Gene gun approches for DNA vaccine and cytokine gene therapy in protozoan parasite infection

The particle-mediated method for gene delivery with a gene gun utilizes a shock wave to accelerate DNA-coated gold particles into target cells or tissues. This gene delivery method is effective in various somatic tissues in vitro and in vivo. We have, herein, applied this gene delivery system to DNA vaccine and cytokine gene therapy for protozoan parasite infections. We used cDNA encoding 47 kDa of Plasmodium falciparum serine repeat antigen (SERA) that is a vaccine candidate antigen and did SERA DNA immunization with mice using gene gun. Significant SERA-specific antibodies (Abs) were observed by SERA DNA immunization. Furthermore, these Ab responses were enhanced and regulated by coinoculation of cytokine expression plasmid. For other application, we examined the effects of in vivo IL-12 gene treatment on the course of infection with obligate protozoa, Trypanosoma cruzi. Transfer with IL-12 expression plasmid in vivo regulated systemic immune responses and furthermore this treatment controlled the progression of experimental trypanosomiasis. Therefore, this gene gun approach may be a useful for DNA vaccine and gene therapy in a wide spectrum of diseases other than the protozoan parasite infection

Cathepsin B-inhibitor promotes the development of Th1 type protective T cells in mice infected with Leishmania major

BALB/c mice are genetically susceptible to infection with Leishmania major (L. major). When such mice infected with L. major were treated with specific inhibitors of cathepsin B, a lysosomal cysteine protease that digests exogenous antigenic proteins, the mice acquired resistance against L. major infection. T cells from these mice produced large amounts of IFN-γ and low amounts of IL-4 as compared with those of untreated BALB/c mice. In addition, the mice treated with cathepsin B inhibitor produced a high titer of IgG2a specific antibodies and only low titers of IgG1 and IgE antibodies. This type of response is in contrast with the high specific IgG1 or IgE antibody responses which are the usual antibody responses in BALB/c mice infected with L. major. These findings indicate that cathepsin B may be critically involved in processing antigens of L. major to promote exclusively the development of Th2 type CD4+T cell responses

Molecular cloning of a cystatin from parasitic intestinal nematode, Nippostrongylus brasiliensis

A novel member of the cystatin family, nippocystatin (NbCys), was identified from excretory-secretory (ES)-products of a nematode Nippostrongylus brasiliensis, and the cDNA was cloned and sequenced. The mRNA of NbCys was confirmed to be expressed in both larvae and adults of the parasite. NbCys was translated as a proform with a single domain for secretion and was detected as a 14-kDa mature form in ES-products of the adult worm. Recombinant protein of NbCys profoundly inhibited the activity of cysteine proteases such as cathepsin L and B, but not that of cathepsin D, an aspartic protease. Furthermore, the ES-products had also been confirmed to inhibit cysteine proteases. Taken together, NbCys may play a role in evasion of N. brasiliensis from host defense systems, since cysteine proteases are known to participate in immune systems of infected hosts

Experimental study of combined treatment with tacrolimus and donor splenocytes via the portal vein in small bowel transplantation

We previously reported that the combined treatment of perioperative administration of donor splenocytes via the recipient's portal vein (DSPV) and a short-course Tacrolimus significantly prolonged the survival of fully allogenic grafts in rat small bowel transplantation (SBTX). In the present study we examined whether this effect depended on the quantity of the administered alloantigens in DSPV. In addition, we examined the expression of the surface antigen on T cells of the splenocytes and the induced toleragenic factor, according to the tolerant recipients which in our previous report had shown the prolongation of allogenic transplant small bowel graft survival by the combined treatment of DSPV (1×108 donor splenocytes) and a short-course Tacrolimus. Donor splenocytes were prepared from Brown-Norway (BN (RT1n)) rat spleens for Lewis (LEW (RT1l)) recipients. The recipients (n=10), treated with a short course of Tacrolimus (0.5mg/kg, 0 to 3 days postoperatively) only showed graft rejection with an average of 6.3±1.0 days postoperatively. However, the combined treatment, consisting of DSPV of 1×108 donor splenocytes and a short course Tacrolimus significantly prolonged graft survival to 12.7±2.1 days (n=12, P<0.01). DSPV of less than 1×108 donor splenocytes (5×107 cells and 2.5×107) could not prolong the graft or animal survival under a short-course Tacrolimus treatment. In the tolerant recipients, the CD4 and CD8 percentages of splenocytes were not significantly different from those of control rats or recipients that were treated with short-course Tacrolimus alone. Neverthless, the percentage of Tcr-αβ+ cells expressing IL-2 receptor (R) was significantly lower than in either control rats or the recipients with short-course Tacrolimus. In the suppression assay to one-way mixed lymphocyte response, a toleragenic factor was suggested to the present in the serum of the tolerant recipients. In the present study, it was suggested that the effects of the combined treatment of DSPV and short-course Tacrolimus for the prolongation of graft survival in the rat allogenic SBTX should depended on the quantity of the antigens administered into the portal vein. The beneficial effects of this treatment were reflected in the suppression of IL-2R on the recipient's splenocytes, and tolerogenic factor(s) might subsequently be induced in the tolerant recipient's serum

Role of innate immune cells in protection against Toxoplasma gondii at inflamed site

The intraperitoneal infection with Toxoplasma gondii (T. gondii) caused accumulation of γδ T, NK, NK1.1+T-like (NKT) cells at inflamed sites. To clarify the roles of these cells in protection against T. gondii at the inflamed sites, BALB/c mice were depleted of γδ T, NK, NK and NKT cells by treatment with antibody against TCR-γδ, asialoGM1 or Interleukin-2 receptor β-chain (IL-2Rβ), respectively, prior to infection. Mice treated with anti-TCR-γδ monoclonal antibody (mAb) became more susceptible to infection, whereas mice treated with anti-IL-2Rβ mAb acquired resistance. Treatment with anti-asialoGM1 Ab showed no effect. We previously reported that heat shock protein 65 (HSP65) in macrophages induced by γδ T cells plays an essential role in protective immunity against T. gondii infection, by preventing apoptotic death of infected macrophages. In the present study, we showed that treatment with anti-IL-2Rβ mAb, but not with anti-asialoGM1 Ab, enhanced the HSP65 induction in macrophages, and inhibited Interleukin-4 (IL-4) expression in nonadherent peritoneal exudate cells. Furthermore, neutralization of endogenous IL-4 by anti-IL-4 mAb enhanced the HSP65 induction in macrophages. These findings suggest that NKT cells, but not NK cells, negatively regulate the protective immunity against T. gondii infection possibly by producing IL-4and suppressing HSP65 induction

Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor–β1

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell–specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-β1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-β1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-β1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-β1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-β1 and IL-10 through modulating STAT3 activation

CD8+ T-cell Activation in Mice Injected with a Plasmid DNA Vaccine Encoding AMA-1 of the Reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique