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Abstract: Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8+T cells. Here, to develop a vaccine strategy taking advantage of activated CD8+T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi. C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-γ secretion by CD8+T cells. Furthermore, the depletion of CD8+T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8+T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28α/β or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8+T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells. [Copyright &y& Elsevier]

Abstract: The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells. In this study, we exploited UPS to induce CD8+ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8+ T cells compared with those from the latter group of mice. [Copyright &y& Elsevier]

Abstract: The ubiquitin–proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28α/β complex. [Copyright &y& Elsevier]

Abstract: Malaria is one of the most life-threatening infectious diseases worldwide. Specific immunity to natural infection is acquired slowly despite a high degree of repeated exposure and rarely continues for a long time even in endemic areas. Malaria parasites have evolved to acquire diverse immune evasion mechanisms that evoke poor immune responses and allow infection of individuals previously exposed. The shrewd schema of malaria parasites also hampers the development of effective vaccines. Furthermore, some of those mechanisms are essential for malaria pathogenesis. In this article, an outline of protective immunity to malaria is given, then strategies used by malaria parasites to evade host immunity, including antigen diversity/polymorphism, antigen variation and total immune suppression, are reviewed. Finally, trials to control malaria based on accumulating insights into the host–parasite relationship are discussed. [Copyright &y& Elsevier]

The carboxyl-terminal region of the merozoite surface protein-1 (MSP1) is a leading candidate for a vaccine against malaria in the erythrocytic stage. In this study, we investigated the utility of interleukin-12 (IL-12) cDNA as an adjuvant for malaria DNA vaccine in a mouse challenge model. We found that co-immunization of expression plasmids encoding a C-terminal 15-kDa fragment of MSP1 (MSP1-15) with the IL-12 gene using a gene gun significantly increased the protective immunity against malaria as compared with MSP1-15 DNA immunization alone. Co-immunization of IL-12 DNA potentiated MSP1-15-specific T helper (Th)1-type immune responses as evaluated by in vivo antibody (Ab) responses and in vitro cytokine profiles. After the Plasmodium yoelii challenge, mice immunized with MSP1-15 plus IL-12 DNA showed a higher level of interferon gamma (IFN-γ) production than did other groups of mice. In vivo neutralization of IFN-γ or depletion of CD4+ T cells completely abolished this protective immunity. Macrophages, but not nitric oxide (NO), were found to play an important role in this effector mechanism. The sera from mice in which the infection had been cleared by the vaccination showed strong protection against P. yoelii infection. Thus, in addition to cellular immune responses, Abs against parasites induced in the course of infection are essential for protection against P. yoelii. The results indicate that combined vaccination with DNA encoding antigenic peptides plus IL-12 DNA provides a strategy for improving the prophylactic efficacy of a vaccine for malaria infection. [Copyright &y& Elsevier]

A blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3) was tested for efficacy in a primate model. Aotus nancymai monkeys were vaccinated with yeast-expressed MSP3 before a lethal challenge with Plasmodium falciparum parasites. Five of 7 control monkeys had acute infections and required treatment to control parasitemia. Only 1 of 7 monkeys vaccinated with MSP3 required this treatment. The efficacy of the MSP3 vaccination appeared to be comparable to that of MSP142, a leading asexual vaccine candidate, in response to which 2 monkeys experienced acute infections. In the MSP3-vaccinated group, protection correlated with prechallenge titers of antibody to MSP3. In the MSP1 and control groups, protection correlated with antibody to MSP3 raised by challenge infection. [ABSTRACT FROM AUTHOR]

We investigated the roles of γδ T, NK, and NK1.1+ T-like (NKT) cells in protective immunity against infection with Toxoplasma gondii. γδ T cells, NKT and NK cells, and NK cells in BALB/c mice were depleted by treatment with anti-TCR-γδ monoclonal antibody (mAb), anti-interleukin-2 receptor beta chain (IL-2Rβ) mAb, and anti-asialoGM1 Ab, respectively, and these mice were infected with T. gondii. Treatment of mice with anti-TCR-γδ mAb aggravated toxoplasmosis, while treatment with anti-asialoGM1 Ab had no effects. Treatment with anti-IL-2Rβ mAb enhanced the expression of heat shock protein 65 (HSP65) and gamma interferon (IFN-γ) mRNA, while it inhibited interleukin-4 (IL-4) mRNA expression, ameliorating toxoplasmosis. In addition to NK cells, anti-IL-2Rβ mAb eliminated cells expressing IL-2Rβ and intermediate levels of CD3 (IL-2Rβ+ CD3int). Mice treated with anti-IL-2Rβ mAb decreased the number of DX5+ CD3int cells, which are considered to be equivalent to NK1.1+T cells in NK1.1 allele-negative strains. IL-2Rβ+ CD3int cells isolated from splenic and hepatic lymphoid cells were confirmed to express the TCR-Vα14 transcript. The magnitude of HSP65 induction in macrophages correlated with the protective potential against T. gondii infection after treatment with the antibodies, supporting our previous finding that γδ T cells play an essential role in the induction of HSP65 in host macrophages. Interestingly, NKT cells suppressed the expression of γδ T cell-induced HSP65 and IFN-γ. Furthermore, depletion of IL-2Rβ+ CD3int cells suppressed the IL-4 mRNA expression. These results suggest that NKT cells may be the cells responsible for suppression of protective immunity against T. gondii infection by interfering with the γδ T cell-induced HSP65 expression, possibly through the generation of IL-4. [Copyright &y& Elsevier]

SummaryImmunization of mice with live bradyzoites of a low-virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T-cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T-cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH-infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H-uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon-γ (IFN-γ) than did naive splenic cells; however, the production of nitric oxide (NO) by RH-infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH-infected macrophages, but it did not affect the production of IFN-γ. Treatment of CD8+ T-enriched splenic cells from the immunized mice with concanamycin A, but not an anti-Fas ligand monoclonal antibody, significantly reduced their anti-proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule-dependent cytotoxic activities. [ABSTRACT FROM AUTHOR]

Infection with malaria parasites frequently induces total immune suppression, which makes it difficult for the host to maintain long-lasting immunity. Here we show that depletion of CD4+CD25+ regulatory T cells (Treg) protects mice from death when infected with a lethal strain of Plasmodium yoelii, and that this protection is associated with an increased T-cell responsiveness against parasite-derived antigens. These results suggest that activation of Treg cells contributes to immune suppression during malaria infection, and helps malaria parasites to escape from host immune responses. [ABSTRACT FROM AUTHOR]

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi , in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans. [Display omitted] • An entomological survey to identify simian malaria in Anopheles vectors. • Six Anopheles spp. were collected at a location of high simian malaria prevalence. • All mosquitos were negative by standard nested PCR, but were 25 positives by ddPCR. • First report of simian malaria parasite DNA detection in An. sawadwongporni. • ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles. [ABSTRACT FROM AUTHOR]

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3 -deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3 , oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes. [Display omitted] • Characterization of two histone H3 proteins in P. berghei. • H3.3 plays a critical role on zygote-to-oocyst development. • Δh3.3 mutants showed no sporozoite production. • The majority of H3.3-HA protein was detected in female gametocytes. • Several male-specific genes were upregulated in Δh3.3 female gametocytes. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic CD8+ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8+ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8+ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4+ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8+ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi. [Copyright &y& Elsevier]

Abstract: We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8+ T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8+ T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8+ T cells. [Copyright &y& Elsevier]

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8+ cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2181−188) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2181−188. GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28α/β. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2181−188, by activating CD8+ CTL specific for TRP-2181−188. TRP-2181−188 fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8+ T cells specific for the CTL epitope. Furthermore, CD4+ T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the ‘ubiquitin-proteasome pathway’. It is noteworthy to document that genetic immunization with pGFP plus pTRP-2181−188 failed to exert the antitumour immunity. [ABSTRACT FROM AUTHOR]

