BACKGROUND: To avoid acute rejection,it is necessary to use imunosuppressive drug regimen for long term to control immune state.However,imunosuppressive drug regimen of allogenic organ transplantation increases infection incidence of recipients,and induction of allograft immunological tolerance might be an ideal method for solving these problems.The long-term immunologic tolerance has been able to be induced in the experimental rodent models.Among these protocols,donor bone marrow cell (DBMC) infusion exerts an important role in the induction of allograft immunological tolerance.OBJECTIVE: To investigate effects of combination of cyclosporine A (CsA) with DBMC infusion on heterotopic rat cardiac allograft survival time.DESIDN: A randomized controlled animal experiment.SETTING: Renal Disease Center,First Affiliated Hospital of Zhejing University School of Medicine.MATERIALS: This study was performed at the Laboratory Animal Center,Zhejiang University School of Medicine between March 2002 and December 2005.Inbred male Lewis rats (n=40,serving as donors) and male BN rats (n=60,serving as recipients) of SPF grade were used in this study.The protocol was approved by the Hospital's Ethic's Committee.METHODS: Forty rats prepared for heterotopic rat cardiac allograft were randomly divided into 4 groups,with 10 rats in each: control group,in which,rats received no treatment,CsA group,in which,rats received CsA infusion for 7 days successively; CsA +DBMC group,in which,rats received DBMCs during and 6 days after the surgery and additional 7 successive days of CsA infusion,and a DBMC group,in which,rats received DBMCs infusion during and 6 days after the surgery.In addition,BN rats that received beterotopic rat cardiac allograft served BN controls.The survival time of heteroropic rat cardiac allograft was investigated.Serum interleukin-2 level and tumor necrosis factor-α mRNA expression level in the transplanted cardiac allograft were measured. The percentage of antigen presenting cells (APC) from donor,CD3+CD25+ cells,CD4+CD25+ cells,CD86+ cells,and the ratio for CD4+CD45RC+ and CD4+CD45RC- in the recipient peripheral blood karyocytes were measured by flow cytometry 6,12 and 18 days after surgery.MAIN OUTCOME MEASURES: The survival time of beteruropic rat cardiac allograft,serum interleukin-2 (IL-2)level,tumor necrosis factor- α (TNF- α ) rnRNA expression level, rejection grading,the percentage of DBMCs in the recipient peripheral blood karyocytes,CD3+CD25+ cells,and CD4+CD25+ cells,as well as CD86 expression,and the ratio for CD4+CD45RC+ and CD4+CD45RC.RESULTS: Forty Lewis male rats and sixty male BN rats were all included in the final analysis. The heterotopic rat cardiac allograft survival time was longer in the CsA +DBMC group than in the control group and DBMC group (P ＜ 0.05). Serum IL-2 level and TNF- α mRNA expression were respectively lower in the CsA +DBMC group than in the control group and DBMC group ( P ＜ 0.05).The rejection was milder in the CsA +DBMC group than in the remaining 3 transplantation groups.In the CsA +DBMC group,CD 86 expression in the recipient peripheral blood karyocytes was markedly inhibited,and 6 and 12 days after surgery,the ratio for CD4+CD45RC+ and CD4+CD45RC- and the percentage of CD3+CD25+ were respectively lower compared to control group and DBMC group.DBMCs in the recipient peripheral blood karyocytes were more in rats that received DBMC infusion compared to rats that received no BDMC infusion.CONCLUSION: Short-term CsA treatment combined with DBMC infusion can lower acute rejection of heterotopic rat cardiac allograft and prolongssurvival time of cardiac allograft.

Objective To investigate clinical and pathological features of idiopathic membranous nephropathy (IMN) accompanied by mesangial dense deposit.Methods Clinical data of 46 patients who were diagnosed as IMN accompanied by mesangial dense deposit admitted to Zhejiang Provincial People's Hospital from January 2013 to December 2014 were retrospectively analyzed.They were compared with those of 29 patients who were diagnosed as IMN without mesangial dense deposit during the same period in the hospital.Analysis of their clinical and pathological features was conducted.Results The IMN accompanied by mesangial dense deposit accounted for IMN 61.3％,and had more hyaline changes of arteriole (43.5％ vs 6.9％,P=0.001) and more obvious arteriolar wall thickening (78.2％ vs 51.7％,P=0.016) than IMN without mesangial dense deposit.Furthermore,the positive rate of IgA deposition in IMN accompanied by mesangial dense deposit was greatly higher than that in IMN without mesangial dense deposit (21.7％ vs 0,P=0.007).In other index,such as serum biochemical parameters,urine protein,glomerular lesion,tubulointerstitium pathological damage and other immunopathologic changes,no statistically significant differences were found between these two groups.Conclusions IMN patients accompanied by mesangial dense deposit have severe intrarenal artery lesions,and high positive rate of IgA deposition.

