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There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell–mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major. We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow–derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ–secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05–0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63–79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major. We generated I-Ab-DLD63–79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63–79–specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

Intracellular infection with the parasite Leishmania major features a state of concomitant immunity in which CD4+ T helper 1 (Th1) cell-mediated immunity against reinfection coincides with a chronic but sub-clinical primary infection. In this setting, the rapidity of the Th1 response at a secondary site of challenge in the skin represents the best correlate of parasite elimination and has been associated with a reversal in Leishmania-mediated modulation of monocytic host cells. Remarkably, the degree to which Th1 cells are absolutely reliant upon the time at which they interact with infected monocytes to mediate their protective effect has not been defined. In the present work, we report that CXCR3-dependent recruitment of Ly6C+ Th1 effector (Th1EFF) cells is indispensable for concomitant immunity and acute (, Author summary Ongoing chronic infection with the intracellular parasite Leishmania major results in robust protective immunity at sites of secondary challenge. However, translation of our understanding of this protective response into an efficient vaccine has remained elusive. This is due, at least in part, to the fact that primary chronic infection maintains multiple effector and memory subsets of lymphocytes, making it difficult to determine the relative importance of any single subset and their functional properties, including the time at which they mediate their effector function. Employing parabiosis, adoptive transfer, and in-vivo blockade of effector functions followed by analysis by intravital microscopy and flow cytometry, we find that the acute availability of circulating CD4+ T helper 1 effector cells (Th1EFF) at the time of secondary challenge is critical for the Th1 immune response to prevent L. major-mediated immunomodulation of host phagocytes and mediate protective immunity. Therefore, the present lack of effective Th1-mediated vaccines that target CD4+ memory T cell generation may be because protective Th1 immunity relies on preactivated Th1EFF cells and IFN-γ availability prior to the establishment of a pathogen niche, which occurs before these memory T cells acquire effector function. To the best of our knowledge, our observations are the first to formally demonstrate the degree to which Th1EFF cells are reliant on the time at which they interact with infected phagocytes to mediate protection and have significant implications for vaccine strategies against phagosomal infections such as Leishmania spp., Mycobacterium tuberculosis, Salmonella enterica, and Crytococcus spp..

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete. Understanding of the persistence mechanisms is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells has been proposed to prevent parasite clearance in vivo. In the presented study, we used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4+ T cell responses and to characterize mechanisms which restrain their effector function. We show that macrophage:T cell interaction dynamics were prolonged upon antigen recognition. This leads to a production of high levels of IFNγ and can be significantly suppressed by PEPCK-specific Tregs in vitro, as compared to polyclonal Treg controls. Treg antigen activation leads to a substantial increase in IL-10 levels, while decreasing IL-12, TNF, and IL-2 production. Intravital microscopy studies characterizing PEPCK-specific CD4+ T cell migration within skin lesions in mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying behaviours consistent with antigen recognition at early stages, but not at chronic stages. Imaging of infected FoxP3-GFP reporter mice show recruitment of Tregs to the lesion site, where they localize in the lesion, unlike the effectors cells which surround it. Collectively, our findings show for the first time that Leishmania-specific Tregs influence effector CD4+ T cell responses and this could be a mechanism that derives antigen persistence in L. major infection. Research funded by a grant from the GSK-CIHR Partnered Chair Program

Summary The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3−/−) mice are highly resistant to L. major and L. braziliensis infections. This enhanced resistance is associated with increases in Th17 and IL-17A responses. The neutralization of IL-17A abolishes this enhanced resistance, while rPTX3 treatment results in decrease in Th17 and IL-17A responses and increases susceptibility. PTX3−/− CD4+ T cells display increased differentiation to Th17 and expression of Th17-specific transcription factors. The addition of rPTX3 suppresses the expression of Th17 transcription factors, Th17 differentiation, and IL-17A production by CD4+ T cells from PTX3−/− mice. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by negatively regulating Th17 and IL-17A responses.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4

The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3

Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity.