• A new method to evaluate the integrity of the blood–cerebrospinal fluid barrier (BCSFB) was developed. • BCSFB permeability was increased specifically in cerebral malaria (CM), but not in non-CM. • CD8+ T cells are involved in the BCSFB breakdown during CM. • Hemozoin was found in the choroid plexus in CM. Cerebral malaria is one of the most severe pathologies of malaria; it induces neuro-cognitive sequelae and has a high mortality rate. Although many factors involved in the development of cerebral malaria have been discovered, its pathogenic mechanisms are still not completely understood. Most studies on cerebral malaria have focused on the blood–brain barrier, despite the importance of the blood–cerebrospinal fluid barrier, which protects the brain from peripheral inflammation. Consequently, the pathological role of the blood–cerebrospinal fluid barrier in cerebral malaria is currently unknown. To examine the status of the blood–cerebrospinal fluid barrier in cerebral malaria and malaria without this pathology (non-cerebral malaria), we developed a new method for evaluating the permeabilization of the blood–cerebrospinal fluid barrier during cerebral malaria in mice, using Evans blue dye and a software-assisted image analysis. Using C57BL/6J (B6) mice infected with Plasmodium berghei ANKA strain as an experimental cerebral malaria model and B6 mice infected with P. berghei NK65 strain or Plasmodium yoelii as non-cerebral malaria models, we revealed that the permeability of the blood–cerebrospinal fluid barrier increased during experimental cerebral malaria but not during non-cerebral malaria. We observed haemorrhaging in the cerebral ventricles and hemozoin-like structures in the choroid plexus, which is a key component of the blood–cerebrospinal fluid barrier, in cerebral malaria mice. Taken together, this evidence indicates that the blood–cerebrospinal fluid barrier is disrupted in experimental cerebral malaria, whereas it remains intact in non-cerebral malaria. We also found that P. berghei ANKA parasites and CD8+ T cells are involved in the blood–cerebrospinal fluid barrier disruption in experimental cerebral malaria. An understanding of the mechanisms underlying cerebral malaria might help in the development of effective strategies to prevent and manage cerebral malaria in humans. [ABSTRACT FROM AUTHOR]

Intestinal helminthes induce immunosuppressive responses as well as type 2 immunity. Their suppressive properties are intended to regulate inflammatory diseases such as allergies and autoimmune diseases. This study evaluated whether helminthic infections suppress obesity, a chronic inflammatory state, using an intestinal nematode, Heligmosomoides polygyrus (Hp). Infection with Hp at the same time as feeding a high‐fat diet (HFD) prevented weight gain, dyslipidaemia and glucose intolerance observed in uninfected obese mice. Immunologically, Hp infection skewed M1 macrophages to M2 macrophages and induced type 2 innate lymphoid cells in adipose tissues. The expression of interleukin (IL)‐33, a potent initiator of type 2 responses, was also increased in association with uncoupled protein 1 (UCP1). To further investigate the anti‐obesity effects of IL‐33 in mice infected with Hp, IL‐33‐deficient mice were fed the HFD and infected with Hp. These mutant mice rapidly gained weight compared with wild‐type mice, indicating the anti‐obesity effect of IL‐33. In the absence of IL‐33, the rapid increase in weight was not prevented, and type 2 responses and UCP1 expression were not observed even during Hp infection. These results suggested that the suppression of obesity by Hp is dependent on IL‐33. [ABSTRACT FROM AUTHOR]

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans. [ABSTRACT FROM AUTHOR]

Cerebral malaria is a fatal complication of malaria. Conventional methods for evaluating experimental cerebral malaria have several drawbacks. Therefore, we aimed to develop an easy-to-use method for evaluating experimental cerebral malaria using OKD48 (Keap1-dependent Oxidative stress Detector, No-48-luciferase) mice to evaluate oxidative stress. OKD48 mice infected with Plasmodium berghei ANKA strain (PbA) suffered from experimental cerebral malaria and oxidative stress was successfully detected in the brains of living OKD48 mice developing experimental cerebral malaria. Oxidative stress in the brain was dependent on the development of experimental cerebral malaria, as prevention of experimental cerebral malaria did not elicit oxidative stress. We provide a novel evaluation method for experimental cerebral malaria using oxidative stress indicator OKD48 mice. [ABSTRACT FROM AUTHOR]

Recent studies show that some human malaria parasite species Plasmodium falciparum and P.vivax parasitize erythroblasts; however, the biological and clinical significance of this is unclear. To investigate further, we generated a rodent malaria parasite (P. yoelii 17XNL) expressing GFP-ovalbumin (OVA). Its infectivity to erythroblasts was confirmed and parasitized erythroblasts were capable of initiating malaria infections. Experiments showed that MHC class I molecules were highly expressed on parasitized erythroblasts. As CD8+ T cells recognize MHC class I and peptide complexes on target cells and are involved in protection or pathology against malaria, we examined whether erythroblasts are targeted by CD8+ T cells. Purified non-parasitized erythroblasts pulsed with OVA peptides were recognized by OVA-specific CD8+ T cells. Crucially, parasitized erythroblasts isolated from GFP-OVA-, but not GFP- infected-mice, activated OT-I CD8+ T cells, indicating that CD8+ T cells recognize parasitized erythroblasts in an antigen-specific manner. [ABSTRACT FROM AUTHOR]

