humanized mice.

Immunomodulating functions of human leukocyte antigen-G and its role in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapeutic strategy to treat several hematological malignancies and non-hematological malignancies. However, graft-versus-host disease (GVHD) is a frequent and serious transplant-related complication which dramatically restrains the curative effect of allo-HSCT and a significant cause of morbidity and mortality in allogeneic HCT recipients. Effective prevention of GVHD mainly depends on the induction of peripheral immune tolerance. Human leukocyte antigen-G (HLA-G) is a non-classical MHC class I molecule with a strong immunosuppressive function, which plays a prominent role in immune tolerance. HLA-G triggers different reactions depending on the activation state of the immune cells and system. It also exerts a long-term immune tolerance mechanism by inducing regulatory cells. In this present review, we demonstrate the immunomodulatory properties of human leukocyte antigen-G and highlight the role of HLA-G as an immune regulator of GVHD. Furthermore, HLA-G could also serve as a good predictor of GVHD and represent a new therapeutic target for GVHD.

TTDA inhibited apoptosis by regulating the p53-Bax/Bcl2 axis in glioma.

The trichothiodystrophy group A protein (TTDA) functions in nucleotide excision repair and basal transcription. TTDA plays a role in cancers and serves as a prognostic and predictive factor in high-grade serous ovarian cancer; however, its role in human glioma remains unknown. Here, we found that TTDA was overexpressed in glioma tissues. In vitro experiments revealed that TTDA overexpression inhibited apoptosis of glioma cells and promoted cell growth, whereas knockdown of TTDA had the opposite effect. Increased TTDA expression significantly decreased the Bax/Bcl2 ratio and the level of cleaved-caspase3. TTDA interacted with the p53 gene at the -1959 bp and -1530 bp region and regulated its transcription, leading to inhibition of the p53-Bax/Bcl2 mitochondrial apoptosis pathway in glioma cells. These results indicate that TTDA is an upstream regulator of p53-mediated apoptosis and acts as an oncogene, suggesting its value as a potential molecular target for the diagnosis and treatment of glioma.

[Clinical characteristics of hemophagocytic syndrome: analysis of 46 cases].

OBJECTIVE: To analyze the clinical manifestations, laboratory findings, treatment and prognosis of patients with hemophagocytic syndrome (HPS). METHODS: A retrospective study was carried out to analyze the underlying disease, clinical characteristics, laboratory findings and outcomes of 46 patients with HPS. RESULTS: This cohort included 19 cases of HPS secondary to cancer, 11 cases of HPS secondary to infection, 10 cases of suspected malignant lymphoma based on PET-CT findings (without biopsy), and 6 cases of unknown etiology. The coincidence rate of the clinical characteristics of the patients with the indices listed in HPS-2004 criteria were: fever (100%), elevated serum ferritin (100%), cytopenias (93.48%), splenomegaly (91.30%), hemophagocytosis in the bone marrow, spleen or lymph nodes (84.78%), hypofibrinogenemia (67.39%), and hypertriglyceridemia (54.05%). The cases of cancer, infections and unknown etiology showed significant differences in serum levels of ferritin and ß2MG (P<0.05), and significant differences were found in triglycerides, LDH, and fibrinogenemia between the nonfatal and fatal cases (P<0.05). CONCLUSION: HPS can be secondary to various underlying diseases, many associated with Epstein-Barr virus infection. Cancer, especially NK/T-cell lymphoma, is the main cause of HPS. Persistent fever, elevated serum ferritin level and cytopenias are the most sensitive indicators for diagnosis of HPS, and early diagnosis and treatment are critical to lower the mortality rate of this disease.

[Immunomodulatory effects of human amniotic versus bone marrow-derived mesenchymal stem cells on peripheral blood T lymphocytes in vitro].

