Induction of alloimmune tolerance in heart transplantation through gene silencing of TLR adaptors.

Toll-like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4(+) CD25(+) FoxP3(+) regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.

Inhibition of allergic responses by CD40 gene silencing.

BACKGROUND: Gene silencing using small interfering RNA (siRNA) is a potent method of specifically knocking down molecular targets. Small interfering RNA is therapeutically promising, however, treatment of allergic diseases with siRNA has not been explored in vivo. The aim of this study was to evaluate therapeutic effects of CD40 siRNA on inhibition of allergic responses. METHODS: Mice sensitized with ovalbumin (OVA) and alum were treated with CD40 siRNA, scrambled siRNA, or phosphate buffer saline (PBS) alone, and then challenged intranasally with OVA. RESULTS: A significant reduction in nasal allergic symptoms was observed in the CD40 siRNA treated OVA-allergic mice compared to the controls of scrambled siRNA and PBS alone, which is correlated with the decrease of local eosinophil accumulation. CD40 siRNA treatment knocked down CD40 expression on dendritic cells (DCs) in vivo and impaired their antigen presenting function. Treatment with CD40 siRNA resulted in inhibition of OVA-specific T cell response and decrease of interleukin-4 (IL-4), IL-5, and interferon-gamma production from T cells stimulated with OVA. Administration of CD40 siRNA also suppressed CD40 expression on B cells, resulting in down-regulation of OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a levels. Additionally, increased regulatory T cells were observed in the CD40 siRNA treated mice. CONCLUSIONS: The present study demonstrates a novel therapeutic use for siRNA in allergy. CD40 siRNA attenuated allergy through inhibition of DC and B cell functions and generation of regulatory T (Treg) cells.

Improved survival of biolistically transfected mouse islet allografts expressing CTLA4-Ig or soluble Fas ligand.

BACKGROUND: Pancreatic islet transplantation is limited because of immune rejection of the transplanted tissue. Long-term survival of allogeneic pancreatic islet grafts in the absence of systemic immunosuppressive agents should be possible by transfecting the islets directly with DNA encoding immunoregulatory molecules. Localized production of these molecules should affect only the immune cells that come into the vicinity of the foreign tissue. We investigated whether local expression of human CTLA4-Ig or soluble human Fas ligand from biolistically transfected mouse islets would have a protective effect on allograft survival. METHODS: Isolated CBA (H2k) islets were biolistically transfected using the gene gun. The experimental groups were naked gold particles (n=6), empty vector DNA (n=5), DNA encoding human CTLA4-Ig (n=8), or soluble human Fas ligand (n=5). Secretion of the transfected gene product was confirmed by screening islet culture supernatants for protein production using a sandwich ELISA. The blasted islets were transplanted under the kidney capsule of alloxan-diabetic BALB/c (H2d) recipients. RESULTS: Control grafts survived for 23 days, on average. CTLA4-Ig-transfected islets showed a bimodal distribution: 50% of cases survived > or = 46 days and 50% were similar to the controls. In the soluble human Fas ligand group, 80% of grafts survived > or = 50 days. There was no correlation between graft survival times and pretransplant levels of protein production. CONCLUSION: Our results indicate that local production of human CTLA4-Ig or soluble human Fas ligand by biolistically transfected islets can promote allograft survival. This approach should be valuable as a potential immunoprotective therapeutic strategy in tissue transplantation.

Identification of an epitope for T-cells correlated with antibody response to hepatitis B surface antigen in vaccinated humans.

Two antigenic T-cell epitopes of HBsAg, designated HBs 16-31 and HBs 81-99, were identified using synthetic peptides and HBsAg-specific T-cell lines. HBs 16-31 was recognized by five HBsAg-specific T-cell lines from vaccinees with both high and low antibody titers, whereas HBs 81-99 was recognized by two T-cell lines derived from vaccinees with high antibody titers. The antibody titer against HBsAg was correlated significantly with the proliferation of vaccinee's PBLs in response to HBs 81-99 (r = 0.47) but not to HBs 16-31, suggesting that HBs 81-99 plays a critical role in anti-HBs antibody production in humans vaccinated with HBsAg.

A survey of Gnathostoma larvae in fresh water fish in the valley of the Yangtze River and morphological characteristics of the recovered larvae.

