BACKGROUND: Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). METHODS: Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. RESULTS: The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). CONCLUSIONS: IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.
Animals ; Antibodies/blood/immunology ; Antigens, CD40/immunology ; Area Under Curve ; CD40 Ligand/immunology ; Disaccharides/*immunology ; Epidermal Growth Factor/blood ; Graft Rejection/*immunology ; Immunoglobulin G/blood ; Immunoglobulin M/*blood ; Immunosuppressive Agents/therapeutic use ; *Islets of Langerhans Transplantation ; Macaca mulatta ; ROC Curve ; Swine ; Transplantation, Heterologous

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.
Animals ; B7-1 Antigen ; metabolism ; Bone Marrow Transplantation ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Immune Tolerance ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Signal Transduction ; Transplantation, Homologous

OBJECTIVETo explore the effect of CD40 blockade in suppressing acute rejection of renal graft in rats.

METHODSWith Wistar rats as the donor and male SD rats as the recipients, rat models of acute renal graft rejection was established. The rat models were divided into therapy group and control group, and in the former group, CD40 ligand (CD40L) monoclonal antibody was injected daily for 4 consecutive days starting on the next day following kidney transplantation. On day 5 after the transplantation, the renal graft was harvested for histological examination, and graft rejection was evaluated semiquantitatively.

RESULTSThe mean semiquantitative score of the renal graft was 0.63∓0.52 in the therapy group, significantly lower than that of the control group (3.72∓1.48, P<0.05).

CONCLUSIONCD40L monoclonal antibody can inhibit acute renal graft in rats.

Animals ; Antibodies, Monoclonal ; pharmacology ; therapeutic use ; CD40 Antigens ; antagonists & inhibitors ; immunology ; CD40 Ligand ; immunology ; Female ; Graft Rejection ; drug therapy ; prevention & control ; Graft Survival ; drug effects ; Kidney Transplantation ; adverse effects ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

Previously, we reported that CD40-induced production of reactive oxygen species (ROS) by NADPH oxidase requires the TNF receptor-associated factor (TRAF) 3, as well as the activities of phosphatidylinositol 3-kinase (PI3K) and Rac1. Here we investigated the possible mechanisms of the production of ROS after CD40 ligation in B cells. We describe an alternative ROS production pathway that is triggered by CD40 ligation, involves 5-lipoxygenase (5-LO), and results in activation of p38 MAPK. Our studies in Raji human B lymphomas revealed that CD40-induced ROS production by 5-LO also requires the activities of PI3K and Rac1. In contrast to the NADPH oxidase pathway, however, TRAF molecules are not required for the CD40-induced ROS production by 5-LO. The association of CD40 with 5-LO is dependent on CD40 ligation in Raji B cells, and co-immunoprecipitation experiments using epitope-tagged proteins transiently expressed in human embryonic kidney 293T cells revealed the role of the regulatory subunit of PI3K, p85, in this association. Collectively, these data suggest a separate pathway for the CD40-induced ROS production in B cells and demonstrate that this pathway requires 5-LO via direct association of p85 with both CD40 and 5-LO.
Antigens, CD40/*metabolism ; Arachidonate 5-Lipoxygenase/*metabolism ; B-Lymphocytes/*enzymology/immunology ; CD40 Ligand/metabolism ; Cell Line, Tumor ; *Enzyme Activation ; HEK293 Cells ; Humans ; Phosphatidylinositol 3-Kinases/metabolism ; Protein Binding ; *Reactive Oxygen Species/metabolism ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/*metabolism ; rac GTP-Binding Proteins/metabolism

OBJECTIVEThe pathogenesis of bronchiolitis has not been fully identified. Immune function abnormality following virus infection may be associated with the pathogenesis. CD40 and CD40 ligand (CD40L) is a pair of co-stimulatory molecules in immunoreaction. They might play an important role in the development of bronchiolitis. This study aimed to investigate the expression of CD40 and CD40L in peripheral blood mononuclear cells (PBMCs) in children with bronchiolitis and explore their possible roles in the disease.

METHODSThirty children with bronchiolitis, 26 children with bronchopneumonia and 30 healthy children (control) were enrolled. Flow cytometry was used to detect CD40 and CD40L expression in PBMCs. Total serum IgE level was measured using ELISA.

RESULTSCompared with the control group, CD40L expression significantly increased in the bronchiolitis and bronchopneumonia groups (P< 0.05). The CD40L expression in the bronchiolitis group was significantly higher than that in the bronchopneumonia group (P< 0.05). A significantly increased CD40 expression was also found in the bronchiolitis group when compared with the bronchopneumonia and the control group (P< 0.01). Total serum IgE level in the bronchiolitis group was significantly higher than the bronchopneumonia and the control groups (P< 0.01). CD40 and CD40L expression was positively correlated with serum IgE level in the bronchiolitis group (r=0.607, r=0.819, respectively; P< 0.01).

CONCLUSIONSCD40 and CD40L expression in PBMCs and serum IgE level increased and there is a positive correlation between CD40 and CD40L expression and serum IgE level in children with bronchiolitis. Over-expression of CD40 and CD40L may play an important role in the development of bronchiolitis.

