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This study aims to explore the biological roles of long non-coding RNA (lncRNA) ZNF281 and KLF15 in regulating cervical carcinoma progression.Differential expressions of ZNF281 in 58 collected cervical carcinoma and normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between ZNF281 and clinicopathologic characteristics in cervical carcinoma patients was analyzed. By generating ZNF281 knockdown model in HeLa and SiHa cells through the transfection of shZNF281, migratory ability changes were examined via transwell and wound healing assay. The role of ZNF281 in in vivo tumorgenicity of cervical carcinoma was examined by implanting xenografted cancers in nude mice. The downstream target of ZNF281 and their interaction were assessed by bioinformatics tool and Dual-Luciferase reporter assay, respectively. Finally, co-regulations of ZNF281 and KLF15 on cervical carcinoma progression were elucidated.ZNF281 was upregulated in cervical carcinoma tissues and cell lines. It was correlated to TNM staging, and incidences of lymphatic metastasis and distant metastasis in cervical carcinoma patients, while it was unrelated to age and tumor size. The knockdown of ZNF281 effectively attenuated migratory ability in HeLa and SiHa cells. Besides, knockdown of ZNF281 also reduced tumorigenicity of cervical carcinoma in nude mice. KLF15 was the downstream gene binding ZNF281, and they were negatively correlated to each other in cervical carcinoma tissues. Notably, KLF15 was responsible for ZNF281-induced regulation on cervical carcinoma migration.LncRNA ZNF281 is upregulated in cervical carcinoma samples, and it is correlated to lymphatic metastasis, distant metastasis, and poor prognosis in cervical carcinoma patients. By targeting KLF15, ZNF281 triggers migratory potential in cervical carcinoma. We believed that ZNF281 is a promising biomarker for cervical carcinoma.

Alkaline water electrolysis becomes increasingly important for the supply of renewable energy, and of raw material for the chemical industry. An attractive choice for the encapsulation of the electrolyte cell is an (advanced) engineering polymer. The objective of this paper is to find a suitable one that can withstand for many years: 30 wt% KOH solution and pure oxygen at a high pressure of 50 bar and at an elevated temperature of 90°C. Using CES EduPack, 12 possible thermoplastic polymers were selected, of which polyphenylene sulfide (PPS) and polysulfone (PSU) were further investigated using accelerated testing. The polymers have been exposed to three KOH concentrations (15, 30 and 45 wt%), two oxygen pressures (pure O2at 5 bar and air with pO2= 20%), and three temperatures (90°C, 120°C, and 170°C). Extensive characterization of the exposed samples has been carried out using various techniques, including weight, tensile, DMA, and creep-recovery measurements, as well as DSC, FTIR, XRD and SEM. After 12 weeks of aging, glass fiber reinforced PPS failed in a strong alkaline solution at high temperatures, due to the dissolution of the glass fibers. The PPS matrix itself and PSU turned out to be resistant to thermo-oxidative and chemical degradation under the conditions tested. Only marginal changes in mechanical, visco-elastic and thermal behavior were observed, which can be ascribed to physical rather than chemical aging. In view of the brittle nature of PPS, it could be concluded that PSU is the most promising candidate for the long-term application in alkaline electrolysis. Extrapolating the data using time-temperature superposition, it is predicted that PSU will retain its integrity and mechanical properties for a period of 20 years of operation.

During influenza A virus (IAV) (H5N1) infection, the levels of inflammatory cytokines are markedly elevated in the lungs of infected hosts. One of them, high-mobility group box 1 protein (HMGB1) functions in regulation of cellular transcription and activation of proinflammatory responses, but little is known about its role in viral infection. In this study, we attempted to address this question. Using an IAV (H5N1) - mouse model, lung tissues were analyzed for virus titer, expression of HMGB1 and other inflammatory cytokines and histopathological changes. Moreover, the effect of administration of HMGB1-specific antibody to the infected mice on these parameters was investigated. The results showed that the HMGB1 expression was induced on days 3-7 post infection (p.i.) and primarily localized to epithelial cells of alveoli and bronchioles. The HMGB1-specific antibody reduced the levels of inflammatory cytokines and chemokines and the survival rate, but did not influence the virus titer. Summing up, these data suggest that HMGB1 contributes to the pathogenesis of IAV (H5N1) infection in mice by inducing extensive inflammatory responses and severe pneumonia.

