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Objective:To construct a transposon mutation library and screen new virulence genes of hypervirulent Klebsiella pneumoniae(hvKp). Methods:The transposon mutation library was constructed and treated with human serum. The changes in the abundance of the genes of library mutant strains were analyzed by Transposon sequencing (Tn-seq). Besides, KEGG (kyoto encyclopedia of genes and genomes) annotation and enrichment analysis were performed on the screened genes.Results:A total of 405 genes were screened out according to the abundance of the genes in the library treated with human serum was 20% lower than that without treated, and 351 genes, 86.7% of these genes were conserved in HS11286, NJST258_1, NTUH-K2044 and RJF293. Ten genes existed in strains NTUH-K2044 and RJF293 with high virulence, while these genes were absent in HS11286 and NJST258_1 with low virulence. The mutants with genes such as glycosyl transferase gene wzy, aggregator protein gene wzi and capsule transporter gene wza, which belong to the capsule polysaccharide gene clusters, could not be detected after serum treatment. The abundance of iron carriers gene clusters such as aerobacterin and salmonellin in each library changed less than one time. KEGG annotation results showed that most annotated genes were involved in amino acid metabolism, cofactor and vitamin metabolism, carbohydrate metabolism, etc. Conclusions:Tn-seq is a reliable method to screen functional genes. In this study, 405 candidate virulence genes of hvKp were successfully screened out, providing an experimental basis for further research on the function and regulation mechanism of new virulence genes of hvKp.
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Pure red cell aplasia (PRCA) is a well-recognized complication of ABO major mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), with a reported incidence of 10%-20% (Zhidong et al., 2012; Busca et al., 2018). It is clinically characterized by anemia, reticulocytopenia, and the absence of erythroblasts in a normal-appearing bone marrow biopsy (Shahan and Hildebrandt, 2015). The mechanism for PRCA has been presumed to be persistence of recipient isoagglutinins, produced by residual host B lymphocytes or plasma cells, which can interfere with the engraftment of donor erythroid cells (Zhidong et al., 2012). Several risk factors of PRCA at presentation are known, such as presence of anti-A isoagglutinins before transplantation, reduced intensity conditioning, absence of acute graft-versus-host disease (GVHD), sibling donors, and cyclosporin A (CsA) as GVHD prophylaxis (Hirokawa et al., 2013). PRCA is not considered to be a barrier to HSCT, as some patients can recover spontaneously or benefit from various approaches including high-dose steroids, erythropoietin (EPO), plasma exchange, immunoadsorption, donor lymphocyte infusion (DLI), treatment with rituximab, bortezomib, or daratumumab, and tapering or discontinuation of immunosuppression (Hirokawa et al., 2013; Bathini et al., 2019). However, there are still some patients who fail to respond even to aggressive treatment; they become red cell transfusion-dependent and iron-overloaded, and their life quality is impaired.
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Objective@#To study the effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia (AL) and its significance as molecular marker to dynamically monitor minimal residual disease (MRD) .@*Methods@#Retrospectively analyzed those AL patients who underwent allo-HSCT in the First Hospital Affiliated to Zhejiang University School of Medicine during Jan 2016 to Dec 2017, a total number of 314 cases, 163 males and 151 females, median age was 30 (9-64) years old. Comparing the difference of WT1 expression at diagnosed, pre-HSCT and after HSCT. Using the receiver operating characteristic (ROC) curve to determine the WT1 threshold at different time so as to predict relapse. The threshold of WT1 expression before transplantation was 1.010%, within 3 months after HSCT was 0.079% and 6 months after HSCT was 0.375%. According to these thresholds, WT1 positive patients were divided into low expression groups and high expression groups. Analyzed the relationship between overall survival (OS) , disease-free survival (DFS) , cumulative incidence of relapse (CIR) and WT1 expression.@*Results@#The OS and DFS of high expression group pre-HSCT were lower than low expression group [69.2% (9/13) vs 89.1% (57/64) , χ2=4.086, P=0.043; 53.8% (7/13) vs 87.5% (56/64) , χ2=9.766, P=0.002], CIR was higher than low expression group [30.8% (4/13) vs 7.8% (5/64) , P=0.017]. There was no significant difference of OS and DFS between high expression and low expression group of 3 months after HSCT (P=0.558, P=0.269) . The OS and DFS of high expression group of 6 months after transplantation were both lower than low expression group (P=0.049, P=0.035) . Multivariate analysis showed that WT1>0.375% when 6 months after transplantation was the only independent prognostic factor for shorter DFS (P=0.022) . There was no statistically significant difference in CIR between the high-expression group and the low-expression group 3 months after transplantation and 6 months after transplantation (P=0.114, P=0.306) .@*Conclusion@#High expression of WT1 before and after HSCT was an adverse prognosis factor. It is of clinical practical value to use WT1 as a transplant recommendation index for patients with acute leukemia and as a marker to monitor MRD dynamically.
