The heterophilicic epitopes in conserved HA regions of human and avian influenza viruses can produce antibodies that bound to kidney tissue.

Influenza virus infection can cause kidney damage. However, the link between influenza infection and disease is still unclear. The purpose of this study was to analyze the relationship between heterophilic epitopes on H5N1 hemagglutinin (HA) and disease. The monoclonal antibody (mAb) against H5N1 was prepared, mAbs binding to human kidney tissue were screened, and the reactivities of mAbs with five different subtypes of influenza virus were detected. Design and synthesize the peptides according to the common amino acid sequence of these antigens, and analyze the distribution of the epitope on the crystal structure of HA. Immunological methods were used to detect whether the heterophilic epitopes could induce the production of antibodies that cross-react with kidney tissue. The results showed that H5-30 mA b binding to human kidney tissue recognized the heterophilic epitope 191-LVLWGIHHP-199 on the head of HA. The key amino acid were V192, L193, W194 and I196, which were highly conserved in human and avian influenza virus HA. The heterophilic epitope could induce mice to produce different mAbs binding to kidney tissue. Such heterophilic antibodies were also detected in the serum of the patients. It can provide materials for the mechanism of renal diseases caused by influenza virus infection.

Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance.

BACKGROUND: Immature dendritic cells (imDCs) play an important role in the induction of donor-specific transplant immunotolerance. However, these cells have limitations, such as rapid maturation and a short lifespan in vivo. In previous studies, induced pluripotent stem cells (iPSCs) differentiated into imDCs, and sinomenine (SN) was used to inhibit the maturation of imDCs. AIM: To study the capacity of SN to maintain iPSC-derived imDCs (SN-iPSCs-imDCs) in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance. METHODS: In this study, mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN (iPSCs-imDCs and SN-iPSCs-imDCs). The imDC-related surface markers, endocytotic capacity of fluorescein isothiocyanate-Dextran and apoptosis were analyzed by flow cytometry. The effects of iPSCs-imDCs and SN-iPSCs-imDCs on T-cell stimulatory function, and regulatory T (Treg) cell proliferative function in vitro were analyzed by mixed lymphocyte reaction. Cytokine expression was detected by ELISA. The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting. The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice. Statistical evaluation of graft survival was performed using Kaplan-Meier curves. RESULTS: Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained, and their biological characteristics and ability to induce immunotolerance were compared. SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs. Reduced major histocompatibility complex II expression, worse T-cell stimulatory function, higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs (P < 0.05). The levels of interleukin (IL)-2, IL-12, interferon-γ in SN-iPSCs-imDCs were lower than those in iPSCs-imDCs, whereas IL-10 and transforming growth factor-ß levels were higher (P < 0.05). The apoptosis rate of these cells was significantly higher (P < 0.05), and the expression levels of cleaved caspase3, Bax and cleaved poly(ADP-ribose) polymerase were higher after treatment with lipopolysaccharides, but Bcl-2 was reduced. In Balb/c mice recipients immunized with iPSCs-imDCs or SN-iPSCs-imDCs 7 d before skin grafting, the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+ T-cell proliferation (P < 0.05) and a higher capacity to induce CD4+CD25+FoxP3+ Treg cell proliferation in the spleen (P < 0.05). The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern. CONCLUSION: This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

Long-term outcomes of arthroscopic debridement of the knee in adults with Kashin-Beck disease: an 18-year follow-up.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic degenerative joint disease with a high disability rate. We retrospectively evaluated the 18-year clinical follow-up outcomes of adult patients with KBD who underwent arthroscopic debridement for knee osteoarthritis. METHODS: Thirty-one patients with KBD (31 knees) underwent arthroscopy for knee osteoarthritis. The visual analog scale (VAS) score, walking distance, knee mobility, and patients' self-evaluated improvement in clinical symptoms were retrospectively evaluated before and 18 years after the operation. RESULTS: The patients' self-evaluated clinical symptoms showed considerable improvement at 2, 6, and 8 years after surgery but deteriorated at 10 and 18 years after surgery. Knee mobility was greater after than before arthroscopy but decreased from 6 to 18 years postoperatively. The VAS score for knee pain was high before the operation, decreased at 2 years postoperatively, increased at 6 years postoperatively, and was significantly lower at 18 years postoperatively than before surgery. The walking distance was significantly longer at 2, 6, and 8 years postoperatively than preoperatively. CONCLUSIONS: Arthroscopic treatment may be an effective therapy for adult patients with KBD who develop knee osteoarthritis. In this study, arthroscopy had a long-term effect on patients with KBD who had Kellgren-Lawrence grade