Investigations of the prevalence of larval gnathostomes in fresh water fishes were carried out at the southeastern Yangtze Valley, People's Republic of China, in the periods of October 1989 and November 1990. Fishes were collected from Shanghai, Chenchiang, Nanching, Chiuchiang and Nanchang districts in 1989. Additional sampling in Shanghai district was done at Kunshan, Tien-shanfu, Chingpu and Nanhui. Species of fishes collected were Channa argus (110), Siniperca chuatsi (24) and Silurus asotus (2). Muscle tissue of the fishes was dissected into small pieces, sliced and then examined under a dissecting microscope. The viscera were pooled by species in groups of 4 or 5 individuals, homogenized, and were then digested overnight in artificial gastric-juice at 37 degrees C. Four encysted larvae were recovered from the muscle tissue of four C. argus. Thirty-four larvae were obtained from digestion of viscera. A total of 38 larvae were recovered. Eighteen of 38 larvae were examined morphologically and they were able to be divided into three types by their body length; 5 early third-stage larvae (0.58-0.86 mm), 12 third-stage larvae (1.12-2.61 mm), and one advanced third-stage larva of 4.86 mm. Light and scanning electron microscopy revealed that the former two types had characteristics of Gnathostoma hispidum and the last one had those of G. spinigerum. In 1990, we investigated fish near Hongtze-hu and Tai-hu lakes. A total of 553 fishes belonging to 12 genera and 12 species were examined. Seventeen larvae were recovered from the viscera of G. argus and Monopterus albus. These larvae were identified as G. hispidum.

Preventing renal ischemia-reperfusion injury using small interfering RNA by targeting complement 3 gene.

The complement system is one of the important mediators of renal ischemia-reperfusion injury (IRI). We hypothesized that efficient silencing of C3, which is the central component on which all complement activation pathways converge, could be achieved using small interfering RNA (siRNA), and that this would result in overall inhibition of complement activation, thereby preventing IRI in kidneys. A series of experiments was conducted, using a mouse model of IRI and vector-delivered C3-specific siRNA. We demonstrated the following: (1) renal expression of C3 increases as a result of IRI; (2) by incorporation into a pRNAT U6.1 vector, siRNA can be delivered to renal cells in vivo; (3) systemically delivered siRNA is effective in reducing the expression of C3 in an experimentally induced mouse kidney model of IRI; (4) similarly, siRNA reduces complement-mediated IRI-related effects, both in terms of renal injury (as evidenced by renal function and histopathology examination) and mouse mortality and (5) silencing the production of C3 diminishes in vivo production of TNF-alpha. This study implies that siRNA represents a novel approach to preventing IRI in kidneys and might be used in a variety of clinical settings, including transplantation and acute tubular necrosis.

Synergy in induction of increased renal allograft survival after portal vein infusion of dendritic cells transduced to express TGFbeta and IL-10, along with administration of CHO cells expressing the regulatory molecule OX-2.

Dendritic cells (DC), generated from C57BL/6 mouse bone marrow cells cultured with GM-CSF and IL-4 for 9 days, were engineered to express constitutively the cytokines TGFbeta, IL-10, and IL-12, using adenovirus vectors constructed using an E1-deleted replication-deficient recombinant adenovirus carrying the appropriate cDNA for the relevant cytokines (Ad-TGFbeta, Ad-IL-12, or Ad-IL-10). C3H mice receiving nontransduced DC or pretransplant infusion of DC-Ad-LacZ showed increased survival of C57BL/6 renal grafts relative to that of control nonimmunized mice. Transfusion of Ad-IL-12-transduced DC abolished this increased survival, leading to a graft survival equivalent to that of controls with no DC. Optimal graft survival was seen in the group receiving a mixture of DC transduced with constructs for both IL-10 and TGFbeta. There was a correlation between increased graft survival and both inhibition of the induction of CTL and enhancement of a polarization to produce type-2 cytokines (IL-4, IL-10, and TGFbeta) on antigen-specific restimulation in vitro. These effects were more pronounced following concomitant infusion of CHO cells transfected with a full-length cDNA for murine OX-2.

An immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that prolongs allo- and xenograft survival.

We have established that, in mice receiving donor-specific immunization by the portal vein, the increased graft survival seen is associated with the increased expression of a molecule (OX-2) on a subpopulation of dendritic cells (DC), and polarization of cytokine production to type 2 cytokines on Ag-specific restimulation of cells from these mice. Furthermore, infusion of a mAb to OX-2 blocks both the increased graft survival and the altered cytokine production seen. We have constructed an immunoadhesin in which the extracellular domain of OX-2 is linked to the murine IgG2a Fc region, and we have expressed this molecule (OX-2:Fc) in a eukaryotic (baculovirus) expression system. Incubation of lymphocytes with 50 ng/ml OX-2:Fc inhibits a primary mixed lymphocyte reaction in vitro, as assayed by proliferation and induction of cytotoxic T cells, and also alters cytokine production with decreased IL-2 (IFN-gamma) production and increased IL-4 (IL-10) production. Similarly, in vivo infusion of OX-2:Fc promotes increased allo- and xenograft (both skin and renal grafts) survival and decreases the Ab response to sheep erythrocytes. Our data suggest this molecule might have clinical importance in allo- and xenotransplantation.

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.