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Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.
Subject(s)
Animals , Mice , Depsipeptides , Endoribonucleases , Hematopoietic Stem Cell Transplantation , Histone Deacetylase Inhibitors/pharmacology , Protein Serine-Threonine Kinases , Skin TransplantationABSTRACT
Objective:To explore a new method of diagnosing and identifying renal transplantation clinical almost tolerance through a diagnostic model using plasma proteomics.Methods:From November 2011 to November 2012, plasma samples from 43 subjects were collected and divided into the groups of clinical almost tolerance(18 cases), rejection(12 cases)and healthy control(13 cases). Protemic analysis of plasma samples was performed by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS). Differential mass spectrometry peaks were screened and diagnostic model was established by Biomarker Wizard and Biomarker Pattern software. Identification of mass spectrometric peaks of nodes in the diagnostic model was carried out by searching the databases of SWISS-PROT and TrEMBL using ExPASy's TagIdent tool.Results:A total of 21 differential proteins peaks were obtained( P<0.05). Diagnostic model was composed of five mass spectrum peaks of 2 565.15, 1 966.28, 6 674.78, 1 103.27, 1 716.69 and 1 966.28.The sensitivity, specificity and area under the ROC curve of model were 83.3%, 92.0% and 0.951 respectively for diagnosing clinical almost tolerance.Bioinformatic identification results of mass spectrometric peaks of nodes in model were proteins of ANFB, MCH, TFF1, PDYN and PSME3. Conclusions:Establishing a diagnostic mode by plasma proteomics may be effectively employed for diagnosing clinical almost tolerance in kidney transplant.
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Objective To analyze the common reasons for misdiagnosis of atypical pulmonary embolism (APE) ,and to im‐prove the identification of APE .Methods The risk factors ,clinical manifestations ,laboratory examinations and radiographic data of 120 cases of APE diagnosed from January 2006 to December 2013 in the department of cardiovascular medicine and respiratory medicine of Xinqiao Hospital and the Affiliated Hospital of Luzhou Medical College were studied retrospectively .Results Among those 120 cases of APE ,39 cases were misdiagnosed on admission (32 .5% ) .8 cases were misdiagnosed as acute coronary syn‐drome ,7 cases as stable angina pectoris ,7 cases as chronic cor pulmonale ,5 cases as pneumonia ,3 cases as pleural effusion ,3 cases as tuberculosis ,3 cases as asthma ,1 case as atrial septal defect ,1 case as acute heart failure ,and 1 case as cardiogenic syncope .Con‐clusion APE is easy to be misdiagnosed for its non‐specific clinical manifestation .Pulmonary enhanced CT or CTPA should be car‐ried out in time for those highly suspected patients ,in order to reduce the misdiagnosis of APE .
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<p><b>OBJECTIVE</b>To investigate the differences in the gene expression profiles of the peripheral blood immune cells between liver and kidney transplantation recipients.</p><p><b>METHODS</b>A dataset containing the gene expression profiles of 27 liver transplantation recipients and 25 kidney transplantation recipients (from GSE22229 and GSE28842, respectively) was downloaded from the GEO database. By combining gene set enrichment analysis (GSEA) and biological network analysis of the differentially expressed genes using Cytoscape software, we analyzed the core genes closely related to liver or kidney transplantations.</p><p><b>RESULTS</b>GSEA identified 20 highly overlapping genes for liver transplantation and another 20 for kidney transplantation using leading edge analysis. Fourteen hub nodes (gene) for liver transplantation and 13 for kidney transplantation were identified by cytoscape software using interaction network analysis. Five core genes related to liver transplantation and 5 to kidney transplantation were obtained by integrating GSEA and biological network analysis.</p><p><b>CONCLUSION</b>Controlling the transcription and translation of the genes of the peripheral blood immune cells is the main immune regulation mechanism in liver transplantation recipients, but in the recipients of kidney transplantation, the protein interaction network plays a more prominent role. Energy metabolism and functional regulation of the immune cells are closely related. The core genes in peripheral blood immune cells related to liver or kidney transplantation may play key roles in regulating immune functions.</p>
Subject(s)
Humans , Gene Regulatory Networks , Kidney Transplantation , Liver Transplantation , Oligonucleotide Array Sequence Analysis , TranscriptomeABSTRACT
Objective To observe the pathologic features on cardiac allograft and to test archived endomyocardial biopsy specimens for antibody-mediated rejection specific marker-C4d deposition and its characteristics by using immunoperoxidase (IP) techniques. Methods From January 2003 to December 2007,10 recipients underwent orthotopic cardiac transplantation and 17 specimens of endomyocardial biopsy were obtained either for a protocol basis (generally at 1 st month,3rd month,1st year and 2nd year post-transplant) and on immediate clinical indications.All specimens of endomyocardial biopsy were collected for histopathological examination and C4d immunohistochemical staining,simultaneously. All pathological diagnoses were done according to 2004 International Society for Heart and Lung Transplantation (ISHLT) recommendation working formulation and AMR Schema,and C4d staining intensity were graded and recorded as 0 to 3 +.Results Except 1 specimen unqualified,all 16 consecutive specimens of endomyocardial biopsy were qualified.There were 4 cases of acute T cell-mediated rejection (all graded 1 ),2 cases of Quilty lesion,and 7 cases of antibody-mediated rejection,who were documented according to ISHLT Schema and C4d deposition.Meanwhile,there were 6 cases showing evidence of antibody-mediated rejection without concurrent acute cellular rejection and only one case concordant with acute T cell-mediated rejection.One case of antibody-mediated rejection died 20 months posttransplantation due to combined transplant coronary artery disease (TCAD). The C4d in the cardiac allograft was deposited in microvasculature diffusively.Conclusion Antibody-mediated rejection is an important clinical entity following orthotopic heart transplantation and is difficult to diagnosis except to perform endomyoeardial biopsy.Immunoperoxidase staining for C4d is a sensitive and specific technique for detecting one marker of antibody-mediated rejection.