 Abstract—Treadmill exercise has been a controversial method to help stroke rat with functional recovery and neuroprotection. In this study, we investigated the effect of treadmill exercise on focal brain ischemia stroke rat in subacute phase. Twelve rats were induced focal ischemic stroke by a 60 min middle cerebral artery occlusion/reperfusion surgery and then randomly divided into two group, control group (CG, n=6) and treadmill group (TG, n=6). TC rats received daily treadmill exercise training during the seven-day experimental period while CG rats did not receive any intervention. Daily functional test results were recorded and all rats were sacrificed for TTC staining on the 7 th day. The increase rate of behaviour score in TG is significantly higher than that in CG (P

Tissue trauma in the peritoneal and pelvic cavities following surgery or bacterial infection results in adhesions that are a debilitating cause of intestinal obstruction, chronic pelvic pain, and infertility in women. We recently demonstrated that CD4+ αβ T cells are essential for development of this process. Using a murine model of experimental adhesion formation, we now demonstrate that adhesion formation is characterized by the selective recruitment of Tim-3+, CCR5+, CXCR3+, IFN-γ+ cells, indicating the presence of a Th1 phenotype. We further demonstrate that adhesion formation is critically dependent on the function of Th1 cells because mice genetically deficient for IFN-γ, T-bet, or treated with Abs to the Th1-selective chemoattractant IL-16 show significantly less adhesion formation than wild-type mice. In addition, disrupting the interaction of the Th1-specific regulatory molecule Tim-3, with its ligand, significantly exacerbates adhesion formation. This enhanced response is associated with increases in the level of neutrophil-attracting chemokines KC and MIP-2, known to play a role in adhesiogenesis. These data demonstrate that the CD4+ T cells orchestrating adhesion formation are of the Th1 phenotype and delineate the central role of T-bet, Tim-3, IFN-γ, and IL-16 in mediating this pathogenic tissue response.

Allograft(dagger) transplant outcome, rejection or tolerance, depends upon striking a balance between the pertinent cytopathic and regulatory T cells. The drug cyclosporine is a widely used immunosuppressive agent among transplant recipients. Previous studies have demonstrated that cyclosporine blocks apoptosis of activated T cells and the ability of costimulation blockade based regimens to create peripheral transplant tolerance. We now test the hypothesis that the mechanism by which cyclosporine blocks tolerance induction is IL-2 dependent, and linked to a detrimental effect upon T(reg) function. Our study demonstrates that cyclosporine blocks IL-2 gene expression and activation induced cell death (AICD) of alloreactive T effector cells. We also show that cyclosporine abolishes the beneficial effects of a donor specific transfusion (DST) plus anti-CD154 monoclonal antibody (alpha CD154) regimen on enhanced T(regs) function and allograft tolerance induction. Interestingly, provision of IL-2/Fc, a long-lived form of IL-2, completely reverses the detrimental effects of this adjunctive cyclosporine treatment on AICD of alloreactive T effectors, T(regs) function and tolerance induction. Furthermore, in a MHC mismatched islet allograft model, the combination of cyclosporine with IL-2/Fc permitted long-term allograft survival and induced alloantigen specific allograft tolerance. The combination of IL-2/Fc and cyclosporine treatment may provide a new clinical strategy to promote transplant tolerance.

Human chronic Chagas disease cardiomyopathy (CCC) is an inflammatory-dilated cardiomyopathy occurring years after infection by the protozoan Trypanosoma cruzi. The heart inflammatory infiltrate in CCC shows a 2:1 predominance of CD8(+) in relation to CD4(+) T cells, with a typical Th1-type cytokine profile. However, in vitro expansion of infiltrating T cells from heart biopsy-derived fragments with interleukin-2 (IL-2) and phytohaemagglutinin leads to the outgrowth of CD4(+) over CD8(+) T cells. We hypothesized that survival cytokines, such as IL-2, IL-7 and IL-15 might be differentially involved in the growth and maintenance of heart-infiltrating and peripheral CD8(+) T cells from CCC patients. We found that IL-7 and IL-15 were superior to IL-2 in the expansion and viability of CD8(+) T cells from both PBMC and heart-infiltrating T-cell lines from CCC patients, and the combination of the three cytokines showed synergic effects. Heart-infiltrating CD8(+) T cells showed higher expression of both IL-15R alpha and gamma(c) chain than CD4(+) T cells, which may explain the improvement of CD8(+) T-cell growth in the presence of IL-2 + IL-7 + IL-15. Immunohistochemical identification of IL-15 and the higher mRNA expression of IL-15R alpha, IL-7 and gamma(c) chain in CCC heart tissues compared with control individuals indicate in situ production of survival cytokines and their receptors in CCC hearts. Together, our results suggest that local production of IL-7 and IL-15 may be associated with the maintenance and predominance of CD8(+) T cells, the cells effecting tissue damage in CCC hearts.

The ability of T helper (TH) precursor cells to differentiate into T effector populations confers the adaptive immune system with a means to protect the host from microbes and react to “foreign” antigenic tissues. T-cell immunoglobulin and mucin domain (TIM) proteins have recently been shown to be novel and critical regulators of T cell subset-driven dependent immune responsiveness. A dichotomy is emerging as to how Tim-3– and Tim-2– related signals respectively impact TH1 and TH2 responses. By comparison, the influence of the Tim-1 pathway seems to be broader and is probably not restricted to a specific type of T helper response. Beyond the mere control of the TH1/TH2 balance, Tim proteins are likely to target other regulatory components of the T cell response. Likewise, it is tempting to speculate that Tim proteins might also modulate the function of other T helper cell subsets such as TH3, TR1 and TH17 cells, among others.

Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

Introduction: Cervical cancer is one of the most common female malignancies and leading cause for high mortality rate. In the present study we made an attempt to determine the extent of angiogenesis, apoptosis, accumulation of mutant p53 protein, cell proliferation rate in the uterine cervical cancer tissues. Materials and Methods: Cervical cancer samples were obtained from patients and they were subjected to PCR analysis and immunocytochemistry. Results: A total of 30 cervical cancer tissue samples were analyzed, by PCR, we found 25 collected cervical cancer samples showed HPV-16 and E6 positive. Further, we observed the increased CD34 expression was associated with HPV-16 and E6 positive cancer tissues when compared to the corresponding control tissues. This elevated level of CD34 confirms the increased extent of angiogenesis in cervical cancer tissues. Further by immunocytochemistry we have demonstrated that the rate of apoptosis is reduced, over expression of bcl-2, Ki 67 and thus increases rate of cell proliferation. Discussion: Therefore, our data suggest that development of new anticancer or antiviral drugs could efficiently compromise the HPV-16 mediated angiogenesis and reduced apoptosis in cervical cancer and thus will improve the survival rate of patients.

Adenovirus-mediated β-galactosidase gene delivery to the liver leads to protein deposition in kidney glomeruli. The many cell types of the kidney, precisely arranged, allow this organ to perform its complex physiologic functions. However, this architectural complexity makes gene transfer into the kidney difficult. One approach to delivering a therapeutic protein to the kidney is to transfer a gene to a non-renal tissue. Release of the protein into the circulation might then result in deposition in the kidney, if the protein has the appropriate molecular properties. In this study, we found that parenterally administered replication deficient adenovirus carrying the β-galactosidase gene resulted in intense β-galactosidase gene expression in hepatocytes. As a result of immune attack on transduced hepatocytes, β-galactosidase protein from these cells is released into the circulation, transported, and deposited almost exclusively in kidney glomeruli. Intense β-galactosidase activity was noted in both kidneys with a peak at two weeks following viral administration, concurrent with loss of β-galactosidase positive hepatocytes. Consistent with our hypothesis of protein transfer, no β-galactosidase mRNA was detected in glomeruli. Moreover, systemically administered protein generated similar glomerular β-galactosidase activity. Finally, co-administration of murine CTLA4 Ig, an immunomodulator of T cell activation, with the adenovirus protected infected hepatocytes and markedly diminished glomerular β-galactosidase activity. Collectively, these findings suggest that a therapeutic protein can be “targeted” to the renal glomerulus, utilizing the liver as a gene transfer organ.

Blockade of CD28-mediated T cell costimulatory signals produces effective immunosuppression of a variety of T cell-dependent in vivo immune responses, including autoimmune disorders and transplant rejection. The soluble fusion protein CTLA4Ig, which competitively blocks CD28 ligands B7-1 and B7-2, can prevent allograft and xenograft rejection and in some circumstances induce transplantation tolerance. To determine the relative roles of B7-1 and B7-2 in graft rejection, we have performed islet and cardiac allografts with normal and B7-1(-/-) mice in conjunction with selective blocking reagents. We found that the absence of B7-1 on donor or recipient tissues leads to a slight prolongation of islet allograft survival, but has minimal or no effect on cardiac allograft survival. Allograft function is further prolonged in the islet model when both donor and recipient lack B7-1, although cardiac allograft survival is not prolonged. In the cardiac model, treatment with CTLA4Ig induces long term survival in B7-1(-/-) recipients regardless of donor status. In contrast, anti-B7-2 mAb leads to indefinite allograft survival only when the recipient and donor both lack B7-1, indicating that even in the absence of available B7-2, B7-1 molecules on the donor or recipient cells alone are sufficient to induce graft rejection. These data also indicate that B7-1 and B7-2 are the only CD28 ligands relevant to cardiac allograft rejection in mice.

In infection with the helminth Schistosoma mansoni, parasite eggs elicit a Th cell-mediated hepatic granulomatous inflammation that leads to scarring, portal hypertension, hemorrhage, and death. On the other hand, the cytokine IL-10 has been shown to decrease egg Ag-specific Th cell responses by down-regulating MHC class II as well as B7 costimulatory molecule expression on accessory cells. We now test the effect of IL-10 in vivo on models of egg Ag-specific hypersensitivity as well as on the natural disease. Systemic administration of IL-10 significantly inhibited delayed-type hypersensitivity reactions to egg Ag as well as primary and secondary granuloma formation to eggs embolized in the lung. However, significant inhibition of hepatic granuloma formation associated with the natural infection required the use of an IL-10/Fc fusion protein with a prolonged in vivo half-life. Lymph node cells from IL-10/Fc-treated mice produced less IL-2 and IFN-gamma, and more IL-4 and IL-10 than control cells, suggesting that reduced egg granuloma formation resulted primarily from down-regulation of Th1 responses. These results indicate that suitable administration of exogenous IL-10 can be effective in ameliorating immunopathologic damage associated with schistosomiasis.