General cellular functions of proteasomes occur through protein degradation, whereas the specific function of immunoproteasomes is the optimization of antigen processing associated with MHC class I. We and others previously reported that deficiency in subunits of immunoproteasomes impaired the activation of antigen-specific CD8+ T cells, resulting in higher susceptibility to tumor and infections. We demonstrated that CD8+ T cells contributed to protection against malaria parasites. In this study, we evaluated the role of immunoproteasomes in the course of infection with rodent malaria parasites. Unexpectedly, Plasmodium yoelii infection of mice deficient in LMP7, a catalytic subunit of immunoproteasomes, showed lower parasite growth in the early phase of infection and lower lethality compared with control mice. The protective characteristics of LMP7-deficient mice were not associated with enhanced immune responses, as the mutant mice showed comparable or diminished activation of innate and acquired immunity. The remarkable difference was observed in erythrocytes instead of immune responses. Parasitized red blood cells (pRBCs) purified from LMP7-deficient mice were more susceptible to phagocytosis by macrophages compared with those from wild-type mice. The susceptibility of pRBC to phagocytosis appeared to correlate with deformity of the membrane structures that were only observed after infection. Our results suggest that RBCs of LMP7-deficient mice were more likely to deform in response to infection with malaria parasites, presumably resulting in higher susceptibility to phagocytosis and in the partial resistance to malaria. [ABSTRACT FROM AUTHOR]

Abstract: Cerebral malaria (CM) is the most severe complication of Plasmodium infection. Although inappropriate immune responses to Plasmodium falciparum are reported as the major causes of CM, the precise mechanisms for development remain unclear. IL-23 and IL-17 have critical roles in the onset of autoimmunity and inflammatory diseases triggered by microbial infections. Thus, we investigated the influence of IL-23 and IL-17 on experimental CM (ECM) using Plasmodium berghei ANKA infection of C57BL/6 mice. Both IL-23 deficient mice and wild-type (WT) mice developed ECM. IL-17 deficient mice also developed ECM, while IL-17 producing cells other than CD4+ T cells (Th17) were increased in WT mice that developed ECM. In conclusion, this study showed that IL-23 and IL-17 are not involved in ECM development. [ABSTRACT FROM AUTHOR]

CD4+CD25+ regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4+CD25+ Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25+ cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25+ cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4+CD25− T cells alone or a combination of CD4+CD25+ and CD4+CD25− T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4+CD25+ Treg cells to CD4+CD25− effector T cells, as demonstrated by the lack of response of CD4+CD25+ T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette–Guérin and M. tuberculosis. Our data show that CD4+CD25+ Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection. [ABSTRACT FROM PUBLISHER]

Abstract: Chagas’ disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8+ T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8+ T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-γ (IFN-γ) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-γ production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas’ disease. [Copyright &y& Elsevier]

Toxoplasmosis is a rare but rapidly fatal complication that can occur following hematopoietic stem cell transplantation (HSCT). Over a 17-yr period at our institutions, a definite diagnosis of toxoplasmosis was made in only two of 925 allogeneic HSCT recipients (0.22%) and none of 641 autologous HSCT recipients. These two patients received a conventional conditioning regimen followed by transplantation from an HLA-matched donor; however, they developed severe graft-vs.-host disease, which required intensive immunosuppressive therapy. Despite prophylactic treatment with trimethoprim/sulfamethoxazole, their immunosuppressive state, as indicated by a low CD4+ cell count, might have resulted in toxoplasmosis encephalitis. Rapid and non-invasive methods such as a polymerase chain reaction (PCR) test of their cerebrospinal fluid for Toxoplasma gondii and magnetic resonance imaging of the brain were useful for providing a definitive diagnosis and prompt therapy in these patients: one patient stabilized and survived after responding to treatment with pyrimethamine/sulfodiazine whereas the other died of bacterial infection. In addition, retrospective PCR analyses of the frozen stored peripheral blood samples disclosed that detection of T. gondii preceded the onset of disease, indicating routine PCR testing of peripheral blood specimens may be an early diagnostic tool. It should be noted that when patients receiving HSCT have an unexplained fever and/or neurological complications, PCR tests should be considered to avoid cerebral lesions and improve the outcome of the patients. [ABSTRACT FROM AUTHOR]

