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Malaria vaccines based on ookinete surface proteins (OSPs) of the malaria parasites block oocyst development in feeding mosquitoes and hence disrupt the parasite life cycle and prevent the disease from being transmitted to other individuals. To investigate whether a noninvasive mucosal vaccination regimen effectively blocks parasite transmission in vivo, Plasmodium yoelii Pys25, a homolog of the Pfs25 and Pvs25 OSPs of Plasmodium falciparum and Plasmodium vivax, respectively, was intranasally (i.n.) administered using a complement-deficient DBA/2 mouse malaria infection model, in which a highly elevated level of oocysts develops in feeding mosquitoes. Vaccinated mice developed a robust antibody response when the vaccine antigen was given together with cholera toxin adjuvant. The induced immune serum was passively transferred to DBA/2 mice 3 days after infection with P. yoelii 17XL, and Anopheles stephensi mosquitoes were allowed to feed on the infected mice before or after serum transfusion. This passive immunization completely blocked oocyst development; however, immune serum induced by the antigen or adjuvant alone did not have such a profound antiparasite effect. Further, when i.n. vaccinated mice were infected with the parasite and then mosquitoes were allowed to directly feed on the infected mice, complete blockage of transmission was again observed. To our knowledge, this is the first time that mucosal vaccination has been demonstrated to be efficacious for directly preventing parasite transmission from vaccinated animals to mosquitoes, and the results may provide important insight into rational design of nonparenteral vaccines for use against human malaria.

ABSTRACTEimeriaspp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles against Eimeriaspp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier during Eimeriasp. infection. Here, we demonstrated two distinct roles of IEL against infection with Eimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and decreased transforming growth factor β (TGF-β) production. Addition of IFN-γ and TNF-α or supernatants obtained from cultured IEL from E. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-β attenuated the damage of epithelial monolayer and changes in TER caused by IFN-γ and TNF-α. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-γ and TNF-α and a low level of TGF-β in E. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium against E. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28α/β, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.

The processing of foreign protein antigens into peptides requires the participation of various endo/lysosomal proteases in antigen-presenting cells (APCs). In this study, a proenzyme of cathepsin L, procathepsin L, was found to be present in the spleens of naive mice, as demonstrated by immunoblotting. Interestingly, the maturation of cathepsin L from procathepsin L was strongly induced when the host BALB/c mice were immunized with ovalbumin or soluble leishmanial antigen, despite the fact that mouse albumin, a kind of self-antigen, did not have such a potential. Furthermore, foreign antigens, but not self-antigens, could increase the activity of cathepsin L, probably being mediated by interferon-γ, as demonstrated by in vivo and in vitro experiments. As cathepsin L matured, the efficiency of antigen processing was increased in APCs. These results suggest that endo/lysosomal cathepsin L plays an important role in the immune regulation via antigen processing even in peripheral lymphoid tissues as well as in the thymus.

ABSTRACTTransmission-blocking vaccines are one strategy for controlling malaria, whereby sexual-stage parasites are inhibited from infecting mosquitoes by human antibodies. To evaluate whether the recently clonedPlasmodium vivaxproteins Pvs25 and Pvs28 are candidates for a transmission-blocking vaccine, the molecules were expressed in yeast as secreted recombinant proteins. Mice vaccinated with these proteins adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens, although for Pvs28, this response was genetically restricted. Antisera against both recombinant Pvs25 and Pvs28 recognized the corresponding molecules expressed by cultured sexual-stage parasites isolated from patients with P. vivaxmalaria. The development of malaria parasites in mosquitoes was completely inhibited when these antisera were ingested with the infected blood meal. Pvs25 and Pvs28, expressed inSaccharomyces cerevisiae, are as yet the only fully characterized transmission-blocking vaccine candidates against P. vivaxthat induce such a potent antiparasite response.

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2d) and in susceptible BALB/c mice (H-2d), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Zhang, M., Hisaeda, H., Tsuboi, T., Torii, M., Sakai, T., Nakano, Y., Ishikawa, H., Maekawa, Y., Good, R. A., and Himeno, K. 1999. Stage-specific expression of heat shock protein 90 in murine malaria parasite Plasmodium yoelii. Experimental Parasitology93, 61–65.