Treatment with rapamycin (RPM) prevents accelerated rejection of (LEW x BN)F1 cardiac allografts in LEW rats presensitized with BN skin grafts. This study analyzed the influence of RPM on cytokine (IL-2, IL-4, IL-10, and IL-12) and alloantibody networks in this model. Accelerated (24-h) rejection was associated with strong expression of intragraft IL-2 and IL-12 (p40) mRNAs, which reached maximal levels 3 to 6 h post-transplantation. IL-4 and IL-10 mRNAs were readily detectable throughout the observation period. RPM therapy abrogated rejection at 24 h and prolonged cardiac allograft survival to about 50 days. This effect was correlated with a profound initial depression of IL-2 mRNA; delayed expression of IL-2 mRNA was detected in well functioning grafts at > 20 days. In RPM-treated hosts, expression of IL-12 (p40) mRNA was low at the early time points (6-24 h), but prominent in long term grafts. The expression of both IL-4 and IL-10 mRNAs was preserved in RPM-conditioned hosts. Immunohistologic analysis of long term allografts revealed an interstitial cellular infiltrate and areas of intimal proliferation within small arteries indicative of early transplant arteriosclerosis. Analysis of cytokine proteins showed dense labeling of mononuclear and some endothelial cells for IL-4 and IL-12 (p70), but not for IL-2 or IFN-gamma alloantibody in the early post-transplant period. However, an increase in circulating and intragraft IgM and, to a lesser extent, IgG, primarily of the IgG2b subclass, was evident in long term recipients. Thus, RPM treatment reduces, but does not completely inhibit, the expression of Th1-type and preserves the expression of Th2-type cytokines. The demonstration of IL-12 in long term allografts after RPM therapy may reflect late activation of macrophages that, coupled with the appearance of IgG2b, may contribute to the chronic rejection of cardiac allografts.

Vascularized composite allografts (VCAs) are unique among transplanted organs in that they are composed of multiple tissues with disparate antigenic and immunologic properties. As the predominant indications for VCAs are non-life-threatening conditions, there is an immediate need to develop tolerance induction strategies and to elucidate the mechanisms of VCA rejection and tolerance using VCA-specific animal models. In this study, we explore the effects of in vitro induced donor antigen-specific CD4(-) CD8(-) double negative (DN) Treg-based therapy, in a fully MHC mismatched mouse VCA such as a vascularized osteomyocutaneous as compared to a non-VCA such as a full thickness skin (FTS) transplantation model to elucidate the unique features of VCA rejection and tolerance. We demonstrate that combined therapy with antigen-induced CD4 derived DN Tregs and a short course of anti-lymphocyte serum, rapamycin and IL-2/Fc fusion protein results in donor-specific tolerance to VCA, but not FTS allografts. Macrochimerism was detected in VCA but not FTS allograft recipients up to >60 days after transplantation. Moreover, a significant increase of CD4(+) Foxp3(+) Tregs was found in the peripheral blood of tolerant VCA recipients. These data suggest that VCA are permissive to tolerance induced by DN Treg-based induction therapy.

B7 + -transfectant tubular epithelial cells induce T cell anergy, ignorance or proliferation. We have previously established that interferon (IFN)-γ stimulated, antigen-pulsed tubular epithelial cells (TEC) stimulate antigen (Ag) specific activation of T cell hybridomas to express IL-2. In contrast, these Ag pulsed TEC do not stimulate T helper 1 (Th1) clones to proliferate, but rather render them unresponsive, since Ag pulsed spleen cells cannot restore these cells to proliferate. The interaction of the T cell CD28 surface protein with its ligand B7 expressed on Ag presenting cells bearing la is a potent co-stimulatory signal capable of inducing T cell proliferation. Hence, the lack of B7 on TEC was hypothesized to be responsible for anergy in these Th1 cells. Therefore, the B7 gene was transfected into a SV40 transformed TEC or Chinese hamster ovary (CHO) cells, and created TEC and CHO cells expressing surface B7 protein. TEC-B7 (IFN-γ stimulated, Ag pulsed) express la and induce IL-2 production by T cell hybridomas. In contrast, T cell proliferation was not induced by TEC-B7 or CHO-B7 cells; however, these Th1 cells were not anergic since they could be stimulated to proliferate to Ag pulsed spleen cells (immunological ignorance). However, co-cultivating TEC- B7 (IFN-γ stimulated, Ag pulsed) with Th1 cells stimulated through the T cell receptor (TCR) using anti-CD3 monoclonal antibody (mAb) caused these Th1 cells to proliferate. Furthermore, anti-CD28 and anti-B7 mAbs blocked this response. These data suggest that the spectrum of Th1 cell activation after encounter with Ag is dictated by: (1) the vigor of TCR/CD3 signal, and (2) presence or absence of co-stimulatory signals through the CD28 pathway. Since expression of Ia on TEC induces anergy, this may serve as a mechanism to thwart—not foster—autoimmunity.

To establish a model in vitro for screening antiepileptic drugs.Perfusing guinea pig hippocampal slice with penicillin offers a stable epileptiform discharge which was tested by two commonly clinically used antiepileptic drugs, phenytoin sodium and phenobarbital sodium at different concentration.The experimental result showed that penicillin induced epileptiform discharge in the hippocampal slice and two antiepileptic drugs, phenytoin sodium and phenobarbital sodium inhibition of this discharge induced by penicillin.This electrophysiological model has the advantage of simplicity, flexibility and ease of quantitation and has potential value for screen antiepileptic drugs and epilepsy research.