Bronchiolitis ; etiology ; immunology ; CD40 Antigens ; blood ; physiology ; CD40 Ligand ; blood ; physiology ; Female ; Humans ; Immunoglobulin E ; blood ; Infant ; Leukocytes, Mononuclear ; chemistry ; Male

OBJECTIVETo investigate the role of CD40 ligand (CD40L) in dendritic cells (DC) of CEA transgenic mice and to evaluate the specific cellular immunity induced by activated DC.

METHODSBone marrow cells of the CEA transgenic mice were used to generate immature dendritic cells under the condition of GM-CSF and IL-4. CD40L was added to activate dendritic cells into mature phenotype. Dendritic cells cancer vaccine was pulsed with CEA526-533 peptide which made the vaccine specific for cancer immunity. The immunophenotype molecules were identified by flow cytometry. The cytokines produced by cells were determined by ELISA. T cells proliferation was measured by (3)H-thymidine essays.

RESULTSImmunophenotype molecules expressions of CD40L-activated dendritic cells were significantly higher than those in control group. IL-12 secretion by CD40L-activated dendritic cells was (937.81+/-51.99) pg/10(6) DC, significantly higher than that in control group [(83.06+/-8.58) pg/10(6) DC, P<0.01]. CD8(+) T cells proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.05), and the secretion of IFN-gamma was(33.900+/-4.550) ng/L, significantly higher than that in control group [(5.226+/-0.460) ng/L, P<0.01]. Splenocytes proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.01), and the secretion of IFN-gamma was (69.802+/-11.407) ng/L, significantly higher than that in control group [(2.912+/-0.562) ng/L, P<0.01].

CONCLUSIONThe method of using CD40L to stimulate bone marrow-delivered dendritic cells promotes the maturation and activation of dendritic cells, which enhances the cellular immunity in CEA transgenic mice.

Animals ; CD40 Ligand ; immunology ; physiology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

OBJECTIVETo examine the in vivo anti-metastatic effect of enhanced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L-OVHM) injected into the spleen on liver metastasis in mice.

METHODSOVHM cells were inoculated into the spleen of 6 to 8 week-old female B6C3F1 (C57BL/6N x C3H/He) mice. The established liver metastasis was identified by histopathology (HE staining). OVHM cells, DNA-pMKITneo-OVHM cells or CD40L-OVHM cells were inoculated into the spleen of female B6C3F1 mice and the expressions of CD11c in splenic cells were detected by flow cytometry. The specific cytotoxicity of splenic cells was detected by MTT assay, and the serum cytokines of IFN-gamma, TNF-alpha, IL-12, IL-4 and IL-10 of the mice were measured by enzyme linked immunoabsorbent assay. The liver metastases and the survival time of the mice were also recorded.

RESULTSThe mouse models with liver metastasis by injecting tumor cells into the spleen of mice were established. The expression of CD11c and the specific killing rate in CD40L-OVHM cells group was significantly higher than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group. The expressions of IFN-gamma, TNF-alpha and IL-12 in the CD40L-OVHM cells group were much more increased than OVHM cells group and DNA-pMKITneo-OVHM cells group, but the expressions of IL-4 and IL-10 in the CD40L-OVHM cells group were decreased significantly (p < 0.05). The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lower than those of DNA-pMKITneo-OVHM cells group and OVHM cells group. The survival time of mice in CD40L-OVHM cells group was also significantly longer than that in the OVHM cells group and DNA-pMKITneo-OVHM cells group (P < 0.05).

CONCLUSIONThe data directly demonstrate that the expression of CD40L in ovarian cancer cells (CD40L-OVHM) can enhance the proliferation and differentiation of dendritic cells in the spleen and induce specific cytotoxic effect of T cells in the spleen, and may regulate the immune function of peripheral blood cells and the immune balance between Th1 cells and Th2 cells, which maybe the possible mechanism induced by CD40L in mice inhibiting the development of liver metastasis.

Animals ; CD11c Antigen ; metabolism ; CD40 Ligand ; genetics ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Liver Neoplasms ; pathology ; secondary ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Ovarian Neoplasms ; metabolism ; pathology ; Spleen ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the effects of gene transfer cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) and anti-cluster of differentiation 154 (CD154) mAb on the rejection of rat islet xenografts.

METHODSHuman islets were infected with the recombinant adenoviruses containing CTLA4-Ig gene. Transduced islets were transplanted under the left kidney capsule of diabetic rats. And then the animal model were treated with anti-CD154 monoclonal antibody. The changes of blood sugar were measured and the survival rates of grafts and transplantation rats were observed after transplantation. The morphological changes of grafts were observed. Expression of CTLA4-Ig and insulin were detected by immunohistochemical staining and cytokines were quantified by ELISA.