Autoimmune processes have been implicated in the development of rheumatoid arthritis (RA); however, specific autoantigens that play a role in the aetiology of RA have been lacking. In this study, we found that sera from RA patients were particularly immunoreactive against the protein tryptase. Compared with osteoarthritis (OA) patients and healthy controls, RA patients had relatively higher levels of tryptase and concomitant anti-tryptase antibodies in their synovial tissues and sera. Similarly, synovial fluid from RA patients, but not from OA patients, contained antibodies that recognized tryptase in vitro. In addition, serum tryptase levels in both early and late RA patients significantly correlated with clinical indices usually used to diagnose RA, such as rheumatoid factor, Disease Activity Score using 28 joint counts and autoantibodies against cyclic citrullinated peptide. Our results identify tryptase as a candidate autoantigen involved in the pathogenesis of RA and monitoring its levels may have diagnostic and prognostic value.

Resolution of cutaneous leishmaniasis (CL), caused by the protozoan parasite Leishmania (L) major, relies on the nature of the host immune response. The long pentraxin 3 (PTX3), a soluble pattern recognition molecule, is critical for wound healing by regulating tissue repair and also in innate and adaptive responses during infection and inflammation. Here, we show that PTX3 regulates disease pathogenesis and immunity to CL. PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3−/− mice were highly resistant to primary and secondary L. major infection. Interestingly, the enhanced resistance of PTX3−/− mice to primary or secondary L. major infection was not associated with enhanced IFN-γ or decreased IL-10 response. Instead, L. major-infected PTX3−/− mice displayed strong IL-17 response and in vivo neutralization of IL-17A abolished their enhanced resistance. Naïve CD4+ T cells from PTX3−/− mice displayed increased differentiation into Th17 cells and this was associated with increased expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and the frequency of Th17 cells in Th17 polarizing cultures of PTX3−/− CD4+ T cells. Similarly, healed PTX3−/− mice had higher frequencies of effector memory CD4+ T cells that rapidly proliferate and differentiate into IL-17A-producing CD4+ T cells upon secondary challenge. Collectively, our results show that PTX3 contributes to pathogenesis of CL by negatively regulating protective Th17 and IL-17 responses.

The breakdown of L-arginine to ornithine and urea by host arginase supports Leishmania proliferation in macrophages. Studies using arginase-null mutants show that Leishmania-derived arginase plays an important role in disease pathogenesis. We investigated the role of parasite-derived arginase in secondary (memory) anti-Leishmania immunity in the resistant C57BL/6 mice. We found that C57BL/6 mice infected with arginase-deficient (arg−) L. major failed to completely resolve their lesion and maintained chronic pathology after 16 wk, a time when the lesion induced by wild-type L. major is completely resolved. This chronic disease was associated with impaired Ag-specific proliferation and IFN-γ production, a concomitant increase in programmed cell death-1 (PD-1) expression on CD4+ T cells, and failure to induce protection against secondary L. major challenge. Treatment with anti–PD-1 mAb restored T cell proliferation and IFN-γ production in vitro and led to complete resolution of chronic lesion in arg− L. major–infected mice. These results show that infection with arg− L. major results in chronic disease due in part to PD-1–mediated clonal exhaustion of T cells, suggesting that parasite-derived arginase contributes to the overall quality of the host immune response and subsequent disease outcome in L. major–infected mice. They also indicate that persistent parasites alone do not regulate the quality of secondary anti-Leishmania immunity in mice and that the quality of the primary immune response may be playing a hitherto unrecognized dominant role in this process.

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.

Despite decades of clinical and biomedical research, the pathogenesis of sepsis and its spectrum of diseases (severe sepsis and septic shock), which are leading causes of death in intensive care units, are still poorly understood. In this article, we show that signaling via the p110δ isoform of PI3K is critical for survival in experimental sepsis. Mice with an inactive knock-in mutation in the p110δ gene (p110δD910A) succumbed acutely to nonlethal dose LPS challenge. The susceptibility of p110δD910A mice to LPS was associated with increased neutrophil numbers and activities in the tissues, due in part to delayed apoptosis resulting mostly from inherent reduced regulatory T cell (Treg) numbers. Adoptive transfer of wild-type or p110δD910A Tregs abrogated exaggerated neutrophil activity, increased neutrophil apoptosis, and rescued p110δD910A mice from mortality after LPS challenge. We confirmed the clinical relevance of these findings by showing that human Tregs also regulate neutrophil function and survival. Collectively, our results show that PI3K δ is essential for survival during sepsis. In addition, our data highlight the importance of Tregs in regulating the pathogenesis of sepsis and septic shock via their effects on neutrophil survival and function, and provide evidence of regulation of innate immunity by cells of the adaptive immune system.