OBJECTIVE: To compare the immunomodulatory effects of human amniotic mesenchymal stem cell (hAMSCs) and human bone marrow mesenchymal stem cells (hBMSCs) on peripheral blood T lymphocytes in an in vitro co-culture system. METHODS: hAMSCs and hBMSCs isolated using enzymatic digestion and Ficoll-Hypaque density gradient centrifugation, respectively, were culture-expanded in vitro to obtain the 4th-generation cells. The two MSCs were co-cultured separately with human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA-PBMSC) to investigate the changes in T lymphocyte subsets using flow cytomety and the production of interleukin-2 (IL-2) and IL-10 by the T lymphocytes using enzyme-linked immunosorbent assay (ELISA). RESULTS: Co-culture with either hAMSCs or hBMSCs significantly increased the proportions of Treg, Th2 and Tc2 and decreased Th1 and Tc1 cell subsets in the PBMCs as compared with the PBMCs cultured alone (P<0.05), and the changes in the PBMCs were similar between the two co-culture systems (P>0.05). In both of the two co-culture systems, IL-2 production by the lymphocytes was significantly lowered (P<0.05) and IL-10 production was significantly increased (P<0.05) as compared with their levels in the PBMCs cultured alone; no significant difference was found in IL-2 or IL-10 levels between the two co-culture systems (P>0.05). CONCLUSION: The MSCs derived from human amnion and bone marrow have similar immunomodulatory effects on the T lymphocytes, suggesting the possibility of using hAMSCs in the treatment of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

[Comparison of Biological Characteristics and Immunosuppressive Activity between Human Amniotic Mesenchymal Stem Cells and Human Bone Marrow Mesenchymal Stem Cells].

OBJECTIVE: To compare the biological characteristics and immunosuppressive activity between human amniotic mesenchymal stem cells (hAMSC) and human bone marrow mesenchymal stem cells (hBMMSC). METHODS: MSC from human amnion and bone marrow were isolated using enzymatic digestion and Ficoll-Hypaque density gradients, respectively. Their biological characteristics were compared by morphology, cell growth curves, cell cycle profile analysis, immunophenotype and immunofluorescence assay. Their immunosuppressive activities were studied on total activated T-cells with phytohemagglutinin (PHA-PBMSC). An in vitro co-culture was performed to compared the lymphocyte proliferation and the supernatant level of IFN-γ were measured by CCK-8 method and ELISA, respectively. RESULTS: Both hAMSC and hBMMSC demonstrated fibroblast-like morphology. The hAMSC were able to be amplified for at least 15 passages, while the hBMMSC only for 6-7 passages. There was no significant difference in the proportion of G2/M phase cells of the 2 cells types (P>0.05). By FACS analysis for immunophenotype, both MSC were shown to be positive for CD105, CD90, CD73 and negative for CD34, CD45, CD11b, CD19, HLA-DR, but hAMSC were positive for Oct-3/4, which was in contrast to hBMMSC. Both of them expressed vimentin. Both the cells exhibited a inhibitory role on the lymphocyte proliferation induced by PHA in co-culture conditions, that was increased with the increase MSC proportion and both the suppressing effecs were enhanced. The supernatant IFN-γ levels of hAMSC co-cultured with lymphocyte at a ratio of 1:1 after 72 hours were measured by ELISA, and the level of IFN-γ was significantly lower than that in the same co-culture system of hBMMSC. In contrast to the IFN-γ in the PHA-stimulated group, the IFN-γ level in both co-culture groups was significantly lower. CONCLUSION: MSC from amnion displayed a higher proliferative capacity and stem cell properties, compared with hBMMSC. Both MSC can inhibit lymphocyte proliferation and suppress IFN-γ secretion induced by PHA in vitro.

[Effect of simulated microgravity on erythroid differentiation of K562 cells and the mechanism].

OBJECTIVE: To investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism. METHODS: The fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, ß-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, ß-tubulin and vimentin protein expression levels. RESULTS: Benzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, ß-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, ß-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, ß-tubulin and vimentin in the cells. CONCLUSION: Simulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.

[Rituximab-induced interstitial pneumonitis: report of two cases and literature review].

