No description available.

Toxoplasma gondii is a protozoan parasite that causes life-threatening disease in congenitally infected infants and immunocompromised patients, such as those inflicted with AIDS. Toxoplasmic encephalitis (TE) is a common presenting condition in an AIDS infection. People become infected with T. gondii by ingesting tissue cysts in undercooked meats or by ingesting oocysts excreted by cats. Methylmercury (MeHg) is a well-documented neurotoxicant that accumulates in the brain and causes severe mental and visual dysfunction, including chronic encephalopathy. Consumption of contaminated fish, grains, and seeds are common sources of human exposure to methylmercury. Studies from our laboratory suggest that oral exposure to a single high dose of 20 mg/kg MeHg does not increase the susceptibility to acute toxoplasmosis in CBA/J mice. Therefore, we further investigated endpoints associated with immunotoxicity and neurotoxicity in 6-week old, female CBA/J mice exposed to both MeHg and T. gondii during a chronic T. gondii infection. We examined both single and multiple doses of MeHg exposure in a chronic parasitic infection model. In the single high dose study, four groups of six-week-old, female CBA/J mice were either fed 25 T. gondii tissue cysts of the ME-49 strain or given vehicle. Six weeks later, two out of the four groups (T. gondii and vehicle control) were orally gavaged with a single dose of 20 mg/kg body weight of MeHg and sacrificed seven days post exposure. Experiments from the multiple MeHg dose study were performed under similar conditions with the same number of groups and dosed by oral gavage with 8 mg/kg body weight of MeHg on days 0, 2,4,7,10,13. These mice were sacrificed on day 17 or 18 after initiating MeHg exposure. Flow cytometry following exposure to a single dose of MeHg in mice with a chronic T. gondii infection revealed significant changes (P < 0.05) within the T cell subpopulation percentages caused by exposure to MeHg. For example, the thymic CD4+CD8+ T cell subpopulations were increased (P <0.05). However, MeHg had no significant effect on the CD4+CD8-, CD4-CD8+, or non-T cell subpopulations in the spleen. Furthermore, MeHg increased splenic cellularity and spleen-to-body-weight ratios with or without a concurrent T. gondii infection. MeHg also caused a significant decrease in mouse body weight. There was a significant (P <0.05) increase in brain tissue cyst counts within the group exposed to both MeHg and T. gondii (16 ± 4, mean ± SE, n=7) versus T. gondii alone (4 ± 1, n=8). Histopathological examination demonstrated that the brain was affected, as lesions, gliosis, and meningitis were notable in mice given T. gondii. Exposure of mice to multiple doses of MeHg also resulted in effects on the immune system of CBA/J mice with and without chronic toxoplasmosis. Total cellularity and numbers of CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T-cell subpopulations show a marked decrease in number in the thymus, while total cellularity was also decreased in the spleen following concurrent exposure to T. gondii and MeHg. Flow cytometric examination of lymphocyte populations (CD4+ and CD8+ lymphocytes) in the spleen and thymus demonstrated differences from control in the groups exposed to T. gondii and MeHg. Histopathological examination did not reveal any significant lesions. The data from experiments in which single or multiple doses of MeHg were given to mice with a chronic T. gondii infection indicate that concurrent exposure, to both MeHg and T. gondii, dependent on dose and time of exposure had notable effects, especially on the immune system (Supported by NIH Grant F36GM20301). / Ph. D.

No description available.