Background: Autologous whole tumor cell-based vaccinations would seem to be ideal since such vaccinations, in contrast to vaccination with a single defined antigen, have the potential to elicit a broad type of T-cell immune response to tumor-associated antigens. Objective: We modified formaldehyde (formalin)-fixed mouse melanoma cells and investigated the utility of those cells as sources of tumor antigens for immunotherapy. Methods: C57BL/6 or the proteasome activator PA28α-knockout mice were intradermally inoculated with 1% formalin-fixed B16 cells three times at weekly intervals either before or after tumor challenge. Simultaneously, interleukin-12 gene was transferred into the skin around immunization sites using gene gun technology. The effects were evaluated by tumor growth, antigen-specific interferon-γ production in splenic lymphocytes, and activation of dendritic cells. Results: Fixed cells directly induced production of tumor necrosis factor-α in dendritic cells more effectively than did frozen and thawed cells. More than 60% of the mice immunized with fixed cells and interleukin-12 rejected the challenged B16 tumor. CD4+ T cells from those mice produced a significant amount of interferon-γ in response to melanoma cells. Furthermore, this combined treatment showed antitumor immunity initiated by CD8+ and CD4+ T cells in the therapeutic experiments. PA28α/β appeared not to be required for the development of CD8+ T cells, although it is known to be essential for the development of CD8+ T cells specific for tyrosinase-related protein-2, one of melanocyte-lineage differentiated antigens. Conclusion: These results suggest that formalin-fixed autologous melanoma cells have a potential to function as effective antigen sources for immunotherapy. [Copyright &y& Elsevier]

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D. [Copyright &y& Elsevier]

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1−/− mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1−/− splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1−/− mice, and IFN-γ that was overproduced in WSX-1−/− mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-α, were produced by liver mononuclear cells in WSX-1−/− mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production. [Copyright &y& Elsevier]

Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control. To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT). Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response). Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes. The observed transmission-blocking effect was dependent on immune sera dilution. This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria. [Copyright &y& Elsevier]

The consumption of dietary acrylamide (ACR), a carcinogen, results in the dysfunction of various organs and the immune system. However, the impact of ACR exposure on the progression of infectious diseases is unknown. This study investigated the effect of ACR on the progression of malaria infection using a mouse model of malaria. C57BL/6 mice were continuously treated with ACR at a dose of 20 mg/kg bodyweight/day for six weeks (long-term exposure) or phosphate-buffered saline (PBS). Next, the mice were infected with the rodent malaria parasite, Plasmodium berghei NK65 (PbNK). Parasitemia and survival rate were analyzed in the different treatment groups. Magnetic resonance imaging (MRI) and histopathological analyses were performed to evaluate the effect of ACR exposure on the morphology of various organs. Long-term ACR exposure exacerbated PbNK-induced multiorgan dysfunction. MRI and histopathological analysis revealed signs of encephalomeningitis and acute respiratory distress syndrome in the PbNK-infected long-term ACR exposure mice, which decreased the survival rate of mice, but not in the PbNK-infected long-term PBS exposure group. These findings enhance our understanding of the impact of ACR on the progression of infectious diseases, such as malaria. [Display omitted] • Long-term ACR exposure caused earlier death in mice with malaria. • Long-term ACR exposure induced brain damage in mice with malaria. • Signs of meningitis appeared in long-term ACR-exposed mice with malaria. • Long-term ACR exposure exacerbates malaria-associated acute respiratory distress syndrome. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Intestinal helminths induce immune suppressive responses thought to regulate inflammatory diseases including allergies and autoimmune diseases. This study was designed to evaluate whether helminthic infections suppress the natural development of systemic lupus erythematosus (SLE) in NZBWF1 mice. Infection of NZBWF1 SLE-prone mice with two nematodes failed to establish long-lasting settlement. However, the Hymenolepis microstoma (Hm) rodent tapeworm successfully established long-term parasitization of NZBWF1 mice and was used to evaluate the suppressive effects of helminth infection. Ten-month-old NZBWF1 mice developed symptoms including autoantibody generation, proteinuria, glomerular histopathology, and splenomegaly, but mice infected with Hm at 2 months of age did not show any clinical signs. Furthermore, infection with Hm reduced lymphocyte activation and increased regulatory T cells in the spleen and mesenteric lymph nodes. These results indicate that infection with Hm protects NZBWF1 mice from naturally developing SLE and suggest that pathological immunity is attenuated, presumably because of the induction of regulatory T cells. Unlabelled Image • SLE-prone NZBWF1 mice were refractory to Hp and Tm but susceptible to Hm. • Hm prevents natural development of SLE and prolonged survival of NZBWF1 mice • Hm infection increases Tregs and decreased plasma cells and Tfh in NZBWF1 mice. [ABSTRACT FROM AUTHOR]