Objectives This study aimed to evaluate the immuno-prophylactic and -therapeutic effect of p110δ-specific pharmacological inhibitors (CAL-101 and IC87114), either alone or in combination with amphotericin B, against experimental cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). Methods Female BALB/c mice were infected intravenously with Leishmania donovani or subcutaneously with Leishmania major Prophylactic treatment was initiated 24 h prior to infection, whereas therapeutic treatments with or without amphotericin B were initiated either 1 week or 2 weeks post-infection. At different times post-infection, mice were sacrificed and parasite burden, regulatory T cell (Treg) numbers and cytokine production were assessed in the liver, spleen, draining lymph nodes and footpads. In addition, direct cytolytic effects of the inhibitors on parasite growth in axenic cultures and inside infected and uninfected macrophages were also assessed. Results Prophylactic and therapeutic administration of p110δ pharmacological inhibitors significantly reduced cutaneous lesion (in CL) and parasite burdens (in VL and CL) in the spleens, livers and footpads of infected mice. The reduction in parasite burden was associated with a concomitant reduction in Treg numbers and cytokine production by liver, spleen and lymph node cells. Combined low-dose CAL-101 and amphotericin B therapy caused complete clearance of parasites in mice infected with L. donovani CONCLUSIONS: Our studies clearly show a novel therapeutic option for leishmaniasis based on CAL-101 monotherapy or CAL-101 and amphotericin B combination therapy. These observations have important and direct implications for antimicrobial immunotherapy and drug/vaccine development against leishmaniasis.

BALB/c mice are highly susceptible to experimental Trypanosoma congolense infections, whereas C57BL/6 mice are relatively resistant. Infected highly susceptible BALB/c mice die of systemic inflammatory response syndrome. Because interleukin-17 (IL-17) and Th17 cells regulate inflammatory responses, we investigated their role in the pathogenesis of experimental African trypanosomiasis in mice. We show that the production of IL-17 by spleen and liver cells and the serum IL-17 level increased after T. congolense infection in mice. Interestingly, infected highly susceptible BALB/c mice produced more IL-17 and had more Th17 cells than infected relatively resistant C57BL/6 mice. Paradoxically, neutralization of IL-17 with anti-IL-17 monoclonal antibody in vivo induced higher parasitemia in both the susceptible and the relatively resistant mice. Interestingly, anti-IL-17 antibody-treated mice had higher serum levels of alanine aminotransferase and aspartate aminotransferase, and the production of IL-10 and nitric oxide by liver cells was markedly decreased. Moreover, recombinant IL-17-treated mice exhibited significantly faster parasite control and lower peak parasitemia compared to control mice. Collectively, these results suggest that the IL-17/Th17 axis plays a protective role in murine experimental African trypanosomiasis.

Sera from cancer patients contain tumor-specific autoantibodies directly against antigenic proteins. The identification of tumor autoantigens may have utility in cancer diagnosis, prognosis, and therapy. In this study, we used immunoproteomics analysis to identify tumor proteins that elicit humoral response in colorectal cancer (CRC).The CRC cell line HCT116 was used as a source of proteins for two-dimensional polyacrylamide gel electrophoresis and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. Proteins that specifically react with sera from cancer patients were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis. In addition, the selected protein expression in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry.An autoantibody against inosine monophosphate dehydrogenase II (IMPDH2) identified by mass spectrometry was detected in eight out of 25 patients with CRC. However, none of the 15 healthy controls demonstrated autoantibody to IMPDH2.The expression of IMPDH2 in tumor tissue was significantly higher in patients with CRC than that in healthy subjects.The result confirmed that the immunoproteomics analysis holds considerable promise for the discovery of tumor-associated antigens. IMPDH2 may be a protein biomarker and novel therapeutic target in CRC.