A infecção crônica das vias aéreas por Pseudomonas aeruginosa (PA) é a principal causa de morbidade e mortalidade em pacientes com fibrose cística (FC), devido à contínua degradação do tecido pulmonar, que leva ao declínio da função pulmonar, gerada pela infecção e pelo processo inflamatório. O objetivo do presente estudo foi analisar características genéticas de PA que levam à sua adaptação às vias aéreas destes pacientes com infecção pulmonar crônica, atendidos no Centro de Referência em FC do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP e relacionar com resultados de tipagem molecular, resistência a antibióticos, cronicidade e dados clínicos dos pacientes em acompanhamento clínico no período de julho/2011 a abril/2014. As características genéticas dos isolados investigados englobam pesquisa de 18 genes de virulência e genes do sistema quorum sensing (genes lasR e rhlR), associação entre mutações e conversão para fenótipo mucoide (operon algTmucABD) e caracterização de linhagens hipermutantes (genes mutS e mutL). A identificação de P. aeruginosa foi realizada por PCR e MALDI-TOF, que mostraram alta concordância. Foram considerados os dados clínicos dos pacientes: índice de massa corpórea, escore de Shwachman, medidas de capacidade vital forçada e volume expiratório forçado no primeiro segundo. A porcentagem de pacientes com infecção pulmonar crônica por PA observada foi similar aos dados disponíveis na literatura, entretanto, a alta incidência em pacientes jovens foi preocupante. O perfil de macrorrestrição do DNA genômico por PFGE se mostrou útil para definição de colonização crônica/intermitente em associação com critérios clínicos e, juntamente com a detecção de mutações nos genes mucA e mucD confirmaram transmissão interpacientes. Foi observada alta ocorrência dos genes de fatores de virulência pesquisados para grande maioria dos isolados de pacientes crônicos. A resistência aos antibióticos pesquisados dos isolados de P. aeruginosa foi baixa e está de acordo com a literatura nacional e internacional e com a antibioticoterapia adotada no hospital. Não foi observada resistência aos carbapenêmicos e às fluoroquinolonas devido à presença de genes de resistência plasmideais. As mutações no gene mucA foram o principal mecanismo de conversão para o fenótipo mucoide e o fenótipo revertente não-mucoide ocorreu principalmente por mutações no gene algT. Foram detectadas novas mutações nos genes mutS e mutL que também suportam a ideia que hipermutação em PA está associada com mutações do sistema mismatch de reparo do DNA. O sistema quorum sensing dos isolados estudados está parcialmente prejudicado devido às várias mutações no gene lasR, mas todos conservam o gene rhlR intacto, que sustenta alguma atividade quorum sensing envolvida na produção de fatores de virulência importantes. Pacientes com infecção pulmonar crônica por PA com isolamento de outros bacilos gram-negativos não-fermentadores apresentaram maior alteração da função pulmonar quando comparados com pacientes com infecção pulmonar crônica por PA com ou sem isolamento de Staphylococcus aureus. As alterações presentes no operon algTmucABD, quorum sensing e hipermutabilidade contribuem para a cronicidade dos pacientes com FC em relação à infecção por P. aeruginosa. / The chronic airway infection by P. aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients, due to continuous degradation of the pulmonary tissue. This leads to decline in lung function, which is generated by the related infection and inflammation. The aim of this study was to analyze genetic characteristics associated with the adaptation of PA to the airways of patients with chronic pulmonary infection attended at the CF Reference Center from the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP; and to correlate these findings with the results of molecular typing, antibiotic resistance, chronicity and clinical data of patients in clinical follow-up from July/2011 to April/2014. The genetic characteristics of isolates investigated includes the research of 18 virulence genes and the quorum sensing system genes (lasR and rhlR genes), association between mutations and conversion to the mucoid phenotype (algTmucABD operon), and characterization of hypermutable strains (mutS and mutL genes). Identification of PA was performed by PCR and MALDI-TOF, which showed a high correlation. The patients\' clinical data considered were: body mass index, Shwachman score, forced vital capacity measures and forced expiratory volume in one second. The percentage of patients with chronic PA infection observed was similar to the data available in the literature; however, a worrying high incidence in young patients was noticed. The macrorestriction profile of genomic DNA by PFGE proved to be useful to define chronic/intermittent colonization in association with clinical criteria and it confirmed interpatient transmission, in combination with the detection of mutations in the mucA and mucD genes. High occurrence of virulence genes was detected for the vast majority of isolates from chronic CF patients. Antibiotic resistance for PA isolates was low and is in accordance with national and international literature and antibiotic therapy adopted in the hospital. There was no resistance to carbapenems and fluoroquinolones by the presence of plasmid mediated resistance genes. Mutations in the mucA gene were the main mechanism to conversion to mucoidy, and the non-mucoid revertants occurred mainly by mutations in the algT gene. New mutations in mutS and mutL genes were detected, which support the idea that hypermutation in PA is associated with mutations in the DNA mismatch repair system. The quorum sensing system of the isolates is partially damaged due to several mutations in the lasR gene, but all isolates maintain an intact rhlR gene, which holds some quorum sensing activity with production of important virulence factors. Patients with chronic PA infection with isolation of other non-fermenting gram-negative rods had greater change in lung function compared with patients with chronic PA infection with or without isolation of Staphylococcus aureus. The changes presented in the algTmucABD operon, quorum sensing and hypermutability contribute to the chronicity of CF patients in relation to infection by P. aeruginosa.