Prediction of acute renal allograft rejection by combined HLA-G 14-bp insertion/deletion genotype analysis and detection of kidney injury molecule-1 and osteopontin in the peripheral blood.

INTRODUCTION: Acute renal rejection usually fails to be diagnosed before the increase in the serum creatinine levels, and the resultant damage to the renal tissues occur in varying degrees. We hypothesized that the combined detection of human leucocyte antigen-G (HLA-G) 14-bp insertion/deletion genotypes and kidney injury molecule-1 (KIM-1) and osteopontin (OPN) levels in serum might facilitate the prediction of acute renal allograft rejections in kidney transplant recipients. METHODS: HLA-G 14-bp insertion/deletion genotypes and the serum KIM-1 and OPN levels of 77 kidney transplant recipients were determined and compared before operation and on days 1, 4, and 7 after the operation (32 in acute rejection [AR] group and 45 in stable allograft function [STA] group). These 3 indicators were combined to establish a model for the early prediction of AR. RESULTS: The KIM-1 levels in the serum of patients were significantly higher in the AR group than in the STA group. The area under the receiver operator characteristics (ROC) curve (AUC) of KIM-1 for the prediction of rejection was maximized on the1st day after operation, with a sensitivity of 84.4% and a specificity of 86.7%. The OPN levels in the serum of patients were significantly higher in the AR group than in the STA group only before operation and on the 7th day after operation. The AUC of OPN for the prediction of rejection was maximized on 7th day after operation, with a sensitivity of 68.8% and a specificity of 88.9%. The HLA-G + 14-bp allele frequency was also significantly higher in the AR group than in the STA group. The results of these three indicators were converted into a qualitative method. If any two of the three indicators show as positive, it was diagnosed as acute rejection, and it has the highest ability to predict acute rejection with a sensitivity and specificity of 84.38% and 91.11%, respectively. CONCLUSIONS: The HLA-G 14-bp insertion/deletion genotype and KIM-1 and OPN levels in the patients' serum were significantly different between the AR and STA groups. The power of predicting acute renal allograft rejection could be improved by combined these three biomarkers.

Outcomes of total knee arthroplasty in the adult Kashin-Beck disease with severe osteoarthritis.

PURPOSE: Kashin-Beck disease (KBD) is an endemic osteoarthropathy, and the severe knee pain and functional limitations were seriously affecting the quality of life in patients with end-stage KBD. We retrospectively evaluated the clinical outcomes and the quality of life in KBD patients with total knee arthroplasty (TKA). METHODS: A total of 22 subjects (25 knees) suffered KBD with severe knee pain and underwent primary TKA. Knee pain was measured by visual analogue scale (VAS), and the knee function was evaluated by Knee Society Clinical Rating System Score (KSS). KBD Quality of Life (KBDQOL) was used to evaluate the quality of life in KBD patients before and after TKA. RESULTS: There were no major complications after TKA. The levels of VAS score were obviously deceased in post-operation than that in pre-operation. The levels of KSS score were increased in one year after TKA compared with the pre-operative values, and it maintained a higher level on three years after TKA. The average KBDQOL score level of each domain in pre-operation and one and three years after TKA was increased accordingly. The average scores of physical function, activity limitation, support of society, mental health, and general health in one year after TKA were significantly higher than those in pre-operation. CONCLUSIONS: TKA can reduce knee pain, improve knee function, and improve the quality life in KBD patients. KBDQOL questionnaire may be a promising instrument for assessing the quality life in KBD patients.

Neuroprotective effects of genistein on SH-SY5Y cells overexpressing A53T mutant α-synuclein.

Genistein, a potent antioxidant compound, protects dopaminergic neurons in a mouse model of Parkinson's disease. However, the mechanism underlying this action remains unknown. This study investigated human SH-SY5Y cells overexpressing the A53T mutant of α-synuclein. Four groups of cells were assayed: a control group (without any treatment), a genistein group (incubated with 20 µM genistein), a rotenone group (treated with 50 µM rotenone), and a rotenone + genistein group (incubated with 20 µM genistein and then treated with 50 µM rotenone). A lactate dehydrogenase release test confirmed the protective effect of genistein, and genistein remarkably reversed mitochondrial oxidative injury caused by rotenone. Western blot assays showed that BCL-2 and Beclin 1 levels were markedly higher in the genistein group than in the rotenone group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed that genistein inhibited rotenone-induced apoptosis in SH-SY5Y cells. Compared with the control group, the expression of NFE2L2 and HMOX1 was significantly increased in the genistein + rotenone group. However, after treatment with estrogen receptor and NFE2L2 channel blockers (ICI-182780 and ML385, respectively), genistein could not elevate NFE2L2 and HMOX1 expression. ICI-182780 effectively prevented genistein-mediated phosphorylation of NFE2L2 and remarkably suppressed phosphorylation of AKT, a protein downstream of the estrogen receptor. These findings confirm that genistein has neuroprotective effects in a cell model of Parkinson's disease. Genistein can reduce oxidative stress damage and cell apoptosis by activating estrogen receptors and NFE2L2 channels.

Multiple-biomarkers provide powerful prediction of early acute renal allograft rejection by combination of serum fractalkine, IFN-γ and IP-10.

Biomarkers are urgently required for predicting rejection so that anti-rejection treatment can be taken early to protect the allograft from irreversible damage. We hypothesized that the combination of circulating fractalkine, IFN-γ and IP-10 might serve as effective biomarkers for predicting early acute renal allograft rejection. We conducted a retrospective study of 87 subjects, who were classified into acute rejection group (ARG; n = 38) and non-rejection group (NRG; n = 49). Serum fractalkine, IFN-γ and IP-10 levels were measured by Luminex. The levels of fractalkine on day 0 and 7th day, IP-10 on 4th and 7th day, and IFN-γ on 7th day in ARG was significantly higher than that in NRG. Kaplan-Meier survival analysis highlighted the higher-levels groups of fractalkine on day 0, 4th and 7th day, IFN-γ on day 0, 1st, 4th, and 7th day and IP-10 on the 4th and 7th day in rejection-free survival probability were significantly lower than low-levels groups. ROC analyses highlight the superiority of fractalkine on day 0, IP-10 on day 0, 4th and 7th day, and IFN-γ on day 0, 1st and 7th day in prediction of acute rejection. We found the combination of fractalkine on day 0, IP-10 on 7th day and IFN-γ on 7th day had the highest AUC (0.866) for predicting rejection with a sensitivity of 86.8% and a specificity of 89.8%. Our findings demonstrated a more powerful prediction of early acute renal allograft rejection during the first month after transplantation by combination of multiple-biomarkers of fractalkine, IFN-γ and IP-10, and the results might help stratify the immunologic risk of acute allograft rejection in recipients.

Investigation of the status quo of massive blood transfusion in China and a synopsis of the proposed guidelines for massive blood transfusion.