Objective To investigate the clinical and pathological features of idiopathic membranous nephropathy (IMN) in young patients.Methods Clinical data of 20 young patients, 16 to 44 years, who were diagnosed as IMN admitted to the Zhejiang Provincial People's Hospital from January 2013 to December 2014 were retrospectively analyzed, comparing to 55 mid-aged patients who were diagnosed as IMN during the same period in the hospital.Clinical and pathological features of above mentioned patients were analyzed.Results Young patients with IMN accounted for 26.7％ of IMN patients.Compared to mid-aged patients, young patients with IMN had lower proportion of hypertension (P=0.003), lower blood glucose level (P=0.010), higher estimated glomerular filtration rate (eGFR) and low-density lipoprotein (LDL) (P=0.012, P=0.038), and lower levels of T3 and T4 (P=0.030, P=0.034).Furthermore, there were less sclerosis glomeruli (P＜ 0.001), hyaline change of arteriole (P=0.040) and arteriolar wall thickening (P ＜ 0.0001), lower positive ratios of IgA (P=0.008),and more without renal tubulointerstitial lesions (P=0.018) in young patients.There were no statistically significant differences between these two groups in other index.Conclusions Compared to mid-aged patients, young patients with IMN have better blood pressure and blood glucose level, higher glomerular filtration rate and LDL.Moreover, thyroid function is significantly affected, meanwhile the lesions of glomerular, interstitial and vascular are mild in young patients.

Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.

OBJECTIVETo investigate the effect of four traditional Chinese medicines distributed along lung meridian, namely Ephedrae Hebra, Zingiberis Rhizoma, Scutellariae Radix and Mori Cortex, on TLR2 and NF-κB expressions in mice with lung heat syndrome, in order to study the intervention effect of the four traditional Chinese medicines (TCMs) on the lung heat syndrome.

METHODOne hundred KM mice were randomly divided into the normal control group, the model group, the Ephedrae Hebra group, the Zingiberis Rhizoma group, the Scutellariae Radix group and the Mori Cortex group (20, 10 g x kg(-1)), nasally dripped with streptococcus pneumoniae to establish the mouse lung heat syndrome model, and then administered with different TCMs. The expressions of TLR2, NF-κB p65 proteins in lung tissues were analyzed by the immunohistochemical method. The expressions of TLR2, NF-κB p65 mRNA were measured by real time PCR.

RESULTCompared with the normal control group, the expressions of TLR2 and NF-κB p65 proteins in lung tissues in the model group were higher (P < 0.01), and the expressions of TLR2 and NF-κB p65 mRNA in lung tissues were up-regulated (P < 0.01 or P < 0.05). Compared with the model group, Ephedrae Hebra high and low dose groups, the Zingiberis Rhizoma low dose group and the Scutellariae Radix high dose group showed decreased expression of TLR2 protein (P < 0.05 or P < 0.01); Ephedrae Hebra high and low dose groups, the Zingiberis Rhizoma low dose group, Scutellariae Radix high and low dose groups and Mori Cortex high and low dose groups showed reduced expression of NF-κB p65 protein (P < 0.05 or P < 0.01). Ephedrae Hebra high and low dose groups, Zingiberis Rhizoma high and low dose groups, Scutellariae Radix high dose group and Mori Cortex high dose group showed down-regulated expression of TLR2 mRNA (P < 0.01 or P < 0.05).

CONCLUSIONEphedrae Hebra, Zingiberis Rhizoma, Scutellariae Radix and Mori Cortex can induce the TLR2/NF-κB inflammatory signal pathways by down-regulating the expressions of TLR2 and NF-κB p65 in protein and mRNA, so as to alleviate the lung tissue injury in mice with lung heat syndrome.

Animals ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; classification ; pharmacology ; Female ; Gene Expression ; drug effects ; Immunohistochemistry ; Lung ; drug effects ; metabolism ; microbiology ; Male ; Medicine, Chinese Traditional ; methods ; Meridians ; Mice ; Phytotherapy ; methods ; Plants, Medicinal ; chemistry ; classification ; Pneumonia, Pneumococcal ; drug therapy ; genetics ; metabolism ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Scutellaria baicalensis ; chemistry ; Species Specificity ; Syndrome ; Toll-Like Receptor 2 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism

Objective To investigate the cell types of renal allograft glomerulitis in posttransplantation patients and its relationship with peritubular capillary (PTC) C4d deposition.Methods Fifty-one allograft kidney transplant recipients diagnosed as acute rejection from June 2006 to June 2009 in our center were enrolled in this retrospective study. Fifty-one biopsy specimens of these patients accompanied with glomerulitis were examined by immunostain for macrophages, T cells, granzyme B, Foxp3 and C4d. Glomeular macrophages, T cells, cytotoxic T cells, regulatory T cells were counted. The recipients were divided into C4d+ and C4d- groups according to the C4d deposition in peritubular capillaries. Results The total number of glomerular infiltrating cells in C4d+ group was significantly higher than that in C4d- group (17.79±7.70 vs 8.17±3.80, P＜0.01). The infiltrating cell was predominantly macrophage in CAd+ group and significantly higher as compared to C4d- group (13.73 ±7.03 vs 2.57 ±1.22, P＜0.01).However, the infiltrating cell was predominantly T cell in C4d- group and significantly higher as compared to C4d+ group (5.60±2.81 vs 4.05±2.60, P=0.023). The infiltrating T cells in both groups were predominantly cytotoxic T cells. Conclusions The total number of infiltrating cells in glomerulitis is related to peritubular capillary C4d deposition. The infiltrating cell is predominantly macrophage in C4d+ group and predominantly T cell in C4d- group. Meanwhile the infiltrating T cell in both groups is predominantly cytotoxic T cell.