Chronic HCV infection causes generalized CD8+T cell impairment, not limited to HCV-specific CD8+ T cells. Infiltrating monocyte-derived macrophages contribute to a micro- environment that could impact CD8+T cells trafficking through the liver. Macrophages can differentiate into pro-inflammatory (M1) and anti-inflammatory (M2a, M2b, and M2c) subsets. Whether macrophage subset generation in chronic HCV infection is altered and if that has a subsequent impact on CD8+T cell functions was not known. I have shown phenotypic alterations in both M1 and M2 macrophages in chronic HCV infection. In particular, M1 from advanced fibrosis patients show increased CD86 expression, reduced spontaneous TNF-α and increased spontaneous IL-10 production. In uninfected controls, co-culturing CD8+T cells with M1 macrophages significantly increased the percentage of CD107a+ and IFN-γ+ CD8+T cells in a contact-dependent manner. Similar autologous co-cultures between M1 and CD8+T cells from patients with chronic HCV infection showed that M1 significantly reduced the percentage of IFN-γ+ CD8+T cells, even though patients displayed elevated IFN-γ+CD8+ T cells at baseline prior to culture. Overall, I demonstrated the altered phenotype of macrophages generated from patients with chronic HCV infection. I also showed the ability of M1 macrophages to induce IFN-γ+CD8+T cells in normal donors and their opposite impact when the cells are derived from chronic HCV infected patients.

Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immune deficiency syndrome (AIDS). HIV-1 is a worldwide epidemic that currently affects over 35 million people worldwide, and continues to spread at an appalling rate. A universal HIV-1 preventive vaccine is considered to be the optimal solution in achieving the ultimate goal of AIDS eradication. Regretfully, most endeavors thus far of developing a prophylactic vaccine have been largely disappointing. Highly Active Anti-Retroviral Therapy (HAART) has been shown to reduce the plasma HIV-1 RNA level to below the detection limit of clinical assays (50 copies/ml); it combines three or more antiretroviral drugs which belong to at least two different classes – targeting distinct steps in the viral life cycle, and inhibiting viral replication. However, unless the infection is eradicated, strict adherence to a lifelong treatment regimen is required. HAART is limited by its high cost, drug availability, complicated administration schedules, serious side effects, and the potential that the virus will ultimately develop drug resistance. A more plausible approach lies in therapeutic vaccines that provide immunity to partially control viral replication postinfection – delaying or minimizing ART, and offering “drug holidays”. The primary goal of a therapeutic vaccine is to effectively induce HIV-1 specific cytotoxic T lymphocyte (CTL) responses, which plays a critical role in control of viral proliferation. Dendritic cells (DCs)-based therapeutic vaccines have been showing the most promising results. However, the therapeutic efficacy of DCs based vaccines is limited. This is partially due to the fact that DCs induced CD8+ T cell responses are largely CD4+ T cell dependent, while HIV-1 infection usually renders the immune system very “helpless” from CD4+ T cells. In addition, infection, impaired function, and physical depletion of DCs are often reported during the early stage. Furthermore, DCs are often found to be inflammatory and immunosuppressive, which is mainly mediated by the interaction between HIV-1 Env gp120 and DC receptors. Thus, the search for a novel therapeutic vaccine strategy is warranted. Using T-APC (T cells-antigen-presenting cells) as a novel T cell-based vaccine has emerged as a potential candidate for a HIV-1 therapeutic vaccine, which aims at boosting HIV-specific CTL responses. Our previous work demonstrated that CD4+ and CD8+ T cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice via co-culture with OVA-pulsed DCs (DCOVA) can be activated, acquiring pMHC I, pMHC II, and costimulatory molecules, thus act as CD4+ T helper-antigen-presenting cells (Th-APCs) and