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 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.

Objective To observe the quantity change of autophagosomes in podocytes and expressions of autophagy-related gene Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3) in different pathological stages of idiopathic membranous nephropathy (IMN),and to explore how autophagy is related to podocyte injury,the occurrence of proteinuria and the disease progression in IMN.Methods Clinical data of 26 patients who were diagnosed as IMN (14 IMN stage 1 and 12 IMN stage 2) admitted to Zhejiang Provincial people's Hospital from January 2013 to December 2014 were retrospectively analyzed.Normal renal tissue from 15 cases of kidney neoplasms with nephrectomy was collected as control.The changes of kidney tissue pathology were detected after PAS and PASM staining by light microscope.The autophagosomes of podocyte were detected by transmission electron microscopy.Expressions of Beclin-1 and LC3 protein were detected by immunohistochemistry.Expressions of LC3 and synaptopodin were detected by immunofluorescence.The correlation of autophagosomes and clinical pathologic factors in IMN patiens was analyzed.Results There were fewer autophagosomes of podocytes and lower expression of Beclin-1 and LC3 protein in IMN group than those in control group (P=0.034,P=0.011,P=0.013,respectively).Moreover,these effects were more obvious with the development of IMN.Compared with those in control group,autophagosomes,Beclin-1 and LC3 protien were reduced in IMN stage 2 group (P=0.009,P=0.030,P=0.015);the number of autophagosomes and the expressions of LC3 and Beclin-1 were decreased in IMN stage 1 group as well,however statistically insignificant (P=0.352,P=0.087,P=0.128);Comparisons between IMN stage 2 patients and IMN stage 1 patients shown significant difference in the number of autophagosomes (P=0.030),but no significant difference in expressions of Beclin-1 and LC3 (P=0.355,P=0.181).Autophagosomes number was not correlated with serum creatinine,serum urea nitrogen,24-hour urinary protein and eGFR (all P ＞ 0.05).The expressions of synaptopodin and LC3 protein were lower in IMN group than those in control group.Conclusion Autophagy may contribute to podocyte injury and the production of protein urine in IMN,and may be closely related to the progression of disease.

[ 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.

Objective To determine the long term outcomes of laryngomalacia infants with anomalies and to determine the clinical practice guideline for these infants.Methods The charts of infants with moderate to severe laryngomalacia,who were admitted to our hospital between January 2013 and December 2015,were retrospectively reviewed.These infants were divided into two groups,anomaly(A) group(n=37) and non-anomaly (NA) group(n=19).Results Fifty-six cases were enrolled.Infants in A group were older at symptom relief than those in NA group[(10.00±3.56) months vs.(7.89±3.03) months,P＜0.05],and the weight percentiles of infants in A group were lower at 3,6 and 12 months than those in NA group(P＜0.05).There was no statistically significant difference between the two groups on the weights percentiles in infants at 24 months after diagnosis.Five of 37 cases in A group and 3 of 19 cases in NA group had supraglottoplasty.One infant in A group had tracheotomy.Conclusion Both breathing difficulty and development retardations of infants with moderate or severe laryngomalacia could gradually improved with age.There is not enough evidence to support the aggressive supraglottoplasty for infants with anomalies and laryngomalacia.

Objective:To investigate the clinical value ofplatelet distribution width ratio (RDW) and neutrophil lymphocyte ratio (NLR) in the prediction of coronary no reflow in patients treated with acute percutaneous coronary intervention (PCI).Methods:66 patients underwent emergency PCI treatment in our hospital from January 2016 December 2016 were selected as the research object,RDW and NLR of the patients before treatment were detected,and coronary no reflow phenomenon after emergency PCI rate were statistically calculated.RDW and NLR of patients with no reflow and normal blood flow were compared,the relationship between RDW and NLR with no reflow and the value of them predicting coronary no reflow were analyzed.Results:The incidence of coronary no reflow after emergency PCI was 30.30％ (20/66).Compared with patients with normal blood flow,the RDW and NLR of patients with coronary no reflow were elevate (P＜0.05).Spearman correlation analysis results showed that RDW and NLR were positively correlated with coronary no reflow (r=0.826,0.878,P＜0.05).Further ROC curve analysis showed that AUC of preoperative RDW combined with NLR predicting the postoperative coronary no reflow in patients treated with emergency PCI were higher than RDW and NLR alone.Conclusion:RDW and NLR in patients treated with emergency PCI were closely related to the postoperative coronary no reflow,which might be used as reference index for evaluation of coronary no reflow after emergency PCI.