A new differentiation pathway for CD4(-)CD8(-) (DN) T cells has recently been identified that exhibits the potent function of peripheral converted DN T cells in suppressing immune responses and provides the potential to treat autoimmune diseases. The aim of this study was to determine if the DN T cells converted from CD4(+) T cells of NOD mice retain the antigen-specific regulatory capacity and prevent autoimmune diabetes in vivo. We also sought to determine if the combination of DN T cells with rapamycin promotes islet allograft survival in autoimmune diabetic NOD recipients.NOD CD4(+) T cells were converted to DN T cells in an in vitro mixed-lymphocyte reaction, with or without GAD65 peptide, as previously reported. The antigen-specific DN T cells were adoptively transferred to NOD/SCID mice, new-onset diabetic NOD mice or islet-allograft-recipient NOD mice as the part of cell-based therapy. The development of diabetes and allograft survival was assessed by monitoring blood glucose levels.NOD CD4(+) T cells were converted in vitro to DN T cells at a rate of 50% and expressed unique cell features. The DN T cells from NOD donors blocked autoimmunity and prevented diabetes in NOD models, and these effects were even greater for GAD65-peptide-primed DN T cells. DN T cells acted in conjunction with rapamycin to suppress alloantigen-triggered T cell proliferation, promoted apoptosis and prolonged islet allograft survival in NOD recipients.Administration of the islet beta cell antigen-specific DN T cells can prevent the development of autoimmune diabetes and promote islet allograft survival in NOD mice.

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3(+)CD25(+)CD4(+) T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-beta expression and IL-4 production. Depletion of recipient CD25(+)CD4(+) T cells using anti-CD25 mAb (250 microg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3(+) Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4(+) and CD8(+) T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3(+)CD25(+)CD4(+)Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-beta and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.

Face transplantation and other composite tissue transplantation (CTA) are permissive to transplantation tolerance. The real reason, that composite tissue containing bone achieves transplantation immune tolerance more easily than the composite tissue without the bone is not clear. The chimerism may be the main mechanism in the progress of inducing the transplantation tolerance by CTA. We currently have established a non-human Primate Vascularized Osteomyocutaneous Allografts Model. To test the chimerism which comes from donor after the transplantation, we developed a method which combined reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qRT-PCR) technique using primers specific for Macaca fascicularis sex determination region on the Y chromosome (SRY) gene. With the method, we estimated the level of the chimerism.

This paper presented an computerized fundus atlas system for WINDOWS. The classical normal and abnormal fundus photoes were digitalized and saved in real color image format. The graphics technology was used to label the area and the feature of the abnormal section with WINDOWS atom functions. When these functions run, the graphics and the texts, which label the area of the selected section and describe their features, were displayed in a carton mode background funds image.

To study the effect of jujuboside A (JuA), one constituent of Chinese herbal medicine Ziziphus jujuba Mill Var spinosa (Bunge) Hu,on the penicillin sodium induced hyperactivity in rat CA1 neurons in vitro.Hippocampal slices were obtained from the Sprague-Dawley rat brain and populational signals were measured from CA1 neurons of hippocampal slices using the extracellular recording technique.Penicillin sodium of 500, 1000, and 2000 kU/L were found to excite hippocampal CA1 neurons in a concentration-dependent manner in vitro. This excitatory effect of penicillin sodium could be inhibited by phenobarbital sodium of 0.02 - 0.05 g/L and JuA of 0.05 - 0.10 g/L.A high dose of JuA can inhibit the hyperactivity of hippocampal CA1 area induced by penicillin sodium. The inhibition of the amplitude of the first population spike (PS) and the latency of PS are more pronounced than the slope of the field excitatory post-synaptic potential.

The purpose of the present study was to investigate transcripts of perforin, granzyme B, and Fas ligand (FasL) in heart transplants undergoing rejection.Quantitative reverse transcriptase-polymerase chain reaction was applied for mRNA detection in 29 endomyocardial biopsy specimens from 11 cardiac allograft recipients.The mRNA levels of granzyme B, perforin, and FasL were higher (P0.05) in biopsy specimens with rejection than in biopsy specimens without rejection (granzyme B, 0.53 vs. 0.09; perforin, 0.34 vs. 0; FasL, 0.57 vs. 0.36). In prerejection biopsy specimens, granzyme B and FasL levels were significantly higher than in biopsy specimens without rejection. Any two of the three transcripts were increased in 100% of prerejection, in 92% of rejection, and in 36% of no rejection biopsy specimens (P0.04).The assessment of intragraft levels of cytotoxic T lymphocyte effector molecule mRNA represents a valuable tool in the monitoring of cardiac allograft rejection, especially considering the predictive value for warning of impending acute rejection.