Fas Ag is a cell surface molecule that transduces the signal for apoptosis. Since mesangial cells (MC) play important roles in regulating glomerulonephritis, we investigated regulatory mechanisms of Fas system in MC. Fas Ag was expressed on MC from normal mice. This Fas Ag expression was down-regulated by inducing proliferation with platelet-derived growth factor or 18% fetal bovine serum, but was reversed when cycloheximide was added to the culture. Anti-Fas Ab alone did not induce apoptosis in MC, but MC became susceptible to apoptosis induced by anti-Fas Ab if actinomycin D or cycloheximide was added. Noteworthy findings are that mRNA of Fas ligand was expressed in MC. Taken together, MC appears to control the proliferation by regulating Fas system-mediated apoptosis, at least in part, through an autoregulatory mechanism with Fas Ag and Fas ligand expressed on their own MC.

We reported that LEC rats are genetically deficient in the development of thymic CD4+8−cells and that this defect is caused by bone marrow (BM)‐derived stem cells. To determine which BM‐derived cells are responsible for the arrest of T‐cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC‐TG and LEA‐TG mice, respectively). We then examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat‐derived CD4+T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC‐TG mice. Furthermore, the peripheral LN cells in LEC‐TG mice appeared to be functional. These cells produced IL‐2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL‐2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC‐TG as well as LEA‐TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC‐TG mice closely resembled that of LEA rat thymus and that in LEA‐TG mice, suggesting that normal T‐cell differentiation occurred in LEC‐TG mice. The results indicated that BM‐derived progenitor T cells of LEC rats could differentiate to functional CD4+T cells.

A mutant strain of Long-Evans cinnamon (LEC) rats was found to have a novel defect in T-cell maturation, i.e., arrest of differentiation from CD4⁺8⁺ to CD4⁺8⁻, but not to CD4⁻8⁺ thymocytes. Despite the maturational arrest of CD4⁺8⁻ thymocytes, some CD4⁺ T cells occurred in peripheral lymphoid organs in LEC rats. In the present study, expression of adhesion molecules was compared between normal and LEC rat T cells. Three-color flow cytometry analysis showed that the expression pattern of LFA-1 and LECAM-1 of LEC rat CD4⁺ T cells was different from that of normal rat CD4⁺ T cells; F344 rat CD4⁺ T cells were LFA-1⁺, LECAM-1⁺⁺, whereas LEC rat CD4⁺ cells were LFA-1⁺⁺, LECAM-1⁻. However, the expression pattern of these adhesion molecules on LEC rat CD8⁺ T cells was the same as that on F344 rat CD8⁺ T cells, suggesting that LEC rat T cells do not have an intrinsic defect in the expression of these molecules. To address the question of whether the abnormal expression of adhesion molecules is correlated with the thid mutation, we generated WKAH.C-thid congenic rats and analyzed these expression patterns. The expression of adhesion molecules on WKAH.C-thid congenic rat CD4⁺ T cells was the same as that of WKAH rat CD4⁺ T cells, suggesting that abnormal expression of adhesion molecules of LEC rat CD4⁺ T cells is independent of the thid mutation. These results indicate that LEC rat CD4⁺ T cells show a unique expression of adhesion molecules, and these phenotypes are independent of the thid mutation that causes maturational arrest of CD4⁺8⁻ thymocytes. J. Trace Elem. Exp. Med. 10:81–88, 1997. © 1997 Wiley-Liss, Inc.

Rat lymphopoiesis did not develop when naive SCID mice were transplanted with rat fetal liver cells. However, when SCID mice were depleted of NK cells by administration of anti-murine IL-2Rβ mAb before transplantation, remarkable reconstitution of both rat T and B cells was observed in these mice without any evidence of graft-versus-host disease macroscopically or histologically. T cells in these reconstituted mice proliferated well in response to Con A and third-party rat and mouse antigens, whereas no response was seen to the stimulation with either donor rat- or host mouse-type cells. When these xenogeneic chimera mice had been immunized with SRBC, these mice exhibited DTH reaction and antibody production against the homologous antigen. These results indicate that rat fetal liver cells can differentiate to functional T and B cells in the xenogeneic microenvironment of SCID mice, if host NK cells are depleted beforehand. These rat-type T cells develop within SCID thymuses and acquire tolerance to either donor F344 rat or host SCID mouse antigens. Copyright 1995, 1999 Academic Press

We recently evaluated the acetaminophen absorption test as a marker of graft rejection for small bowel transplantation(SBTX). Randomly bred male Wistar rats were used as recipients and donors. Rats (n=45) received heterotopic small intestinal transplants and were divided into three groups (n=15 for each group). In group A, a 10-cm segment of jejunum of was exteriorized as a Thiry-Vella loop. In group B, immunosuppression was not given after SBTX. In group C, rats were treated with FK506 after SBTX (0.3 mg/kg body weight, 06 postoperative days). Serum acetaminophen concentrations were measured 15 min after instillation of 0.15 g/kg acetaminophen into the intestinal loop on postoperative days 1, 3, and 7 (n=5 for each group). Blood flow and histology of the graft were also evaluated. In the SBTX group only, the grafts showed the histological change after acute rejection. On day 3, plasma acetaminophen concentrations in this group showed a significant decrease, which correlated with the mild histological changes of graft rejection. Graft blood flow of the SBTX group decreased significantly on day 7, following the severe graft destruction of advanced rejection. No remarkable changes were observed in the other two groups. The acetaminophen absorption test appears to be useful for the early detection of SBTX graft rejection.