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed. Unlabelled Image • The details of morphological differences between malaria parasites in oocyst stage. • The protruding oocyst with a connected mosquito basal membrane in P. cynomolgi. • The 3D EM analysis reveals endopolygeny-like nucleus in P. cynomolgi oocyst stage. [ABSTRACT FROM AUTHOR]

Summary Mast cells are important for eradication of intestinal nematodes; however, their precise mechanisms of action have remained elusive, especially in the early phase of infection. We found that Spi-B-deficient mice had increased numbers of mast cells and rapidly expelled the Heligmosomoides polygyrus (Hp) nematode. This was accompanied by induction of interleukin-13 (IL-13)-producing group 2 innate lymphoid cells (ILC2) and goblet cell hyperplasia. Immediately after Hp infection, mast cells were rapidly activated to produce IL-33 in response to ATP released from apoptotic intestinal epithelial cells. In vivo inhibition of the P2X7 ATP receptor rendered the Spi-B-deficient mice susceptible to Hp, concomitant with elimination of mast cell activation and IL-13-producing ILC2 induction. These results uncover a previously unknown role for mast cells in innate immunity in that activation of mast cells by ATP orchestrates the development of a protective type 2 immune response, in part by producing IL-33, which contributes to ILC2 activation. [ABSTRACT FROM AUTHOR]

Gastrointestinal symptoms, such as abdominal pain and diarrhea, are frequently observed in patients with Plasmodium falciparum malaria. However, the correlation between malaria intestinal pathology and intestinal microbiota has not been investigated. In the present study, infection of C57BL/6 mice with P. berghei ANKA (PbA) caused intestinal pathological changes, such as detachment of epithelia in the small intestines and increased intestinal permeability, which correlated with development with experimental cerebral malaria (ECM). Notably, an apparent dysbiosis occurred, characterized by a reduction of Firmicutes and an increase in Proteobacteria. Furthermore, some genera of microbiota correlated with parasite growth and/or ECM development. By contrast, BALB/c mice are resistant to ECM and exhibit milder intestinal pathology and dysbiosis. These results indicate that the severity of cerebral and intestinal pathology coincides with the degree of alteration in microbiota. This is the first report demonstrating that malaria affects intestinal microbiota and causes dysbiosis. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica, the parasitic amoeba responsible for amoebiasis, causes approximately 100,000 deaths every year. There is currently no vaccine against this parasite. We have previously shown that intracecal inoculation of E. histolytica trophozoites leads to chronic and non-healing cecitis in mice. Entamoeba moshkovskii, a closely related amoeba, also causes diarrhea and other intestinal disorders in this model. Here, we investigated the effect of infection followed by drug-cure of these species on the induction of immunity against homologous or heterologous species challenge. Mice were infected with E. histolytica or E. moshkovskii and treated with metronidazole 14 days later. Re-challenge with E. histolytica or E. moshkovskii was conducted seven or 28 days following confirmation of the clearance of amoebae, and the degree of protection compared to non-exposed control mice was evaluated. We show that primary infection with these amoebae induces a species-specific immune response which protects against challenge with the homologous, but not a heterologous species. These findings pave the way, therefore, for the identification of novel amoebae antigens that may become the targets of vaccines and provide a useful platform to investigate host protective immunity to Entamoeba infections. [ABSTRACT FROM AUTHOR]