In order to investigate the role of beta-endorphin in the regulation of humoral immune function of rats during acute hypoxia, the effects of beta-endorphin on mitogen-induced spleen lymphocyte DNA synthesis and hemolysin formation as well as IgG production to chicken egg albumen were observed. It was found that after rats received icv injection of beta-endorphin (1 ng/rat), the T-lymphocyte DNA content, the hemolysin-forming capacity of SRBC-sensitized rats and the IgG level were reduced significantly compared with the control (icv normal saline). Similar suppressive effects on immune function could be found after rats were exposed to 7 km altitude in a hypobaric chamber for 48 h. Pretreatment with icv injection of naltrexone, the immunosuppressive effects of acute hypoxia were partially blocked. Icv administration of beta-endorphin produced an increase in splenic cathcholamines, similar to those of hypoxia treated group (7 km for 12 h). All the above findings suggest that beta-endorphin may modulate the immune response to hypoxia stress via opioid receptor and its inhibitory action may be mediated by activating sympathetic nervous system.

The effects of arginine-vasopressin (AVP) on some immune parameters of rats were studied by using icv of AVP. The hemolysin to sheep red blood cell and IgG production were enhanced by icv of 100, 800 ng AVP. Both the actions were partly blocked by icv preinjection of the V1 receptor antagonist DPAVP. It was also found that the NE and corticosterone levels in the serum of rats were significantly reduced after icv of AVP. The above results suggest that AVP executes immunofunctions through V1 receptors in the paraventricular nucleus of the brain. Considering our previous results, the AVP enhanced immunofunction may be related to its inhibition of corticotropin releasing factor release or the excitatory activity of sympathetic nerves.

Health is a global concern. Although nursing is a global profession, most schools of nursing concentrate on teaching health exclusively within the context of their own nation. Sister-school partnerships that cross national boundaries are one way of extending the learning opportunities of faculties and students. An example of a 5-year partnership is described and analysed.

Although treatment of LEW rats with Rapamycin (RPM) prolongs the survival of LBNF1 cardiac allografts to ca. 50 days, it fails to prevent late activation of macrophage/monocyte-associated chemokines.A 7-day course with RPM or CsA was introduced at 1-week posttransplant in RPM-pretreated hosts. At day 35, intragraft mRNA expression of IL-2, IFN-gamma, TGF-beta, IL-12 (p40), MCP-1, and RANTES was evaluated.RPM as a sequential treatment markedly inhibited mRNA levels coding for IL-2/IFN-gamma, and MCP-1. However, RPM monotherapy failed to prevent the expression of IL-12 (p40) and RANTES. Adjunctive treatment with CsA markedly depressed IL-12 (p40) and RANTES, and to a lesser extent MCP-1 mRNA, as compared with RPM-treated groups. Significant increase of TGF-beta mRNA expression was revealed after sequential RPM and adjunctive CsA treatment.A combination of RPM and CsA is more effective in restraining mRNA expression than RPM alone; however, either therapy is associated with TGF-beta hyperexpression.

Severity of inflammatory bowel disease in IL-10 gene-targeted mice is in part determined by genetic background. In the current study, a targeted IL-10 gene was transferred into the C3H/HeJBir substrain, known to exhibit high T-cell and B-cell responses to enteric flora, and to be highly sensitive to colitigenic stress. IL-10-deficient C3H/HeJBir mice developed early onset colitis in contrast to IL-10-deficient C57BL/6J congenic mice. Histopathologic analysis of disease in C3H/HeJBir.Il10-/- and C57BL/6J.Il10-/- mice showed significant differences at all ages studied. Hybrids of these congenic strains (F1.Il10-/-) were produced to study the mode of inheritance as well as subphenotypes that correlated with histopathology. Lesions in F1 mice were intermediate between parental strains. C3H-contributed subphenotypes that correlated best with histopathology were peripheral blood granulocyte percentage, serum amyloid A concentration, spleen weight/body weight ratio, and mesenteric lymph node weight/ body weight ratio. Neither enhanced humoral immunity (secretory IgA, anti-Escherichia coli cellular membrane Ig) characteristic of C3H/HeJBir, nor T-cell percentages in peripheral blood correlated as well. This study represents a necessary step in elucidating murine genetic modifiers controlling colitis sensitivity.

In an attempt to identify potential markers of steroid-resistance in focal segmental glomerulosclerosis (FSGS) we evaluated intra-graft gene expression of IkappaBalpha, nuclear factor-kappaB (NF-kappaB), and angiotensinogen in 60 biopsies from 27 pediatric renal transplant recipients. Intra-graft NF-kappaB expression was significantly elevated in recurrent FSGS (R-FSGS) (218.3 + 55.6 ag/fg versus NON-FSGS 121.1 + 19.9, P=0.04) but not in acute rejection. NF-kappaB:IkappaBalpha ratios were higher in cadaveric donor versus living related donor recipients (15.7 + 2.8 vs. 8.8 + 1.3, respectively, P=0.015), and in African-American versus Caucasian recipients (15.6 + 2.9 vs. 9.1 + 1.3, respectively, P=0.03). Intra-graft angiotensinogen gene expression was significantly elevated in R-FSGS (30.5 + 8.8 ag/fg R-FSGS vs. 16.0 + 4.7 NON-FSGS, P=0.009). We conclude that increased NF-kappaB and angiotensinogen gene expression are associated with R-FSGS. Increased NF-kappaB:IkappaBalpha ratios are associated with cadaveric donor recipients and African-American race.