CD8+ cytotoxic T-antigen-presenting cells (Tc-APCs). We also elucidated that DC-derived exosomes (EXO), which are 50- to 90-nm diameter vesicles containing antigen-presenting, tetraspan, adhesion, and costimulatory molecules, can transfer the antigen-presenting activity of DCs to activated CD4+T cells through EXO uptake. EXOOVA-targeted activated CD4+T (aTexo) cells can (1) stimulate more efficient central memory CD8+ CTL responses and T cell memory than EXOOVA or DCOVA, (2) activate CD8+ CTL responses independent of CD4+Th cells, and (3) counteract CD4+25+regulatory T (Tr) cell-mediated immune suppression. These results formed the new concept of novel EXO-targeted CD4+ T cell vaccines. In this study, we tailored EXO-targeted T cells vaccine by using polyclonal activated CD8+ T cells instead of CD4+ T cells, as CD4+ T cells served as the primary target for HIV-1 infection. We showed that (1) OVA-specific exosome-targeted CD8+ T cell-based vaccine (OVA-Texo) can stimulate efficient OVA-specific CD8+ CTL and memory responses, inducing sufficient antitumor immunity against OVA-expressing tumor cells in mouse models. (2) This exosome-targeted CD8+ T cell-based vaccine strategy could be applied to HIV-1-Gag protein, provoking effective Gag-specific CD8+ CTL, T cell memory, and antitumor immunity against Gag-expressing tumor cells. (3) Engineering Gag-Texo with up-regulated 4-1BBL (APC derived costimulatory molecule) expression could improve the performance of Gag-Texo vaccine. (4) OVA-Texo is able to evoke a successful immune response in bystander chronic infection, converting CD8+ T cell exhaustion, restoring effector functions of exhausted CD8+ T cells. Moreover, combination of OVA-Texo vaccine with PD-L1 blockage in a dual treatment could result in a synergistic effect in rescuing CTLs exhaustion in chronic infection. Those desired features make EXO-targeted CD8+ T cells vaccine an appealing novel strategy in HIV-1 infection. The EXO-targeted CD8+ T cells vaccine may be applicable to therapeutic HIV treatment through the use of autologous T cells with uptake of EXOs derived from engineered DCs.

A hallmark of chronic human immunodeficiency virus (HIV) infection is the impairment of CD8 T cell survival and effector functions, which likely contributes to HIV pathogenesis. A number of factors could be attributed to this impairment, including the declining number of CD4 T cells, progressive destruction of secondary lymphoid tissues and an increasingly inhibitory environment. As highly active antiretroviral therapy shows limited efficacy in improving CD8 T cell functions, this thesis explores the therapeutic application of costimulatory molecules in directly stimulating non-functional HIV-specific CD8 T cells and ultimately their relevance to the control of chronic HIV infection. Costimulatory molecules are adjuvants for functional activation of T cells that act in concert with the antigen-specific signal. The Tumor Necrosis Factor (TNF) family member, 4-1BBL, emerges as the most effective costimulatory molecule in the antigen-specific expansion of human memory CD8 T cells as compared to the related TNF family members CD70 and LIGHT. As well, 4-1BBL improves the cytolytic function of T lymphocytes on a per cell basis. Furthermore, 4-1BBL is identified as a key component in the therapeutic rescue of CD8 T cell function and its effect is at least partially dependent on its signaling adaptor TNF receptor associated factor 1 (TRAF1), both in vitro and in vivo. This thesis also identifies the loss of TRAF1 as a new mechanism of immune dysregulation of HIV-specific CD8 T cells during the chronic phase of HIV infection and offers a means to correct it. The loss of TRAF1 has functional relevance in HIV suppression and HIV-specific CD8 T cell responses. Finally, a combination therapy involving agonistic anti-4-1BB antibody is shown to be successful in a proof of concept treatment of chronic lymphocytic chroriomeningitis virus (LCMV) infection in mice, resulting in sustained reduction in viral load. A new model of HIV-specific CD8 T cell dysfunction is constructed based on these findings.