Objective To investigate the cell types of renal allograft intimal arteritis in posttransplantation patients and its relationship with peritubular capillaries (PTCs)C4d deposition. Methods Twenty allograft kidney transplant recipients from Jun 2006 to Jun 2008 were enrolled in the retrospective study. Twenty-one biopsy specimens with acute vascular rejection were immunostained for macrophages, T cells and C4d. In each biopsy specimen, arterial intimal macmphages and T cells were counted, and mean number of macrophages per "artery and T cells per artery were determined. The recipients were divided into C4d + and C4d- group according to whether C4d deposition in the PTCs. Results In the intimal arteritis of biopsies diagnosed as acute vascular rejection, the infiltrating cells were predominantly macrophages including C4d+ and C4d- group. T cells were the minority. The mean macrophage number per artery cross-section was significantly higher than the mean T cell number per artery cross-section in the C4d + group (12.45±9.86 vs 3.91±3.03, P=0.007) and in the C4d- group (3.47±1.89 vs 1.45±1.37, P=0.006). Forther more, the mean number of macrophage per artery cross-section in the C4d+ group was significantly higher than that in the C4d- group (P=0.007). Conclusions In the intimal arteritis of biopsies diagnosed as acute vascular rejection, the infiltrating cells are predominantly macrophages which has relationship with peritubular capillary C4d deposition. The mean number of macmphage per artery cross-section in the C4d+ biopsies is significantly higher than that in the C4d- biopses.

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.

Objective To investigate the clinical and pathological characteristics of IgA nephropathy (IgAN) in association with active tubular interstitial lesions. Methods 148 patients who were diagnosed as IgAN by renal biopsy and admitted to Zhejiang Provincial People's Hospital from March 2014 to December 2014 were enrolled. They were divided into IgAN with active tubular interstitial lesions group (IgAN?ATIL group, 23 patients) and IgAN without active tubular interstitial lesions group (control group, 125 patients). Clinical and pathological characteristics were retrospectively analyzed. Multivariate logistic regression analysis was used to analyze the influence factors. Results The prevalence of ATIL in 148 IgAN patients was 15.5%. IgAN?ATIL group showed an older average age and more higher proportion of medication history (antibiotics, diuretics, nonsteroidal anti ?inflammatory drugs, etc) than those in control group. There were significant differences in Alb, eGFR, Scr, BUN, 24?hour urinary protein quantity, urinary NGAL and urinary RBC count between two groups (P<0.05, respectively). A moderate of tubulointerstitial lesions of IgAN?ATIL group was shown, while the control group was mainly mild lesions. Multivariate logistic regression analysis showed that age, medication history and the urinary NGAL level were independent risk factors of IgAN ? ATIL. Conclusions IgAN patients with active tubular interstitial lesions had more severe clinical manifestations and chronic interstitial lesions. The age, medication history (antibiotics, diuretics, nonsteroidal anti?inflammatory drugs, etc) and the urinary NGAL level were independent risk factors of IgAN?ATIL.

[ ABSTRACT] AIM:To investigate the effect of storage temperature in renal biopsy tissue frozen section on immu-nofluorescence results.METHODS:One hundred renal biopsy samples of Zhejiang Province People’ s Hospital from Janu-ary to June, 2015 were enrolled.Two sets of cutting slices were stored in -70 ℃ low temperature refrigerator as experi-mental group and in -12 ℃ freezing cryostat as control group.The immunoglobulins ( IgG, IgA and IgM) and comple-ments (C3, C4 and C1q) as well as fibrinogen were detected with direct immunofluorescence in the next day.The typeⅣcollagen a3 and a5 chains were also detected by indirect immunofluorescence, and the qualitative and semi-quantitative re-sults were observed under the fluorescence microscope.RESULTS:There were 16 cases of 3+~4+IgG in the experi-mental group, while the corresponding IgG was all 1+~2+in control group, significantly weaker than that in experimen-tal group.There were 99 cases of 3+~4+a3 and 3+~4+a5 in experimental group, white there were 92 cases of 1+~2+a3/a5 and 8 cases negative in control group.The positive intensity was decreased in control group with statistical difference between the 2 groups ( P<0.05) .There were 15 cases of 4+IgA, 19 cases of 3+IgA, 15 cases of 3+IgM, 11 cases of 2+IgM, 13 cases of 4+C3, and 12 cases of 3+C3 in experimental group.The control group were similar to the results of experimental group, and no difference between the 2 groups was observed.CONCLUSION:The immunoflu-orescence results of renal biopsy frozen sections are highly affected by the section storage temperature, which has greater in-fluence on the immunofluorescence positive intensity of IgG and typeⅣcollagen.The renal biopsy frozen section should be stored in -70 ℃low temperature refrigerator.