Background/Aims: Sinomenine, a pure alkaloid extracted from the Chinese medicinal plant Sinomenium acutum, and sinomenine hydrochloride (SN) has been successfully used for the therapy of rheumatoid arthritis (RA) and kidney diseases. Autophagy is a cytoprotective mechanism used by podocytes and other cells to alleviate the effects of oxidative stress, and angiotensin II (Ang II) significantly promotes podocyte autophagy. However, excessive autophagy may lead to cell death and podocyte depletion. The present study evaluated the effect of SN in podocytes induced by Ang II. Methods: Podocytes were pretreated with graded concentrations (10-8 M ∼ 10-4 M) of SN and then stimulated with Ang II. The LC3B protein and the p47-phox membrane fraction were measured by Western blot. Autolysosomes were assessed by transmission electron microscopy. FACS was used to quantify the ROS produced by podocytes. The translocation of p47-phox to the membrane was investigated by immunofluorescence. Results: The 10-8 M ∼ 10-4 M of SN alone did not effect ROS generation or podocyte autophagy. The 10-8 M and 10-6 M SN attenuated Ang II-induced autophagy in podocytes. Furthermore, SN decreased the level of ROS generation in Ang II-induced podocytes via inhibition of NOX subunit p47-phox translocation to the membrane. Conclusion: The appropriate concentration of SN attenuated Ang II-induced podocyte autophagy through ROS generation, at least in part, by regulating NOX subunit p47-phox translocation to the membrane.

International audience; There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised similar to 20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-gamma and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δD910A) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show for the first time, that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-β, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani–infected HSCs induce CD4+ T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. Conclusion: Our results demonstrate for the first time, the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δD910A mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver. (Hepatology 2015)

Cancer stem cells (CSCs) can invade and metastasize by epithelial-to-mesenchymal transition (EMT). However, how they escape immune surveillance is unclear. B7H1 is crucial negative co-stimulatory molecule but little information about whether it works in CSCs. Therefore, we determined the expression of B7H1 and EMT-associated markers in colorectal cancer stem-like cells to investigate a possible immunoevasion way of CSCs. We enriched CD133+ colorectal cancer cells which manifested the CSCs-like properties such as higher levels of other stem cell markers Oct-4 and Sox-2, tumor sphere forming ability and more tumorigenic in NOD/SCID mice. These CD133+ cells possess EMT gene expression profile including higher level of Snail, Twist, vimentin, fibronectin and lower level of E-cadherin. Moreover, CD133+ cells in both cell line and colorectal cancer tissues expressed high level of negative co-stimulate molecule B7H1. Furthermore, some B7H1+ cancer cells also showed the characteristic of EMT, indicating EMT cells could escape immune attack during metastasis. B7H1 expression and EMT phenotypes on CSCs indicates a possible immunoevasion way.

Major efforts have been put forth for the development of effective rotavirus vaccines including transgenic plant vaccines. Previous studies have reported that rotavirus VP7 maintains its neutralizing immunity when it is transformed into the potato genome. The present study was aimed at investigating the hereditary stability of VP7-transformed potatoes over fifty generations. The VP7 gene was stably transcribed and expressed in potato cells as detected by RT-PCR and Western blotting. Humeral and mucosal responses were successfully induced in BALB/c mice fed with the fiftieth generation transformed potato tubers. There were no significant differences in serum IgG and fecal IgA between the mice fed with the first and fiftieth generation potatoes (P>0.05). Profiles of cytokines such as IFN-gamma, IL-2, IL-4, IL-5 and TGF-beta in immunized mice showed a naive T-cells bias to Th1 and Th3 polarization. Moreover, specific CTL responses were also detected in C57BL/6 mice fed with transformed potatoes. This research represents a significant step towards the development of rotavirus vaccines derived from a transgenic plant that can be obtained by long-term and large-scale vegetative reproduction. To our knowledge, this is the first finding regarding vaccines derived from plants that can be propagated for many generations.

