Objective To establish TaqMan probe real‐time Fluorescent Quantitative PCR in detecting Cryptococcus neoformans genomic DNA ,and to provide important method for detection of cryptococcal meningitis .Methods According to the Cryptococcus neoformans ITS‐rDNA sequences obtained from NCBI ,specific primers and probe were designed based on the conserved sequences , a specific 114 bp fragment was amplified by primers and probe ,then recombinant plasmid was constructed .Eight different concen‐trations from 1 .42 × 10 copy/μL to 1 .42 × 10 copy/μL were used as templates by 2 μL .In the optimum reaction condition ,the sen‐sitivity ,specificity and repeatability were evaluated and standard curve was established .15 clinical cryptococcal meningitis strains i‐solated from clinical diagnosis patients were detected .Results The real‐time PCR showed high sensitivity and specificity and was a‐ble to detect 2 .84 × 102 copies of plasmid DNA .The detection sensitivity was 2 .84 × 102 copies plasmid DNA by real‐time PCR ,no amplification curve was detected with human genomic DNA ,other fungus ,bacterias and viruses .The CV of inter‐assay were 2 .86% ,1 .48% and 1 .36% respectively with excellent reproducibility .Fifteen clinical isolated strains could be detected accurately . Conclusion A method of detection of Cryptococcus neoformans DNA by real‐time PCR is established successfully with high sensi‐tivity and specificity ,and the results are stable ,could diagnose cryptococcal meningitis rapidly and early .

Objective To investigate the influence of BK virus (BKV) activation in renal transplant recipients on the renal allograft function.Method Recipients receiving renal transplantation during 2010.3-2011.4 were sdected as objectives,the urine and peripheral blood samples of them were taken and real-time PCR assays were performed to detect BKV DNA at 0.5,1,3,6,9,and 12 months post-transplantation.Results Among 88 recipients,BKV viruria occurred in 27 (30.68％) patients,and sustained viruria occurred in 17 patients.37.0％ (10/27) of patients with BKV viruria developed inot BKV viremia,and sustained viremia occurred in 5 patients.The viral load in plasma was higher in patients with sustained viremia than in those with transient viremia (P＜0.05),and serum creatinine concentrations were higher when BK viremia occurred (P＜0.05).Conclusion Graft function was impaired among patients with BK viremia,and regularly monitoring BK virus in renal transplant recipients and clinical imervention based on plasma PCR results can prevent transplant kidney damage effectively.

OBJECTIVETo establish real-time PCR and loop-mediated isothermal amplification (LAMP) systems for detecting Cryptococcus neoformans CAP10 gene.

METHODSSpecific primers were designed targeting CAP10 gene of Cryptococcus neoformans, and the plasmid was constructed. After optimization of the reaction condition, the plasmid was quantitatively detected using real-time PCR and LAMP, and the detection sensitivity and specificity were evaluated. Clinical samples were also detected using the two methods.

RESULTSThe detection sensitivity of real-time PCR and LAMP was 6.8×10(1) and 6.8×10(3) copies, respectively. Real-time PCR yielded a higher positivity rate than LAMP. Both of the two methods showed a high detection specificity and produced negative results in the detection of Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli.

CONCLUSIONReal-time PCR is highly sensitive and specific for detecting Cryptococcus neoformans CAP10 gene but requires sophisticated equipment. LAMP, though with a relatively lower sensitivity, is simple to operate without the need of special equipment, and the result can be conveniently observed. Both of the two methods are suitable for detecting Cryptococcus neoformans and evaluating the treatment outcomes.

Cryptococcus neoformans ; genetics ; Fungal Proteins ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Plasmids ; RNA, Fungal ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Objective To analyze C4d deposition in the patients with late acute renal allograft rejection,and explore the role of C4d in grafts survival and grafts loss. Methods Thirty-six patients clinical and pathologically diagnosed as having acute rejection more than one year post-transplant were selected. C4d was detected by immunohistochemistry in renal allograft biopsies. The effect of C4d deposition on long-term graft survival was studied. Results Among 36 recipients with late acute renal allograft rejection, 16 cases were positive for C4d (44.4 %) and 20 negative for C4d (55.6 %). Five cases experienced graft loss in C4d positive group (31.3 %), while 6 cases in C4d negative group (30.0%). There was no significant difference in the graft loss rate between C4d-positive group and C4d-negative group. Log-Rank test demonstrated there was no significant difference in graft survival between C4d-positive group and C4d-negative group. The count of the interstitial infiltrated eosinophils in renal allograft was (9.4 + 4.5) and (2.6 + 1.8) respectively in the C4d-positive group and C4dnegative group (P＜0.05). Conclusion C4d deposition in peritubular capillary of the recipients with late acute renal allograft rejection might not be a prognostic marker for graft outcome.

Objective To measure the cytokines levels in peripheral blood from kidney transplantation recipients by using cytometric bead array and to analyze their change and the clinical significance in pre- and post- kidney transplantation, inducting with basiliximab and graft rejection. Methods A total of 72 renal transplantation recipients were divided into two groups, kidney function stable group(n =53) and acute rejection group (n = 19). And they were also grouped by induction with basiliximab or not,32 in basiliximab group and 40 in without basilixmab group. The levels of IFN-γ, TNF-α, IL-10, IL-5,IL-4, IL-2 were measured by cytometric bead array in peripheral blood of 72 kidney transplantation recipients and 30 healthy donors at differential time. The data was analyzed according to the following grouping:donors and recipients, kidney function stable group and acute rejection group post transplantation and with or without basiliximab group. Results The levels of TNF-α, IL-10, IL-5, IL-4, IL-2 in recipients before transplantation were ( 1.65 ±0. 10) ,(2. 55 ±0. 19) ,( 1.88 ±0. 14) ,(1.85 ±0. 12) ,(2. 12 ±0. 09) ng/L,respectively. While they were (3.04 ±0. 17), (3.33 ±0. 26), (4.03 ±0.25), (2.73 ±0. 16), (4.03 ±0. 26) ng/L respectively in healthy donors. There was statistical significance between the two groups ( t =6. 890, 2. 375, 7. 851,3.955,7.153, P<0. 01, <0. 05, <0.01, <0.01, <0.01). While the level of IFN-γ in recipients before transplantation was (2. 50 ±0. 18) ng/L,compared with (3. 00 ±0. 24) ng/L in healthy donors. There was no statistical significance between the two groups( t = 1. 625, P > 0. 05 ). The levels of IFN-γ and IL-10 in kidney function stable group were (2. 71 ± 0. 11 ) ng/L and (3.91 ± 0. 52) ng/L,while they were ( 3.30 ± 0. 36 ) ng/L and ( 12. 01 ± 5.35 ) ng/L in acute rejection group. There were statistical dirrerences between the two groups ( t = 5. 061, 11. 465, P < 0. 01, < 0. 05 ). Before induction with basiliximab, the levels of IFN-γ, TNF-α, IL-10 in recipients were (2.90 ±0. 21 ), ( 1.67 ±0. 12),(2. 45 ± 0. 16) ng/L respectively. But they were ( 2. 78 ± 0. 17 ), ( 1.58 ± 0. 07 ), ( 2. 77 ± 0. 24 ) ng/L respectively after induction with basiliximab, which showed significantly different ( t = 5. 605, 6.011,4. 126, P <0. 01, <0. 01, <0. 05). Four weeks after kidney transplantation in recipients with basiliximab,the levels of IFN-γ, IL-10, IL-4 were (2. 90 ± 0. 31 ), (9. 08 ± 0. 16), (2. 73 ± 0. 11 ) ng/L. While they were (3.28 ±0. 11 ), (4. 17 ±0. 21 ), (2. 11 ±0. 20) ng/L respectively in recipients without basiliximab induction, which were significantly different from those with basiliximab induction (t = 4. 268,4. 263,3.762, P <0. 01, <0. 01, < 0. 05 ). Conclusions Six kinds of cytokines can be measured by cytometric bead array simultaneously and accurately. The data suggests that the detection of multiple cytokines in kidney transplantation recipients by cytometric bead array can provide more guidance for clinical diagnosis and therapy.

ObjectiveTo determine the correlation of human leukocyte antigen-G (HLA-G)expression with CMV active infection after kidney transplantation. MethodsA total of 215 first-time kidney transplantation recipients in one transplantation center were divided into CMV ( + ) group and CMV ( - ) group according to whether they had active CMV infection. mhla-g1 expression on leukocytes was analyzed by flow cytometry. The concentrations of soluble HLA-G5 were detected by using ELISA. The sHLA-G5 cutoff levels by ROC curve was employed to predict the active CMV infection. The expression of sHLA-G5 mRNA and protein in leukocytes was analyzed by using RTPCR and Western blotting respectively. Immunohistochemical staining was used to detect the HLA-G expression in kidney biopsies of 12 cases. ResultsThe expression of mHLA-G1 in peripheral blood was low in both CMV ( + ) group and CMV ( - ) group. Also when CMV-PP65 was positive, there was no significant change in mHLA-G1. In CMV ( + ) group, the proportion of CD14+ mHLA-G1 +cells[(45. 53 ± 17.32)％]in peripheral blood was increased as compared with that in CMV (-)group[(10. 22 ± 5.78)％]. The expression of sHLA-G5 was increased significantly in CMV ( + )group. The optimal cutoff value of sHLA-G5 predicting the active CMV infection was 202. 9 μg/L,with high diagnostic accuracy. HLA-G was positive in the kidney tissue of 10 patients out of 12 patients with active CMV infection. Both RT-PCR and Western blot analysis showed that sHLA-G5 was significantly higher in CMV ( + ) group than that in CMV ( - ) group. ConclusionROC curve analysis of sHLA-G5 with the cutoff value of 202. 9 μg/L can be used to predict the active CMV infection. The HLA-G levels in peripheral blood were significantly increased and HLA-G expression in the tubular epithelial cells of the graft could be a protection mechanism of the kidney function.

