Objective:To study the cytotoxic effects of granulysin on Candida albicans.Methods: Candida albicans were cultured with different concentrations of granulysin peptides and the colonies of Candida albicans on Sabouraud dextrose agar were calculated.Broth microdilution method was used to determine the minimum inhibitory concentrations(MIC) of granulysin and fluconazole on Candida albicans.Results: When granulysin peptides 2 and 3(corresponding to G2 and G3 peptides) were at 20 ?g/ml,the average colonies of Candida albicans decreased from 838 and 927 to 203 and 218,respectively;G1,G4 and G5 did not reduce the average colony of Candida albicans even at 40 ?g/ml.Three strains of Candida albicans were sensitive to granulysin,with their MICs being 26,22,29 ?g/ml,and their MICs to fluconazole were 4,20 and 128 ?g/ml.Conclusion: Granulysin has cytotoxic effects on Candida albicans and is one of the natural anti-fungi proteins in human body;it has a promising future for anti-fungi drug development.

Objective:To investigate the interactions between natural killer(NK) cells and Cryptococcus neoformans(C.neoformans) in patients with cryptococcosis,so as to pave a way for treatment and prevention of cryptococcosis.Methods: The peripheral blood samples of 40 cryptococcosis patients and 40 healthy controls were collected.Expression of CD2,CD3,CD4,CD8,CD28,CD18,CD19,and CD56 in patients peripheral blood mononuclear cells(PBMC) were detected by FACS.Cytotoxic activity of NK cells was analyzed by MTT using K562 cells as target cells and the influence of IFN-? and IL-2 on NK cell activity was also studied.The level of IFN-? in the culture supernatant was assayed by ELISA and the cytotoxic activity of the supernatant was determined.The transcription levels of perforin,granzyme B and granulysin were examined by quantative real-time PCR.C.neoformans and NK cells were cocultured to investigate the inhibition of NK cells on C.neoformans.Results: Compared with that in healthy controls,CD56~+ cells decreased significantly(P

Objective:To study the effects of glucose,mannose and galactose on CAP10 promoter activity of Cryptococcus neoformans capsule-associated gene.Methods: Yeast cells were transfected with plasmid containing a 951 bp length of 5′ upstream flanking sequence of CAP10 coding region and a reporter gene,chloramphenicol acetyl transferase(CAT);the transfectants were treated with different concentrations(10,20,40,60,80 mg/ml) of glucose,mannose and galactose.CAT activity was assessed by ELISA method and CAT activities of different groups were compared.Results: Different concentrations of glucose and mannose had no obvious influence on CAT activity;different concentrations of galactose had obvious influence on CAT activity and the influence was positively dependent with its dose within the experimental concentration range.Conclusion: Glucose and mannose have no obvious effect on the activity of CAP10 promoter;galactose has obvious inductive effect on activity of CAP10 promoter,suggesting that CAP10 gene might be related with galactose metabolism.

Objective:To analyze the effect of lymphocyte in patients with psoriasis on proliferation of keratinocytes.Methods:Lymphocytes in lesion and peripheral blood were isolated and multiplied,then cultured together with normal keratinocytes. By MTT method,the living cells were counted in the mixed culture.Results:Compared with normal controls, lymphocytes in lesion and peripheral blood of psoriasis both promote the proliferation of keratinocyte(P

Objective:In view of analysis of role of autoantigen Dsg3 in specific T cell response to understand the molecular mechanism of autoimmune diseases.Methods:Based on the sequence analysis of autoantigen Dsg3,the five fractions cDNAs of Dsg3 were cloned and Dsg3-GST fusion proteins induced by IPTG in E.coli.JM109 were purified,then mixed-cultured with T cell from PV patients.The T cell proliferation response were analyzed by MTT.Results:Dsg3 E1,E2 and E4,E5 stimulated T cell from PV patients,not controls.Conclusion:Dsg3 E1,E2 and E4,E5 contained the epitopes relevant to T-B cell interaction,which play an important role in the pathogenesis of pemphigus vulgaris.

AIM:To cultivate the exoerythrocytic stage of Plasmodium yoelii in vitro and to study some involved affecting factors.METHOD:In vitro cultivation.RESULTS:The monolayer hepatocytes grown in 1 6 - mm plastic cell culture dishes were inoculated with sporozoite sus- pension prepared from Anopheles stephensi mosquitoes for48hours.At a final density of2? 1 0 4 cells per well,the infection rate of hepatocyte,cultured in medium supplemented wit15 % bovine serum,was 0 .0 35? 0 .0 1 3% ,the diameter of the nearly mature EEF of Plas- modium yoelii was up to40 .3? 31 .6 ?m,and contained more than1 0 0 nuclei,the number of EEF might be4- 1 0 /cm2 .An intraperitoneal inoculation of the EE schizonts to mice could induce parasitemia.At a final density of0 .5? 1 0 4 or4? 1 0 4 cells per well or the hepatocytes cultured in medium supplemented with1 0 % bovine serum,no EEF could be observed.CON- CL USION:The density of hepatocytes and culture medium are important for the cultivation of the EE stage of Plasmodium yoelii.This procedure will lay foundation for the further studies of the sporozoite invasion,the development of EEF and the affecting factors in- volved.