To study the function of S100A9 protein in HL-60 cells treated with all-trans retinoic acid (ATRA), we had designed and constructed retroviral vectors for expression of small hairpin RNAs (shRNAs), and silenced S100A9 expression in HL-60 cells treated with ATRA. The silence efficiency of siRNA was detected with RT-PCR and Western blotting. The differentiation of HL-60 was monitored by nitro blue tetrazolium (NBT) reduction experiment. Western blot showed that shRNAs remarkably reduce of S100A9 expression in HL-60 cells when they were induced to differentiation by ATRA. But NBT positive percentage of differentiated HL-60 cells was no remarked change with S100A9 expression. The data indicated S100A9 could be no important action during differentiation of HL-60 treated with ATRA.

To characterize the alteration of protein expression during tumor cell differentiation induced by all-trans retinoic acid (ATRA) and to understand downstream signaling and molecular mechanism of ATRA action, we compared the protein expression profiles in HL-60 cells with ATRA treatment using two-dimensional electrophoresis (2-DE). Although many changes in protein expression were found in 2-DE maps, here we identified two protein spots remarkably expressed in the differentiated cells by nanoelectrospray ionization mass spectrometry and database searching. These two protein spots were found to be the same protein, namely S100 calcium-binding protein A9 (S100A9). Further study will be done to ascertain whether S100A9 plays a role in the regulation of differentiation or just a consequence of differentiation.

Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230–240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines.

We previously showed that CD8+ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8+ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8+ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8+ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4+ T cells. This differential activation of CD4+ and CD8+ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4+ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8+ cells had no effect. Collectively, our results show that although CD8+ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity., Author Summary It is known that CD8+ T cells are important for primary immunity to low dose L. major infection, but whether low dose-induced immunity is long lasting and whether CD8+ T cells are also important for memory immune response to low dose L. major is unknown. We studied whether infectious dose affects primary anti-Leishmania immunity and the contribution of CD8+ T cells in immunity following recovery from low and high dose infections. We found that low and high dose infections preferentially induced proliferation and cytokine production by CD8+ and CD4+ T cells, respectively, during early and late stages of infections. Also, both low and high dose-infected mice were solidly protected against secondary L. major challenge. Depletion of CD4+ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, but depletion of CD8+ cells had no effect. Together, our results show that although CD8+ T cells are selectively activated and contribute to optimal primary immunity after low dose infection, they are not required for secondary immunity. This research further enhances our understanding of the immunobiology of cutaneous leishmaniasis.

It is well established that CD4CD25 regulatory T cells (Tregs) downregulate inflammatory immune responses and help to maintain immune homeostasis. Recent reports have shown that ligation of germline encoded pattern recognition receptors such as Toll-like receptors can stimulate Tregs and therefore implicate Tregs in the pathophysiology of sepsis and other inflammatory diseases. In this report, we show that injection of lipopolysaccharide (LPS) leads to expansion of CD4CD25FoxP3 Tregs, suggesting that these cells may play an important role in immune regulation in LPS-induced acute inflammation. Indeed, genetic or immunological inhibition of Treg function using mice lacking functional Tregs (CD25 KO mice) or anti-CD25 monoclonal antibody (anti-CD25 mAb), respectively, led to acute death in an otherwise nonlethal LPS challenge. This was accompanied by exaggerated production of proinflammatory cytokines. Strikingly, adoptive transfer of CD4CD25 Tregs to CD25 KO mice before LPS challenge rescues mice from death. Unlike LPS, depletion of Tregs followed by concanavalin A (Con A) challenge does not result in mortality, suggesting that Treg depletion does not globally influence all models of acute inflammation. We authenticate our findings by showing that depletion of Tregs leads to mortality in a nonlethal Escherichia coli challenge accompanied by elevated serum levels of proinflammatory cytokines. Collectively, our results indicate that in addition to regulation of LPS-induced acute inflammation, Tregs help to improve bacterial clearance and promote survival in an acute model of bacterial infection.

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals.