We report two cases of rituximab (RTX)-induced interstitial pneumonia in two lymphoma patients receiving RTX treatment. Interstitial pneumonia was successfully managed in these two cases after a one-week-long intervention with corresponding treatments without affecting further treatment of the primary disease. RTX-induced interstitial pneumonia is characterized by a latent onset with an unclear pathological mechanism and absence of typical symptoms. High-resolution CT scan can provide valuable evidence for early diagnosis of RTX-induced interstitial pneumonia, which might be attributed partially to an increased susceptibility to P. jirovecii and fungal infection due to prolonged RTX treatment.

Second- versus first-generation azoles for antifungal prophylaxis in hematology patients: a systematic review and meta-analysis.

Second-generation azoles may be more effective than first-generation azoles in the prevention of fungal infections in hematology patients. We performed a systematic review with meta-analysis of randomized controlled trials comparing second- with first-generation azoles in hematology patients with respect to proven or probable invasive fungal infections, invasive aspergillosis, receipt of empirical antifungal therapy, overall mortality, and withdrawal from the studies due to the development of adverse effects. We searched the Medline, Embase, and Cochrane Registry of Controlled Trials electronic databases as well as conference proceedings from 2002 to 2012 for randomized controlled trials comparing second-generation azoles (voriconazole, posaconazole) versus first-generation azoles (fluconazole, itraconazole). Treatment effect measures for all outcomes were expressed as odds ratio with 95 % confidence interval. Meta-analysis was performed using Review Manager, version 5.1. Data from four randomized clinical trials representing a large population of patients demonstrated that antifungal prophylaxis with second-generation azoles reduces proven or probable invasive fungal infections, invasive aspergillosis, and receipt of empirical antifungal therapy in high-risk hematology patients, while there were no differences between second- and first-generation azoles with regard to overall mortality and patients or withdrawal from the studies due to the development of adverse effects. In conclusion, antifungal prophylaxis with second-generation azoles can significantly reduce the incidence of invasive fungal infections and invasive aspergillosis but with no risk of an increase in adverse events.

[Clinical characteristics and outcomes of 31 patients with primary bone lymphoma].

OBJECTIVE: To investigate the clinical characteristics, outcomes and prognostic factors of primary bone lymphoma (PBL). METHODS: We retrospectively analyzed 31 consecutive patients with the diagnosis of PBL initially treated at our hospital between 1992 and 2010. Kaplan-Meier method was used for survival analysis and Cox regression model used for analyzing the prognostic factors. RESULTS: The median age of the patients was 46 years. The most common sites of involvement were the femur (29%) and the spine (29%). Sixteen (52%) patients underwent chemoradiotherapy, and the other 15 (48%) received chemotherapy. With a median follow-up of 49 months, the patients showed an overall response rate of 94% (including a complete response rate of 68% and a partial response rate of 26%). The median overall survival (OS) of the patients was 71 months (95% CI: 36-106 months) with a median progression-free survival (PFS) of 47 months (95% CI: 30-64 months). Univariate analysis identified the use of rituximab, radiotherapy, and an international prognostic index (IPI) score of 0-2 as the favorable prognostic factors for OS and PFS. Multivariate analysis showed that the use of rituximab and IPI score were independent prognostic factors of the OS and PFS, and radiotherapy was the predicting factor for PFS but not for OS. CONCLUSION: The use of rituximab can improve the OS or PFS of patients with PBL, and radiotherapy offers additional benefits for PFS but not for OS.

[Decitabine for treatment of myelodysplastic syndrome in an elderly patient and review of literature].

Findings in epigenetic changes in meylodysplastic syndromes (MDS) and the development of demethylating drugs provide a new approach to the treatment of MDS. We used standard-dose decitabine for treatment of MDS in an elderly patient with an International Prostate Symptom Score (IPSS) of moderate risk group 2, and achieved a complete response in the first course. We report our experience with this case and review the relevant literatures.