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With the progress of medical technology, the development of new drugs and the improvement of the therapeutic effect of graft-versus host disease in the last two decades, the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) have been greatly improved. However, graft failure is still a rare but serious complication of allo-HSCT. HLA incompatibility, virus infection, elderly donor, uncontrolled primary disease, damage of bone marrow hematopoietic microenvironment, ABO blood group incompatibility, T cell depletion, reduced intensity conditioning, and low nucleated cell number are all risk factors for graft failure. In recent years, with the implementation of HLA haplo-identical hematopoietic stem cell transplantation, the role of donor-specific antibodies in graft failure has attracted attention increasingly. This article reviews the recent studies involving the mechanism, risk factors and prevention measures of graft failure in allo-HSCT.
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Humans , Graft vs Host Disease , Epidemiology , Hematopoietic Stem Cell Transplantation , Risk Factors , Transplantation Conditioning , Transplantation, Homologous , Reference StandardsABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of acrylonitrile on T lymphocyte subsets, expression of toll-like receptor 4 and related cytokines in rats.</p><p><b>METHODS</b>Sixty-four Sprague-Dawley rats were randomly divided into 4 female groups and 4 male groups, and there were 8 rats in each group. Rats in each group were respectively given a single dose of 0, 5, 10 and 20 mg/kg acrylonitrile by gavage, once a day, 5 days a week, for 13 weeks. Blood and spleen T lymphocyte subsets was detected by flow cytometry, the mRNA expression of TLR4, IL-1β and TNF-α was analyzed by real-time quantitative PCR, the protein expression of TLR4 was evaluated by Western blot.</p><p><b>RESULTS</b>Compared with control group, the percentages of blood CD3, CD4 T cells in 20 mg/kg female group and CD4/CD8 ratio in 5, 10 and 20 mg/kg female groups was significantly decreased, CD8 T cells in 20 mg/kg group was significantly increased (P < 0.05 or P < 0.01), blood CD3 T cells in 5 mg/kg male group, CD4 T cells and CD4/CD8 ratio in 20 mg/kg male groups were lower than that of control group, CD8 T cells in 20 mg/kg make group was significantly in oreased (P < 0.05 or P < 0.01). Spleen CD4, CD8 T lymphocyte percentages and CD4/CD8 ratio in 20 mg/kg female group decreased significantly, CD8 T cells in 20 mg/kg male group was significantly increased (P < 0.05 or P < 0.01), spleen CD3, CD4, CD8 T cells in 20 mg/kg male group and CD4/CD8 ratio in 10, 20 mg/kg male groups was also significantly decreased, CD3 T cells in 20 mg/kg and CD8 T cells in 10, 20 mg/kg male groups were significantly increased (P < 0.05 or P < 0.01) (TLR4 mRNA was lower expressed in 5, 10 and 20 mg/kg male groups and 10 mg/kg female group (P < 0.05 or P < 0.01), and TLR4 protein in 5 mg/kg female group and 20 mg/kg male group was significantly lower than control group (P < 0.05). The expression level of IL-1β mRNA was significantly decreased in 5, 10 and 20 mg/kg female group and 5, 10 mg/kg male group (P < 0.05 or P < 0.01), TNF-α mRNA was lower expressed in 10, 20 mg/kg female groups and 5, 10 mg/kg male groups (P < 0.01).</p><p><b>CONCLUSION</b>Acrylonitrile may lead to the changes of CD3, CD4, CD8 T lymphocyte percentages and CD4/CD8 ratio in rat blood and spleen, and also significantly effected the expression level of TLR4 mRNA and protein together with the secretion of IL-1β, TNF-α. This may cause effects on the cellular immune function.</p>