Xenograft rejection is a complex response in which macrophages and other effector cells are activated by CD4+ T cells. Initiation and regulation of this response is in part mediated by cytokines. In this study we test the hypothesis that xenograft destruction is an interleukin- (IL) 10 responsive, macrophage-mediated event.To study the effect of the systemic administration of IL-10 on pancreatic islet xenograft rejection, a fusion protein of IL-10/Fc was used. This immunoligand possesses the bioavailability of IL-10 and the long circulating t1/2 in vivo, characteristic of Ig. Wistar rat islets were transplanted into C57BL6 mice. IL-10/Fc was administered either immediately before transplantation or in the posttransplant period.Both therapeutic protocols prolonged xenograft survival. Macrophage effector function was reduced in IL-10/Fc-treated mice, with a reduced macrophage infiltrate, reduced IL-12 and tumor necrosis factor-alpha gene expression and reduced serum NO2- levels. Although the number of T cells infiltrating islet grafts was not reduced, T cell effector function was inhibited in IL-10/Fc-treated animals with reduced interferon-gamma and IL-4 gene expression, reduced anti-donor cytotoxicity by recipient splenocytes and reduced anti-donor IgG1 antibody production. Ultimate rejection of the xenografts appears to be mediated by a CD4+ T cell dependent mechanism probably as a result of inadequate inhibition of IL-12 production by macrophages.IL-10/Fc prolonged rat pancreatic islet xenograft survival by inhibiting macrophage mediated immune responses. The effectiveness of this agent when administered pretransplant suggests it may have a role as an induction agent with potential clinical application.

High affinity IL-2R5 is present on recently activated but not on resting or memory T cells. Selective targeting of T cells bearing high affinity IL-2R is an attractive therapy for many T cell-dependent cytopathic disease processes. A variety of rodent mAbs directed against the alpha-chain of the IL-2R, as well as IL-2 fusion toxins, have been used in animals and humans to achieve selective immunosuppression. Here we report on the development of a novel IL-2R targeting agent, a cytolytic chimeric IL-2/Fc fusion protein. This immunoligand binds specifically and with high affinity to IL-2R and is structurally capable of recruiting host Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activities. The Ig component ensures an extended circulating t1/2 of 25 h following systemic administration. To subsequently explore the mechanisms of the antidiabetogenic effects of IL-2/Fc, we have mutated the FcR binding and complement C1q binding (Fc-/-) domains of the Fc fragment to render the Fc unable to direct Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activities. In a model of passive transfer of diabetes in nonobese diabetic mice, lytic IL-2/Fc, but not nonlytic IL-2/Fc-/-, exhibited striking antidiabetogenic effects. Together with the negligible potential of IL-2/Fc for immunogenicity, this finding forecasts that cytolytic IL-2/Fc may offer a new therapeutic approach for selective targeting of auto and alloimmune T cells.

Using certain immunosuppressive regimens, IL-2 knockout (KO) mice, in contrast to wild-type (wt) controls, are resistant to the induction of allograft tolerance. The mechanism by which IL-2 regulates allograft tolerance is uncertain. As IL-2 KO mice have a profound defect in Fas-mediated apoptosis, we hypothesized that Fas-mediated apoptosis of alloreactive T cells may be critical in the acquisition of allograft tolerance. To definitively study the role of Fas in the induction of transplantation tolerance, we used Fas mutant B6.MRL-lpr mice as allograft recipients of islet and vascularized cardiac transplants. Alloantigen-stimulated proliferation and apoptosis of Fas-deficient cells were also studied in vivo. Fas mutant B6.MRL-lpr (H-2b) mice rapidly rejected fully MHC-mismatched DBA/2 (H-2d) islet allografts and vascularized cardiac allografts with a tempo that is comparable to wt control mice. Both wt and B6.MRL-lpr mice transplanted with fully MHC-mismatched islet allografts or cardiac allografts can be readily tolerized by either rapamycin or combined costimulation blockade (CTLA-4Ig plus anti-CD40L mAb). Despite the profound defect of Fas-mediated apoptosis, Fas-deficient T cells can still undergo apoptotic cell death in vivo in response to alloantigen stimulation. Our study suggests that: 1) Fas is not necessarily essential for allograft tolerance, and 2) Fas-mediated apoptosis is not central to the IL-2-dependent mechanism governing the acquisition of allograft tolerance.

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.