Lupus-related vascular events are becoming a formidable obstacle to the improvement of long-term prognosis of systemic lupus erythematosus (SLE) and the existent findings lack for systematization. Proteomics is a strategic approach but its applications in this regard are rare and primarily involve proteome acquisition or biomarker screening, rather than functional identification. To provide further insight, we investigated the proteomic diversity of peripheral blood mononuclear cells (PBMCs) in SLE and the possible role of the identified Annexin A5 (AnxA5) in pathogenesis. The study involved 214 SLE and 183 healthy women. The two-dimensional electrophoresis gel images showed 649 ± 25 and 676 ± 19 protein spots from the PBMCs of the patients and controls, respectively. From these protein spots, 30 differentially expressed proteins were chosen, and 16 of these proteins were identified by mass spectrometer. Western blotting confirmed the over-expressed candidate, AnxA5, from the PBMCs of the patients (SLE:control=1.607:1, P=0.0004), but ELISAs indicated decreased levels of sera AnxA5 in the patients compared to healthy donors (SLE vs. control=26.8 ± 3.0 vs. 49.0 ± 3.3 ng/mL, P

Background BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines. Methodology/Findings BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 103 T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25+ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4+CD25+Foxp3+ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25+ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25+ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines. Conclusion Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice., Author Summary BALB/c mice are highly susceptible while C57BL/6 is relatively resistant to experimental Trypanosoma congolense infection. Acute death observed in infected BALB/c mice is usually associated with the excessive production of pro-inflammatory cytokines. Regulatory T cells (Tregs) have been shown to play a significant role in the pathogenesis of many diseases including those caused by parasites. However, the role of Tregs in the pathogenesis of T. congolense infection remains unclear. We were interested in addressing the following questions: Do Tregs contribute to the distinct phenotypic outcomes observed in T. congolense-infected BALB/c and C57BL/6 mice? If so, where and how do they operate with respect to parasitemia and cytokine response? By selectively altering the numbers of these cells either by targeted depletion with monoclonal antibody or adoptive transfer of highly enriched naïve CD25+ cells prior to infection, we show that Tregs impairs efficient parasite control and impacts on production of disease-exacerbating proinflammatory cytokines. Collectively, our findings suggest that Tregs contribute to enhanced susceptibility to experimental T. congolense infection in mice.

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2-DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor-associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.

The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

Proteome analysis has rapidly developed in the post-genome era and is widely accepted as a complementary technology to genetic profiling. Sera from tumor patients contain tumor specific antibodies directly against antigenic proteins. Immunoproteomics, which defines the subset of proteins involved in the immune response, holds considerable promise for the discovery of tumor-associated antigens, suggesting that this approach may be a useful tool for identification of prognostic, diagnostic markers and novel therapeutic targets. Recently, various different approaches are developed to identify tumor-associated antigens by autoantibodies from tumor patients' sera. More than one hundred tumor-associated antigens have been found in various tumors by with antibody-based strategy nowadays. However, some autoantibodies are also found in health subjects, even some of them with high antibody occurrence. So, it is necessary to undergo sufficiently detailed validation schemes for these novel tumor-associated antigens.

Dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin (DC-SIGN) is a potential target receptor for vaccination purposes. In the present study, we employed Lewis X (Le(x)) oligosaccharides, which mimic natural ligands, to target ovalbumin (OVA) to human dendritic cells (DCs) via DC-SIGN, to investigate the effect of this DC-SIGN-targeting strategy on the OVA-specific immune response. We demonstrated that Le(x) oligosaccharides could enhance the OVA-specific immune response as determined by enzyme-linked immunospot assay (ELISPOT), intracellular interferon-gamma staining and (51)Cr-release assay. An almost 300-fold lower dose of Le(x)-OVA induced balanced interferon-gamma-secreting cells compared to OVA alone. Furthermore, secretion of interleukin-10, a reported mediator of immune suppression related to DC-SIGN, was not increased by Le(x)-OVA, either alone or together with sCD40L-stimulated groups. A blocking antibody against DC-SIGN (12507) reduced the numbers of interferon-gamma-secreting cells during Le(x)-OVA stimulation, yet it did not prevent Le(x) oligosaccharides from promoting the secretion of interleukin-10 that was induced by ultra-pure lipopolysaccharide. These results suggested that the strategy of DC-SIGN targeting mediated by Le(x) oligosaccharides could promote a T-cell response. This DC-targeting may imply a novel vaccination strategy.