A hallmark of chronic human immunodeficiency virus (HIV) infection is the impairment of CD8 T cell survival and effector functions, which likely contributes to HIV pathogenesis. A number of factors could be attributed to this impairment, including the declining number of CD4 T cells, progressive destruction of secondary lymphoid tissues and an increasingly inhibitory environment. As highly active antiretroviral therapy shows limited efficacy in improving CD8 T cell functions, this thesis explores the therapeutic application of costimulatory molecules in directly stimulating non-functional HIV-specific CD8 T cells and ultimately their relevance to the control of chronic HIV infection. Costimulatory molecules are adjuvants for functional activation of T cells that act in concert with the antigen-specific signal. The Tumor Necrosis Factor (TNF) family member, 4-1BBL, emerges as the most effective costimulatory molecule in the antigen-specific expansion of human memory CD8 T cells as compared to the related TNF family members CD70 and LIGHT. As well, 4-1BBL improves the cytolytic function of T lymphocytes on a per cell basis. Furthermore, 4-1BBL is identified as a key component in the therapeutic rescue of CD8 T cell function and its effect is at least partially dependent on its signaling adaptor TNF receptor associated factor 1 (TRAF1), both in vitro and in vivo. This thesis also identifies the loss of TRAF1 as a new mechanism of immune dysregulation of HIV-specific CD8 T cells during the chronic phase of HIV infection and offers a means to correct it. The loss of TRAF1 has functional relevance in HIV suppression and HIV-specific CD8 T cell responses. Finally, a combination therapy involving agonistic anti-4-1BB antibody is shown to be successful in a proof of concept treatment of chronic lymphocytic chroriomeningitis virus (LCMV) infection in mice, resulting in sustained reduction in viral load. A new model of HIV-specific CD8 T cell dysfunction is constructed based on these findings.

The asymptomatic nature of chlamydial infection renders the differential diagnosis of acute and chronic infection difficult. An untreated Chlamydia trachomatis infection can become chronic, result in disease sequelae such as salpingitis and pelvic inflammatory disease (PID), and ultimately culminate in tubal occlusion and infertility. Diagnostic tests for C. trachomatis such as nucleic acid amplification testing (PCR), antigen detection and serological methods have variable performance capabilities with respect to sensitivity, specificity and stage of infection. The use of PCR as a diagnostic tool is somewhat limited, as specimen collection is routinely sampled from the lower genital tract; hence, infections in the fallopian tube where inflammatory damage is most significant, escape detection. Furthermore, PCR can only detect selected Chlamydia DNA sequences from readily accessible sites of the genital tract, and therefore cannot differentiate between acute and chronic infection. Other serological assays aim to discriminate the various stages of C. trachomatis infection through identification of key antigens. The efficacy of these assays however is impeded due to cross-reactivity between chlamydial species and the subsequent antibody response against the target antigen is not restricted to patients with a specific stage of infection. To identify antibody responses capable of differentiating various states of chlamydial infection, samples were collected from both men and women given the variability of immune responses between the two genders. Samples were assigned to a patient group according to infection status and then probed against protein extracts of HEp-2 cells infected with C. trachomatis serovar L2 and HEp-2 cells pre-treated with IFN-γ and infected with C. trachomatis serovar L2. (persistence cell culture) Serological analysis revealed the presence of five antigens (denoted bands A, B, C, D and M) which were shown to be differential between patient groups. Identification of bands B and C by N-terminal sequencing provided two possible candidates for each antigen, ie. CT727 and CT396 (band B) and CT157 and CT423 (band C). In contrast, band M which was unique to males was a PmpB (probable outer membrane protein B) fragment. The four target antigens (CT157, CT423, CT727 and CT396) were expressed as recombinant proteins using autoinduction media and were subsequently probed by both male and female sera to evaluate their diagnostic potential. Results showed that two chlamydial antigenic targets (CT157 and CT727) have the potential to discriminate between acute and chronic C. trachomatis infection. However, since only a small number of samples (n = 3) were used for this aspect of the study, the findings should simply be viewed as preliminary. In females, sensitivity and specificity values were derived using various combinations of the four target antigens into a panel format for the purpose of detecting chronic C. trachomatis infections. The preferred format was B + C with a sensitivity and specificity of 80% and 84% respectively. Using the IFN-γ-mediated persistence model, only two of the five antigenic targets were shown to be differentially expressed. PmpB in males and CT157 (the most likely band C candidate) in females were shown to be up-regulated to varying degrees in samples across the patient groups. We also demonstrated that no other chlamydial antigens are up-regulated during a persistent C. trachomatis infection. In conclusion, although combinations of bands A, B, C, D and M differentiate between male and female patient groups under normal chlamydial growth conditions, during IFN-γ-induced persistence, only bands C (CT157) and M (CT413 - PmpB) are up-regulated thus suggesting a potential role in chronic C. trachomatis infection.