Objective To investigate the application of PBL teaching mode in laboratory diagnosis teaching. Methods 80 clinical medicine undergraduate students were divided into two groups. Tradition lecture was conducted to control group in laboratory diagnosis teaching,while PBL teaching mode PBL teaching mode was carried out to experiment group,and then the mastery of knowledge and appreciation on these two teaching modes was compared. Results The students in the experiment group mastered knowledge better than those in the control group,and the gratification of appreciation on PBL teaching mode was higher than that on the control group. Conclusion The activeness,energeticity,ability of self-study and combination diathesis of the students were improved through PBL teaching. There was good mutual communication between teacher and students and the teaching quality was improved.

Objective To explore the expression of T cell immunoglobulin and mucin-3(TIM-3)in peripheral blood mononu-clear cells from patients with primary Sj¨ogren’s syndrome(pSS)and its clinical significance.Methods Enrolled 33 pSS pa-tients from Shanghai Changzheng Hospital and Changhai Hospital in October 2012 and July 2013.The relative expression of TIM-3 in the peripheral blood mononuclear cells(PBMC)in 33 patients and 33 healthy individuals was detected by RT-PCR. The association between TIM-3 mRNA level and clinical characteristics were analyzed,for example,serum RF,CRP and anti-SSA/SSB antibody.1 2 of the pSS patients were followed up and their TIM-3 mRNA level was determined after glucocorti-coid treatment for 2 months.Results The expression of TIM-3 in PBMCs from patients with pSS was increased significantly than that in healthy individuals (0.55±0.12 vs 0.13±0.10,t=15.44,P<0.000 1),but its expression between anti-SSA/anti-SSB positive and negative patients were not statistical difference (0.45±0.21 vs 0.54±0.31;0.46±0.15 vs 0.51± 0.24).The TIM-3 mRNA expression in pSS patients was positively correlated with serum RF and CRP (R=0.558 5,R=0.414 7,P>0.05),but was not associated the anti-SSA/SSB antibody (P=0.298 2).TIM-3 mRNA level was decreased in the patients who were treated with glucocorticoids for 2 months (0.62±0.10 vs 0.38±0.13,t=5.07,P<0.05).Conclusion TIM-3 may play a role in the pathogenesis of pSS and it may be regarded as a biomarker to pSS.

Objective To analyze the affection and clinical significance of CD26/DPP4 on CD4+T cells and its cytokines in patients withCrytococcalMeningitis.Methods Peripheral blood was collected from 36 patients diagnosed withCrytococcal Meningitis in Changhai Hospital and Changzheng Hospital,Shanghai from August,2011 to December,2015,meanwhile 36 health controls’was also acquired.Peripheral blood mononuclear cell (PBMC)was separated by density gradient centrifuga-tion,CD26+CD4+T and CD26-CD4+T cell groups were classified by Flow Cytometry,the expression level of cytokines was tested by reverse transcriptase-polymerase chain reaction (RT-PCR).The correlation between DPP4 activity,CD26+CD4+T (%)and APACHE II score,IL-17,TNF-α,IL-4,IFN-γwas measured by Pearson coefficient.Results CD26+CD4+T(%)between experimental and control groups was 13.35±3.83 vs 8.39±2.14 (t=6.78,P<0.000 1).DPP4 activity was 50.89±17.21 mU/ml vs 73.83±20.24 mU/ml (t=5.18,P<0.000 1),with statistically significant differences.In ex-perimental groups,CD26+CD4+T (%)was positively related with APACHE II score,IL-17,TNF-α(r=0.431,0.564, 0.688,P=0.003 8,0.001,0.004 6).DPP4 activity was negatively interrelated with APACHE II score,IL-17,TNF-α,IFN-γ(r=-0.544,-0.489,-0.678,-0.734;P<0.001).Conclusion CD26/DPP4 may be involved in the pathogenesis of Crytococcal Meningitis through regulation of Th subgroups,and it was the potential therapeutic target and the predicted marker of the disease.

Objective To test the expression of MIF in peripheral blood mononuclear cells (PBMCs)from patients with Cry-tococcal Meningitis and further discuss its clinical significance.Methods Peripheral blood from 42 patients with Crytococcal Meningitis diagnosed in Changhai Hospital,Shanghai and 42 healthy individuals examined at the same time was collected from August,2012 to November,2015.PBMCs were separated by density gradient centrifugation method,mRNA relative expression of MIF in PBMCs was measured by PCR,the level of MIF,IL-17,IL-1β,TNF-α,IFN-γand IL-4 in plasma was tested by Enzyme-linked immunosorbent assay (ELISA).The comparison for expression level of cytokines between the two groups was by two-independent samples t test.Pearson correlation coefficient was used to measure the relation between MIF and other cytokines.Results The protein levels of MIF in experimental and controlled groups were 34.17±7.88 ng/ml vs 10.89±2.76 ng/ml(t=18.07,P<0.0001),while relative expression of RNA was 2.87±0.94 vs 1.95±0.89(t=4.606,P<0.0001),and there was statistical significance (P<0.005).Pearson correlation analysis showed that MIF was positively related with IL-1β,IL-17 (r=0.467,0.401,P<0.01),with statisticaldifference.Conclusion MIF may involve in the im-mune regulation for Crytococcal Meningitis by affecting the secretion and function of cytokines as IL-1β,IL-17,and it was potential target and monitored biomarker for this disease.