Entamoeba histolyticais a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota.

Obesity is increasingly causing lifestyle diseases in developed countries where helminthic infections are rarely seen. Here, we investigated whether an intestinal nematode, Heligmosomoides polygyrus, has a suppressive role in diet-induced obesity in mice.

MicroRNA (miRNA) is noncoding short RNA which plays important roles for cell death, proliferation, development and differentiation. MiRNA binds to 3' UTR region of target messenger RNA(mRNA), and negatively regulates gene expressions by inducing instability of mRNAs or translation block. This function of miRNA depends on the presence of cytoplasmic protein complex, RNA induced silencing complex(RISC). A main component of RISC is argonoute2(Ago2), of which deficiency or knockout results in severe cellular dysfunction. We and several groups reported erythroid-specific expressions of miRNAs(Masaki et al., BBRC 346:509-514, 2007). Among those miRNAs, miR-451 is especially specific to erythroid cells. Knockout of miR-451 results in reduction of red blood cell(RBC) production, and a high amount of miR-451 is contained in RBCs which are enucleated, and previously seem to be genetically inactive. Roles of miRNAs in mature RBCs for the biology of RBC are entirely unclear. In this study, we analyzed amounts of miRNA-Ago2 complexes in normal human RBCs.Circulating blood cells were obtained from 5 normal subjects, and fractionated using density centrifugation and ultracentrifugation methods. RNA was isolated directly from each cell fraction, or isolated after immunoprecipitation by anti-Ago2 antibody(Wako, Osaka, Japan). MiRNA was measured by reverse transcriptase-based quantitative real time polymerase chain reaction(RT-qPCR), using microRNA assay kits(ABI).Packed RBCs contained miR-451 at the concentrations of 10,000 folds higher than granulocytes and miR-16 at the same level, whereas miR-155, -152, -221, -223 were contained at very low levels. After ultracentrifugation of circulating RBCs, packed RBCs were fractionated into 4 fractions:buffy coat cells(BFC), upper, middle, lower fractions of packed RBC(BFC, UF, MF, LF, respectively). Numbers of leukocytes were 21.9 and 0.88 per 104 RBCs in BFC and UF, respectively. They were low in MF and LF, 0.20 and 0.10. Coexisting reticulocytes in each fractions were 15.3, 5.50, 3.75 and 1.75 per 106 RBCs in BFC, UF, MF, LF, respectively. We used let-7a as an internal control for RT-qPCR assays, since expression of let-7a was relatively constant between 4 fractions. Analysis of total RNA isolated directly from each fraction showed constantly high expression of miR-16 and -451 in RBCs from each fraction. Analysis of immunoprecipitated miRNAs showed that the almost same amounts of miR-16 and -451 were present as miRNA-Ago2 complex in RBCs from the lower fraction(mature RBCs).We report, for the first time, the presence of miRNA-RISC complex in mature RBCs. Ratio of miRNA bound to Ago2 in total miRNAs is depend on species of miRNA. Our observation showed that almost all miRNA in mature RBCs is present as a miRNA-RISC complex. This finding clearly suggests that miRNAs in mature RBCs are active molecules, and also suggests that enucleated mature RBCs are still using epigenetic controlling system for some biological purpose. The well-known target gene of miR-16 is BCL2. There is a possibility that miR-16 negatively regulates expression of immortalize gene, BCL2, resulting in suppression of proliferation, and in keeping a differentiated state. On the other hand, the target gene of miR-451 is not well determined. Experiments using Zebra fishes showed knockout of miR-451 induced severe reduction of RBC production, suggesting that miR-451 is one of key molecules for erythropoiesis. In conclusion, our observations suggest that miRNA-Ago2 complexes in mature RBCs may be playing roles for homeostasis of normal erythropoiesis, and the analysis of miRNAs-Ago2 complex in RBCs is relevant for understanding normal and pathological erythropoiesis.No relevant conflicts of interest to declare.