Objective To study the correlation of HLA-G levels with acute rejection and CMV active infection post-kidney transplantation.Methods A total of 132 initial kidney transplantation recipients were divided into kidney function stable group (F),acute rejection group (AR),CMV group according to whether they had active CMV infection and acute rejection.Forty-one healthy donors served as control group (H).HLA-G levels and mRNA expression were analyzed by using flow cytometry,ELISA,RT-PCR and Western blotting.Immunohistochemical staining was used to detect the HLA-G expression in kidney biopsies.Results The expression levels of mHLA-G1 were low in all 4 groups pre-transplantation.Only CMV group had significantly more CD14+ mHLA-G1+ cells post-transplantation (P＜0.05).sHLA-G5 levels were higher in F group than in H group (P＜0.05),but there was no significant difference among other groups pre-transplantation (P＞0.05).sHLA-G5 levels were increased significantly in CMV group as compared with F group (P＜0.05),and those in F group were higher than in H and AR groups (P＜0.05).Renal tissue biopsies from 21 renal transplantation recipients with AR indicated that HLA-G5 was expressed negatively in 17 patients,positively in 3 patients and 1 weakly positively.HLA-G was positive in the kidney tissue of 9 patients out of 9 patients with active CMV infection.In total 132 recipients,AR incidence was significantly lower in CMV ( + ) group (7.1 ％,2/28) than that in CMV ( - ) group (24.0 ％,25/104).Conclusion The sHLA-G5 may contribute to predict AR and CMV active infection; AR and CMV active infection may be correlation with immune balance in kidney transplantation recipients.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can prolong the survival time of mice and baboons' alloskin graft and degrade acute and chronic graft-versus-host disease (GVHD) incidence after hematopoietic stem cell transplantation. But, at present there is no report that rat umbilical cord mesenchymal stem cells (UC-MSCs) reduced rejection response following heart transplantation. OBJECTIVE: To study the immunomodulatory effects of rat UC-MSCs on a model of allogeneic heart transplantation. METHODS: A total of 20 DA rats served as donors, and 20 Lewis rats as recipients. They were equally and randomly assigned to 2 groups: drug intervention and control groups (n=10). Using double cannulation, the left pulmonary artery and innominate artery of rat donors and external jugular vein and common carotid artery of rat recipients received end-to-end anastomosis under a microscope to establish heterotopic cardiac transplantation. One Wistar pregnant rat was selected to harvest UC-MSCs by collagenase digestion method. Following model establishment, rats in the cell transplantation group received UC-MSCs via caudal vein. Rats in the control group received sodium chloride. Survival time of the transplanted heart was determined. The transplanted heart received histopathology score using the acute rejection diagnosis criteria. Lymphocyte infiltration of transplanted heart was observed using hematoxylin-eosin staining. RESULTS AND CONCLUSION: Compared with the control group, the survival time of the transplanted heart was significantly longer in the cell transplantation group (P = 0.001), and the pathological score of acute rejection was significantly reduced (P = 0.000 4). There were lots of lymphocyte and monocyte infiltration in the myocardium in the control group. Little lymphocyte infiltration was detected in the myocardium in the cell transplantation group, with the presence of mild edema of myocardial interstitial substance. Results verified that rat UC-MSCs can induce immune tolerance of heart transplantation, soften immunological rejection and prolong xenograft survival.

OBJECTIVETo apply pyrosequencing technique in the detection of the common pathogens in sepsis.

METHODSThe primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained.

RESULTSPyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus.

CONCLUSIONPyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

Bacteria ; classification ; isolation & purification ; DNA Primers ; DNA, Bacterial ; genetics ; Microbiological Techniques ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sepsis ; microbiology

BACKGROUND:Cytotoxic T lymphocyte-associated antigen 4 is a newly discovered costimulatory molecule. It has been studied more in tumor and autoimmune diseases, less in the field of kidney transplantation.
OBJECTIVE:To explore the role of cytotoxic T lymphocyte-associated antigen 4 in acute rejection after renal transplantation.
METHODS:Fifty patients undergoing renal transplantation were divided into acute rejection group (20 cases) and stable graft function group (30 cases). Another 30 healthy persons served as control group. Blood samples were extracted from the peripheral blood. Cytotoxic T lymphocyte-associated antigen 4 was detected by enzyme linked immunosorbent assay and flow cytometry.
RESULTS AND CONCLUSION:The expression of cytotoxic T lymphocyte-associated antigen 4 in the serum showed significant differences in the acute rejection group, stable graft function group and healthy control group (F=70.008 1, P=0.000 0), but showed no difference in peripheral blood lymphocytes of three groups (F=1.865 6, P=0.161 7). Compared with the healthy control group, the expression levels of cytotoxic T lymphocyte-associated antigen 4 in peripheral blood lymphocytes of acute rejection group and stable graft function group were significantly decreased (P=0.000 0). In addition, the acute rejection group had a lower cytotoxic T lymphocyte-associated antigen 4 expression than the stable graft function group (P=0.000 0). In renal transplant rejection, the expression of cytotoxic T lymphocyte-associated antigen 4 in serum was reduced, showing some correlation with acute rejection after renal transplnatation. Cytotoxic T lymphocyte-associated antigen 4 might be involved in the rejection.