The aim of this study was to provide an overview of massive transfusion in Chinese hospitals, identify the important indications for massive transfusion and corrective therapies based on clinical evidence and supporting experimental studies, and propose guidelines for the management of massive transfusion. This multiregion, multicenter retrospective study involved a Massive Blood Transfusion Coordination Group composed of 50 clinical experts specializing in blood transfusion, cardiac surgery, anesthesiology, obstetrics, general surgery, and medical statistics from 20 tertiary general hospitals across 5 regions in China. Data were collected for all patients who received ≥10 U red blood cell transfusion within 24 hours in the participating hospitals from January 1 2009 to December 31 2010, including patient demographics, pre-, peri-, and post-operative clinical characteristics, laboratory test results before, during, and after transfusion, and patient mortality at post-transfusion and discharge. We also designed an in vitro hemodilution model to investigate the changes of blood coagulation indices during massive transfusion and the correction of coagulopathy through supplement blood components under different hemodilutions. The experimental data in combination with the clinical evidence were used to determine the optimal proportion and timing for blood component supplementation during massive transfusion. Based on the findings from the present study, together with an extensive review of domestic and international transfusion-related literature and consensus feedback from the 50 experts, we drafted the guidelines on massive blood transfusion that will help Chinese hospitals to develop standardized protocols for massive blood transfusion.

Investigation of the current situation of massive blood transfusion in different surgical departments: a large multicenter study in China.

OBJECTIVE: This study aims to learn about the current situation of surgical massive blood transfusion of different surgical departments in China's Tertiary hospitals, which could provide the basis for the formulation of guidelines on massive blood transfusion. METHOD: A multicenter retrospective research on the application status of blood constituents during massive blood transfusion was conducted and a comparative analyses of survival and length of hospitalization in patients from different departments (trauma, cardiac surgery, obstetric conditions, or other common surgeries), were performed. RESULT: In China, during massive blood transfusion the ratio of the dosage of fresh frozen plasma to the dosage of red blood cell suspension reached 1:1-2, while the dosage of platelet and cryoprecipitate appeared to be very small. The risk of in-hospital death were associated with the primary disease in patients receiving massive blood transfusion (Log-Rank P = 0.000), cardiac surgery and trauma patients who received massive blood transfusion have a higher risk of death rate. CONCLUSIONS: Patients undergoing massive blood transfusion among different surgical departments have a certain difference in use of blood transfusion, mortality rate and the time of death. Our findings suggested that we should set up an independent transfusion program in cardiac surgery and trauma patients of massive blood transfusion.

The plasma and platelet are important in reducing the mortality in surgical massive blood transfusion: a large multicenter study in China.

OBJECTIVE: The aim of this study was to learn the current situation of surgical massive transfusion of death and survival groups in China, which could provide the basis for the formulation of guidelines on massive transfusion. METHODS: A multicenter retrospective research for the application status of blood constituents during massive blood transfusion was conducted, the differences of fresh frozen plasma and platelet application between death group and survival group were compared, and the transfusion volume and the distribution of other blood constituents were analyses at different periods of time when red blood cells are infused between death group and survival group. RESULTS: The patients with fresh frozen plasma compare the patients with red blood cell was 1:1-2 during massive transfusion, while the dosage of platelet and cryocepitate were transfused very small. Results showed that the average amount of platelet and plasma in death group was significantly lower than those in survival group. CONCLUSION: During massive transfusion, clinicians in 20 Chinese hospitals paid more attention to the infusion of fresh frozen plasma while making the infusion of red blood cells. However, they paid little attention to the supplement of platelet and cryocepitate. The average quantity of plasma and platelet in survival group were also higher than those in death group.

Changes in platelet function following cold storage of RBC suspensions.

OBJECTIVE: To provide a basis for the cold-storage of human platelets as a way to assess changes in platelet function. METHODS: Red blood cell suspensions (11 U and 50 U) were randomly selected at different storage times (3-28 days) and evidence of platelet activation (CD62P) and thromboelastography (TEG) reaction times were investigated. RESULTS: After 21 days of storage at 4°C, a large number of activated platelets (PAC1+62P+, PAC1-62P+) within the red blood cell suspension (RBCs) retained their function and had TEG-maximum amplitude (TEG-MA) indices in the normal range. CONCLUSION: We report that platelets in RBC suspensions retain high activity when stored at 4°C for 21 days. The results provide important information for studies that involve storing platelets under cold conditions.

Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients.