RESULTS(1) The blood glucose of transplantation rats decreased to normal level on 2nd day post-transplantation. The average level blood glucose of control group A, anti-CD154mAb treatment group B, transfected group C and associated treatment group D increased on day 8, 18, 25, 36, post-transplantation respectively. (2) The grafts of group A, B, C and D survived for (10.0 +/- 2.1) d, (22.0 +/- 8.2) d, (28.0 +/- 6.5) d and (37.0 +/- 9.3) d respectively. The survival of grafts in group D was significant longer than that in group A, B and C, respectively; The survival of group B and C were significantly prolonged compared with group A and the survival of group B was significantly different with group C (P < 0.05). The survival of transplantation rats were (21.0 +/- 5.7) d, (35.0 +/- 6.5) d, (48.0 +/- 8.5) d and (65.0 +/- 12.5) d in group A, B, C and D, respectively. The survival of transplantation rats compared each other among four groups were same as the survival of grafts (P < 0.05). (3) In control animals (group A), serum IL-2 and TNF-alpha concentration were elevated to a high level within seven days post-transplantation and significantly increased compared with that before transplantation (P < 0.01). (4) Hematoxylin-eosin staining of grafts showed a lot of islets under the kidney capsule of transplantation rats, no inflammatory cell infiltrate and immunohistochemical staining of grafts demonstrated expression of insulin protein at islets in group B, C and D. These grafts positively stained for CTLA4-Ig in group C and D.

CONCLUSIONSGene transfer CTLA4-Ig and anti-CD154mAb treatment can inhibit the rejection of rat islet xenografts and treatment Ad-CTLA4-Ig and anti-CD154 mAb could induce immune tolerance of islet xenografts.

Adenoviridae ; genetics ; Animals ; Antibodies, Monoclonal ; therapeutic use ; CD40 Ligand ; immunology ; Diabetes Mellitus, Experimental ; surgery ; Genetic Vectors ; Graft Rejection ; immunology ; prevention & control ; Humans ; Immunoconjugates ; genetics ; Islets of Langerhans Transplantation ; Rats ; Rats, Wistar ; Transfection ; Transplantation, Heterologous

The study was aimed to detect the expressions of costimulatory molecules on CD3(+)CD4(+) T cells so as to accumulate informations for investigation of mechanism of myelodysplastic syndrome. 11 healthy blood donors as control and 38 patients with MDS de novo were studied. 38 MDS patients were divided into RA/RARS group and RAEB/RAEB-t group according to FAB classification. The expressions of CD28, CD154, CTLA-4, PD-1, CD25 on CD3(+)CD4(+) T cells in peripheral blood were detected by FCM. The results indicated that as compared with normal controls, the expression of CD28 in MDS patients decreased, and CD154 increased. The percentages of CTLA-4, PD-1 and CD25 in MDS were obviously higher than that in normal controls; the differences of CTLA-4, PD-1 and the ratio of CTLA-4/CD28 between RAEB/RAEB-t and RA/RARS were more significant with progressing of MDS. In conclusion, the expressions of costimulatory molecule in MDS patients were abnormal, which may be involved in the pathogenesis of MDS.
Adult ; Antigens, CD ; metabolism ; Apoptosis Regulatory Proteins ; metabolism ; CD28 Antigens ; metabolism ; CD40 Ligand ; metabolism ; CTLA-4 Antigen ; Case-Control Studies ; Female ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lymphocyte Count ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; metabolism ; Programmed Cell Death 1 Receptor ; T-Lymphocytes ; immunology ; metabolism ; Young Adult

Pancreatic islet transplantation can correct the abnormal glucose metabolism of Type 1 diabetes. Although immunosuppressants greatly reduce the acute rejection rate in transplant patients, the long-term side effects can be debilitating. Therefore, researchers are seeking to develop new immunosuppressive regimens that induce maximal levels of immunosuppression with minor side effects. Rosmarinic acid (Ros A) is a secondary metabolite of certain herbs and has multiple biological activities, including anti-inflammatory effects. Here, we have investigated whether treatment of mice with a combination of Ros A and anti-CD154 monoclonal antibody (MR1) improves islet allograft survival in a murine model. After transplantation, the mice were treated with either Ros A, MR1, or both (the "double" treatment). Allograft survival was prolonged in the double-treated animals compared to animals that received only Ros A or MR1. As is the case with the single-treated animals at 15 days after transplantation, the double-treated recipients did not display a significant decrease in the expression of cytokines or the population of activated T cells. Infiltrating CD3+ T cells were reduced in the MR1- or double therapy relative to control or RosA group. However, at the same time point, double-treated graft showed fewer apoptotic cells and increased expression of insulin and glucagons, compared to the single-treatment groups. Furthermore, long-term (>150 days) allografts that were received with double therapy exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to the MR1-treated grafts. In conclusion, treatment with both Ros A and MR1 has a synergistic effect in murine islet allotransplantation.
Animals ; Antibodies, Monoclonal/*pharmacology ; Apoptosis/drug effects ; CD40 Ligand/*immunology ; Cinnamates/*pharmacology ; Cytokines/biosynthesis ; Depsides/*pharmacology ; Diabetes Mellitus, Experimental ; Flow Cytometry ; Glucose/metabolism ; Glucose Tolerance Test ; Graft Survival/*drug effects ; In Situ Nick-End Labeling ; Injections, Intraperitoneal ; Islets of Langerhans/drug effects/pathology ; *Islets of Langerhans Transplantation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Time Factors ; Transplantation, Homologous