We have shown previously that heightened expression of the cytotoxic lymphocyte (CL) effector genes perforin (P), granzyme B (GB), and Fas ligand (FasL), is closely correlated with acute allograft rejection, particularly when two or more target genes are up-regulated.We used quantitative reverse transcription-polymerase chain reaction to analyze CL gene expression from peripheral blood leukocytes (PBLs) and renal allograft biopsies in 31 paired samples of PBLs and renal tissue from 25 renal allograft recipients. Our aims were (1) to determine whether the expression of CL gene expression in PBLs correlates with expression of these genes in renal allograft biopsy tissue and (2) to determine whether CL gene expression in PBLs correlates with the histological diagnosis.Coordinate gene expression in PBLs and acutely rejecting allografts was found in 9/11 (82%) for P, 07/11 (64%) for GB, and 10/11 (91%) for FasL. Coordinate absence was found in 15/20 (75%) for P, 17/20 (85%) for GB, and 16/20 (80%) for FasL in nonrejecting allografts. Furthermore, up-regulation of any two genes in PBLs correlated with pathological diagnosis of rejection with excellent positive (100%) and negative (95%) predictive values.Coordinate CL gene expression in PBLs and the allograft is usually detected. CL gene expression in PBLs is closely associated with a pathologic diagnosis of rejection. CL gene expression in PBLs may serve as a noninvasive method of monitoring for renal allograft rejection.

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.

T cell growth factors (TCGFs) play a critical role in allograft rejection by promoting the activation and proliferation of alloreactive T cells. To determine whether IL-2 and IL-4 are of quintessential importance in allograft rejection and to identify possible alternative TCGFs, we have bred IL-2(-/-) and IL-4(-/-) double knockout (DKO) mice and studied islet allograft rejection using the DKO mice as allograft recipients. Although mononuclear leukocytes from DKO mice did not mount a proliferative response in vitro in response to anti-CD3 stimulation, crude islet allografts were vigorously rejected by DKO mice (mean survival time 17 +/- 7, n = 8) as compared with wild-type controls (mean survival time 13 +/- 4, n = 7). Treatment of DKO mice with anti-CD3 or rapamycin markedly prolonged the islet allograft survival. An analysis of intragraft cytokine gene transcripts showed robust expression of IL-7 and IL-15. In contrast, intragraft IL-9 gene transcripts were not detected in either wild-type or DKO mice. Provision of exogenous IL-2, IL-4, IL-7, or IL-15, but not IL-9, supports the proliferation of anti-CD3 activated DKO splenic leukocytes in vitro. Blocking the common gamma c of IL-2 receptor, a shared essential signaling component by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, prolonged the survival of islet allografts in DKO mice. Hence, a T cell dependent allograft rejection enabled by rapamycin-sensitive signals or signals mediated by binding of the gamma c chain occurs in the absence of both IL-2 and IL-4. Non-T cell-derived TCGFs, especially IL-7 and IL-15, may play an active role in supporting allograft rejection.

The interaction between CD40 and its ligand CD40L is essential for the development and maintenance of vigorous immunity. We have sought to determine: (i) whether a heightened level of CD40L transcripts is evident during acute allograft rejection and (ii) the kinetics of CD40L gene expression during allograft rejection.By using quantitative reverse transcriptase-assisted polymerase chain reaction techniques, we found that heightened CD40L gene expression is a correlate of acute human renal allograft rejection (P0.01).In a murine model of MHC-mismatched islet allografts, our results showed that CD40L transcripts were rarely detected at day 2 after transplantation, but were remarkably heightened at day 5 after transplantation. The transcript levels then steadily increased and peaked at the time of rejection.These data suggest that therapy aimed at blocking the CD40 to CD40L interaction should be applied during the immediate posttransplant period.

Sensitization to donor antigens is a problem of growing magnitude in clinical transplantation. At a molecular level, this is due to the interaction between antigen bearing antigen-presenting cells and recipient T cells and involves both antigen presentation and co-stimulation.Allogeneic islet transplantation was performed using DBA/2J donors and B6AF1 recipients. Four weeks before transplantation, recipient animals were given donor-specific transfusion (DST) alone, DST + CTLA4Ig, DST + control IgG, or no treatment. Graft loss was defined as a blood glucose300 mg/100 ml.Administration of DST + control IgG 4 weeks before transplantation resulted in accelerated rejection due to presensitization (median survival time of 8 days, compared with 14.5 days for the no-treatment group). Animals treated with CTLA4Ig in combination with DST had a median survival time of 12 days, compared with 8 days for DST + IgG.CTLA4Ig attenuates the tempo of accelerated rejection in this islet allograft model of presensitization, but does not prolong allograft survival as compared with no treatment.

We have been successful in our efforts to develop a long lived noncytolytic murine IL-10/Fc fusion protein. In the nonobese diabetic mouse (NOD) model, administration of IL-10/Fc from 5 to 25 wk of age completely prevented the occurrence of diabetes. Moreover, these mice remained disease-free long after cessation of IL-10/Fc therapy. Immunohistochemistry studies show that IL-10/Fc treatment inhibits expression of TNF-alpha, proinflammatory cytokine, as well as Th1-type cytokines, IL-2 and IFN-gamma, but promotes expression of IL-4 and IL-10, Th2-type cytokines, by islet-infiltrating leukocytes. In an adoptive transfer model of diabetes in NOD mice, we found that: 1) IL-10/Fc treated hosts bear leukocytes that block expression of diabetes and 2) these leukocytes persisted even 8 wk after cessation of IL-10/Fc treatment. The potent antidiabetogenic effects provided by IL-10/Fc in the NOD model, together with its apparent lack of systemic toxicity, are notable.