BACKGROUND: Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. PATIENTS AND METHODS: In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28days, and at 2, 3, 4, 5 and 6months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. RESULTS: The ATP level detected on the day AR occurred (627.07±149.85ng/ml) was obviously higher than that of the stable group (320.48±149.11ng/ml, P<0.05). ATP value decreased to 265.35±84.33ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87±162.85ng/ml, P<0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55ng/ml. A positive correlation between the intracellular ATP level and absolute CD4+ T cell number (r=0.656, P<0.001) was found in the patients with CD4+ T cell counts <200/µl.

Kidney injury molecule-1 and osteopontin: new markers for prediction of early kidney transplant rejection.

BACKGROUND: Kidney injury molecule-1 (KIM-1) and osteopontin (OPN) play important roles in immune regulation. We hypothesized that serum KIM-1 and OPN might serve as biomarkers for predicting early acute rejection after kidney transplantation (KTx). METHODS: We conducted a single-center study of 155 subjects, who were classified into acute rejection group (ARG, n=32), non-rejection group (NRG, n=45) and healthy controls (HC, n=78). Serum KIM-1 and OPN levels were measured by Luminex. RESULTS: The pre-transplant levels of serum KIM-1 and OPN in all KTx recipients were higher than those of HC (P<0.01). Compared with NRG, ARG showed significantly high serum levels of KIM-1 on day 0 (pre-KTx) and on the 1st, 4th, and 7th post-KTx days, and significantly high OPN levels on day 0 and the 7th day. Kaplan-Meier survival analysis showed that the higher levels of KIM-1 on day 0, the 1st and 4th days and OPN on day 0 and the 7th day were significantly associated with the lower probabilities of rejection-free survival. ROC analyses highlight the superiority of KIM-1 on the 1st day and OPN on the 7th day over those on other post-KTx days in prediction of acute rejection episodes. Multivariate logistic analysis revealed that the serum KIM-1 levels on the 1st post-KTx day and the OPN level on the 7th day were independent and powerful predictors of acute rejection episodes. An optimal predictive model was built by combining KIM-1 on the 1st day and OPN on the 7th day, and this model had the highest AUC (0.922). CONCLUSIONS: This study was the first to demonstrate that serum KIM-1 and OPN may be the promising and elegant markers for prediction of early acute kidney allograft rejection.

Impact of HLA-G 14-bp polymorphism on acute rejection and cytomegalovirus infection in kidney transplant recipients from northwestern China.

Human leukocyte antigen (HLA)-G plays an important role in promoting transplant tolerance and helping human cytomegalovirus (CMV) to subvert host defenses. Strong evidence suggests that HLA-G 14-bp insertion/deletion polymorphism influences the stability of HLA-G mRNAs and levels of protein expression. We hypothesized that HLA-G 14-bp polymorphism of recipients has an influence on the risk of acute rejection (AR) and CMV infection. We investigated the impact of HLA-G 14-bp polymorphism on a total of 363 unrelated Chinese Han individuals who included 42 kidney transplant recipients with AR, 43 recipients with CMV infection, 102 recipients with stable allograft function (STA), and 176 healthy controls (HC). No statistically significant difference was found between all kidney transplant patients and HC (P=0.149). But, our data showed an increased frequency of homozygous genotype +14/+14 bp (P(c)=0.004) and allele +14 bp (P(c)=0.002) in patients with AR when compared with STA, with the odds ratio of 3.17 and 2.28, respectively. Moreover, we found that the frequency of the -14/-14 bp genotype (P(c)=0.008) and the -14 bp allele (P(c)=0.016) was increased in patients with CMV infection when compared with STA, with the OR of 2.66 and 1.96, respectively. Multivariate analysis further demonstrated that HLA-G homozygous +14 bp and -14 bp genotypes were an independent risk factor for allograft rejection and CMV infection, respectively. In conclusion, this study identified an important genetic risk factor for acute allograft rejection, and it was the first to show a significant correlation between HLA-G 14-bp polymorphism and CMV infection after kidney transplantation from northwestern China.