Subject(s)
Animals , Female , Male , Rats , Acrylonitrile , Toxicity , Interleukin-1beta , Metabolism , Rats, Sprague-Dawley , T-Lymphocyte Subsets , Allergy and Immunology , Toll-Like Receptor 4 , Metabolism , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Objective To determine the frequency of Toll-like receptor 4 (TLR4)polymorphisms Asp299Gly and Thr399Ile in a cohort of hematopoietic stem cell transplant recipients and their donors in China.Method We examined the polymorphisms in 208 peripheral blood samples collected from 104 recipients and their donors in a single center between 2007-2012 in Zhejiang Province,China,and Asp299Gly and Thr399Ile TLR4 gene polymorphisms were detected using the sample DNA amplification products direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.Result Both methods didn't demonstrate TLR4 polymorphisms Asp299Gly or Thr399Ile base mutation in our samples.Conclusion The TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in our part of the population of hematopoietic stem cell transplantation.
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Objective To explore the relationship between tumor necrosis factor (TNF) gene polymorphisms in donors and recipients and the incidence and severity of acute graft-versus-host diseases (aGVHD) after unrelated allogeneic hematopoietic stem cell transplantation (alIo-HSCT). Methods Single nucleotide polymorphisms (SNPs) of TNFα-238 (G/A), TNFα-857 (C/T), TNFα-863 (C/A), TNFα-1031 (T/C), TNFβ + 252 (A/G) were analyzed by Multiplex SNaPshot analysis in 76 pairs of donors and recipients. Results Transplantation involving donors with TNFα-857 CC genotype resulted in a higher incidence of grade Ⅱ-Ⅳ aGVHD than donors with CT genotype (91.3% vs 8. 7% , P =0. 039). In the 23 patients with grade Ⅱ-Ⅳ aGVHD, no patients had TNFβ +252 AA genotype, 19 (82.6%) had GA genotype and 4 (17.4%) had GG genotype. There was a significant difference in the distribution pattern of the TNFβ +252 (AA, GA and GG) genotypes in these patients (P =0.03). There was no significant association of TNFα-238 (G/A), TNFα-863 (C/A) and TNFα-1031 (T/C) polymorphisms with the risk of aGVHD. Conclusion These results suggest donor TNFα-857 CC genotype is related to a higher incidence of grade Ⅱ -Ⅳ aGVHD, and patients with TNFβ +252 AA genotype have protection against the risk of grade Ⅱ -Ⅳ aGVHD.
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<p><b>OBJECTIVE</b>To prepare a monoclonal antibody against human telomeric repeat binding factor 1 (TRF1) protein and explore its biological characteristics.</p><p><b>METHODS</b>BALB/c mice were immunized with GST-TRF1(33-277) fusion protein for the preparation of monoclonal antibody by hybridoma technique. The obtained antibody was used for clinical assay by Western-blot and immunohistochemical staining.</p><p><b>RESULTS</b>One strain of hybridoma was obtained. It was confirmed by Western-blot that the antibody specifically recognized the 60 kD TRF1 protein. Immunohistochemical staining of the antibody showed that TRF1 protein located in the cytoplasm of epithelial cells and bone marrow cells.</p><p><b>CONCLUSION</b>A TRF1 monoclonal antibody, with high specificity was developed. It is useful for detection of TRF1 protein in tissue specimens.</p>