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In vivo imaging system (IVIS) is a new and rapidly expanding technology, which has a wide range of applications in life science such as cell tracing. By counting the number of photons emitted from a specimen, IVIS can quantify biological events such as tumor growth. We used B16F10-luc-G5 tumor cells and 20 Babl/C mice injected subcutaneously with B16F10-luc-G5 tumor cells (1x10(6) in 100 muL) to develop a method to quantitatively analyze cells traced by IVIS in vitro and in vivo, respectively. The results showed a strong correlation between the number of tumor cells and the intensity of bioluminescence signal (R (2)=0.99) under different exposure conditions in in vitro assay. The results derived from the in vivo experiments showed that tumor luminescence was observed in all mice by IVIS at all days, and there was significant difference (P<0.01) between every two days from day 3 to day 14. Moreover, tumor dynamic morphology could be monitored by IVIS when it was invisible. There was a strong correlation between tumor volume and bioluminescence signal (R (2)=0.97) by IVIS. In summary, we demonstrated a way to accurately carry out the quantitative analysis of cells using IVIS both in vitro and in vivo. The data indicate that IVIS can be used as an effective and quantitative method for cell tracing both in vitro and in vivo.
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Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9's pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.
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Objective To explore the expression level of Tim-3,the marker of activated T_H 1 cells.in T lymphocytes in different sites from recipients with acute rejection.Methods The model of cervical heterotopic heart transplantation was established in mice Two groups were get up:the isograft group(C57BL/6→C57BL/6) and the allograft group (Balb/c→C57BL/6).Lymphocytes were isolated from peripheral blood,spleens,draining lymph nodes and grafts 3 or 6 days after transplantation.The expression of TIM-3 in CD4~+ and CD8~+ T subsets was detected by flow cytometry.Results There was no significant difference in Tim-3~+/CD4~+ and Tim-3~+/CD8~+ ratio in peripheral blood or spleens between two groups.As compared with the isograft group,the proportion of Tirn-3~+/CD4~+ cells was slightly elevated in draining lymph node(P<0.05),but the percentage of Tim-3~+/CD4~+ cells had no significant change between 3 days and 6 days in the allograft group(P>0.05).The expression of Tim-3 in CD4~+ and CD8~+ of graft infiltrating T cells was obviously increased in allograft group(P<0.01),and it was significantly (P<0.01) up-regulated on the 6th day as compared with that on the 3rd day.Conclusion The dynamic changes of Tim-3 expression in T lymphocytes in draining lymph node and graft were correlated with the progresston oi acute rejection in mice.
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To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.
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Objective To explore the subcellular localization of Galectin-9 and its effect on allogeneic immune response.Methods The plasmid pEGFP-N1 was inserted with Galectin-9 fragment which was amplified from pBKCMV-Galectin-9 by PCR.The recombinant plagmid wag then transfected into CHO cells using JetPEI in vitro.The cells were cultured in G418 selecting mediam to obtain the stably-transfected cells.The transcription and expression of Galectin-9 gene were verified by immunohistochemical staining and RT-PCR.The solid-phase transgenic CHO cells or freshly-cultured supernatant wag added into the mixed lymphocyte response system to detect the inhibitory effect of Galectin-9.Galectin-9 protein wag administered intraperitoneally for 7d consecutively.Results The expression of Galectin-9 wag localized in the cytosol of CHO.The allogeneic mix lymphocyte proliferation was dose-dependently inhibited by the freshly-cultured supernatant from stably-transfected CHO cells.Furthermore,the supernatant from stably-transfected CHO cells dose-dependently inhibited the level of IL-2.The inhibitory effect could be reversed by Tim-3-Fc blocking.Administration of Galectin-9 significantly prolonged the survival of allogeneic cardiac transplants[(22.7±1.2)d vs(7.2±0.4)d)].Conclusion Galectin-9 may be secreted in physical situation to exert its immunomodulatory function on allogeneic immune response.Furthermore.Galectin-9 may be a novel therapeutic drug in transplant medicine.
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Aim To investigate the protective effects and mechanism of astragaloside Ⅳ on hypertrophy induced by angiotensinⅡ(AngⅡ)in primary cultured cardiomyocytes of neonatal rats.Methods Cardiomyo-cytes were incubated with AngⅡ to establish the hypertrophy model of primary cultured cardiomyocytes in neonatal rats.AST 10 mg?L-1 or 20 mg?L-1 were added with AngⅡ simultaneously to observe its protective effects on cardiomyocytes.The diameter and the total protein content of cardiomyocyte were measured.The intracellular free calcium concentration([Ca2+]i),activity of sarcoplasmic reticulum Ca2+-ATPases(SERCA)and activity of calcineurin(CaN)were determined.Results Compared with control group,diameter and total protein content of cardiomyocyte induced by AngⅡ were increased significantly,which were inhibited by AST 10 mg?L-1 and 20 mg?L-1,respectively(P
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Residents(medical specialists)programmed education is an important aspect to fulfill the post-graduated medical education. It can remedy the clinical practice of young doctors,which ac-cords with the trend of international medical education .This article is to give advice for the resi-dents (medical specialists)programmed education according to the work and existing problems in our hospital.