[The influence of rapamycin on BTLA expression of human peripheral blood T lymphocytes].

AIM: To explore the effect of rapamycin (RPM) on the expression of B and T lymphocyte attenuate (BTLA) on human peripheral blood T lymphocytes, providing a experimental basis for application of RPM to organ transplantation and autoimmune diseases. METHODS: Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and then the T cells were isolated from PBMCs with immunomagnetic beads. Concanavalin A (Con A) used to stimulate and activate the human peripheral blood T cells. The proliferation of T cells was detected by MTT colorimetry. The levels of IL-2 and IFN-γ in cell culture supernatant were detected by ELISA. The expression of BTLA on T cells was assayed by Flow cytometry. RESULTS: The expression of BTLA on T cells treated with various concentrations (10-1 000 ng/L) of RPM had no significant difference, while had significant difference (P<0.01) by compared with RPM non-treatment group. ELISA detection manifested that compared with the untreated group different concentrations of RPM could significantly inhibit the IL-2 and IFN-γ secretion and significantly inhibited T lymphocyte proliferation (P<0.01). CONCLUSION: RPM has little effect on expression BTLA, but has stronger inhibition on lymphocyte proliferation and inflammatory cytokines secretion. Suggesting that RPM is suitable for the treated of organ transplant rejection and autoimmune diseases.

[Evaluation of immune status of kidney transplant recipients by combined HLA-G5 and sCD30].

OBJECTIVE: to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. METHODS: from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. RESULTS: the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L. CONCLUSION: the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells.

OBJECTIVE: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4(+) CD25(+) regulatory T (Treg) cells, in order to inform further studies in Treg cell function. METHODS: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4(+) CD25(+) Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4(+) CD25(+) Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4(+) CD25(+) Treg cells in the in vitro proliferation experiments. RESULTS: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4(+) CD25(+) Treg cells, however, obtained with both methods have similar immunosuppressive capacities. CONCLUSION: The result suggests that both methods can be used in isolating CD4(+) CD25(+) Treg cells, and one can select the best method according to specific needs and availability of the methodologies.

[Clinical application and exploration on mechanism of action of Cordyceps sinensis mycelia preparation for renal transplantation recipients].

OBJECTIVE: To retrospectively study and analyse the immune regulatory effect of Bailing Capsule (BLC, a dry powder preparation of Cordyceps sinensis mycelia) on patients after renal transplantation, its influences on various systems of organism, and to explore its possible acting mechanism. METHODS: In accordance with the entry criteria, 67 recipients of renal homo-allograft were assigned to two groups. The 42 cases in the control group were treated with mycophenolate mofetil (MMF) plus cyclosporine A (CsA), or tacrolimus (FK506) plus prednisone (Pred); the 25 in the treated group treated with the chemotherapy the same as in the control group plus BLC. They were followed up for 48 weeks by checking up blood routine, urine routine, hepatic and renal function, total serum protein, serum albumin, uric acid, etc., and the dosage of immunoinhibitory used was recorded periodically. RESULTS: Comparison showed no significant difference in graft survival rate, occurrence of reject reaction and renal function recovery between the two groups; but levels of urinary erythrocytes and leucocytes, blood alanine transaminase, aspartate amino transferase, uric acid, total bilirubin, direct bilirubin, as well as the incidence of infection were significantly lower, and serum total protein and albumin were significantly higher in the treated group (all P < 0.01); moreover, counts of erythrocyte and leukocyte from 12 to 48 weeks, T-lymphocyte from 4 to 48 weeks after transplantation were significantly higher in the treated group (P < 0.05 and P < 0.01), and the recovery appeared earlier, the dosage of CsA or FK506 used 12 weeks after operation was significantly lower in the treated group than in the control group (P < 0.05, P < 0.01). CONCLUSIONS: BLC could effectively protect liver and kidney, stimulate hemopoietic function, improve hypoproteinemia, as well as reduce the incidence of infection and the dosage of CsA and FK506 used, etc. Therefore, it is a useful drug for immunoregulation after organ transplantation.