L'infection chronique par le virus de l’hépatite B (HBV) est un problème de santé majeur dans le monde et peut conduire à la cirrhose et à l’hépatocarcinome. Des réponses défectives des cellules T ont été démontrées, mais les événements précis qui peuvent contribuer à l'insuffisance des réponses des cellules B restent à déterminer. L'expansion et la différenciation optimales des cellules B reposent sur l'activité du récepteur Toll Like Receptor 9 (TLR9) qui détecte l'ADNdb et est exprimée chez l'homme principalement par les cellules dendritiques plasmacytoïdes (pDC) et les cellules B. L'impact de HBV sur TLR9 dans les cellules B humaines reste à explorer. Dans cette étude, nous avons exploré les effets de HBV sur l’expression et les fonctions médiées par TLR9 dans les cellules B humaines en utilisant une lignée cellulaire de cellules B ainsi que des lymphocytes B primaires. De plus, on a corroboré nos résultats dans une cohorte de patients HBV chroniques. L'expression de TLR9 a été réduite chez les toutes les sous-populations de cellules B du sang périphérique, exposées au VHB. Les fonctions des cellules B médiées par TLR9 comme la prolifération et la sécrétion de cytokines pro-inflammatoires ont été abrogées en présence de HBV. Nos résultats montrent que l’antigène de surface HBsAg inhibe la phosphorylation du facteur de transcription CREB qui ne peut plus se lier sur son site CRE situé sur le promoteur TLR9 ce qui affecte la transcription de ce dernier. Enfin, nous avons corroboré nos résultats in vitro dans une cohorte de porteurs chroniques du HBV et constaté que l'expression et la fonction de TLR9 étaient significativement diminuées. Nos résultats révèlent le mécanisme qui induit une réponse immunosuppressive par HBV sur la fonction TLR9 dans les cellules B humaines, ce qui peut contribuer à la persistance de ce virus chez l'hôte et la complication de la phase chronique de la maladie / Chronic HBV infection is a major health problem worldwide. Ineffective T cell and antibody responses have been demonstrated, yet the precise events that may contribute to insufficient B cell responses remain to be determined. Optimal B cell function, expansion and differentiation rely on Toll Like Receptor 9 (TLR9) activity which senses dsDNA and is expressed in human mainly by plasmacytoid dendritic ( pDC) and B cells. The impact of HBV on TLR9 in human B cell subsets remains to be explored.Here, we investigated the effects of HBV on TLR9 function in human B cells. Both primary and B cell lines were used to analyze the effect of HBV on TLR9 expression and function. These results were corroborated in a cohort of chronically infected HBV patients. TLR9 expression was reduced in all peripheral blood B cells subsets exposed to HBV. B cell function mediated by TLR9, such as proliferation and pro-inflammatory cytokines secretion were abrogated in the presence of HBV. Our results show that the viral surface antigen HBsAg inhibited the phosphorylation of the transcription factor CREB which could no longer bind the CRE site located on the TLR9 promoter. Finally, we corroborated our in vitro findings in a cohort of chronic HBV carriers (CHB) and found that TLR9 expression and function were significantly suppressed.Our findings reveal the mechanism that induces an immunosuppressive response by HBV on TLR9 function in human B cells, which may contribute to HBV persistence in the host