Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS. / Ph. D.

Brucella are Gram-negative intracellular bacteria that cause abortion and infertility in livestock and chronic disease in humans. The Centers for Disease Control and Prevention (CDC) categorizes them as class B pathogens due to their zoonotic potential. Currently, there are no efficacious Brucella vaccines for humans available. Very few studies have focused on identifying protective vaccines against respiratory exposure. Protection by B. abortus rough vaccine strains RB51 and RB51SOD is through strong CD4⁺ Th₁ and CD8⁺ Tc₁ adaptive immunity. However, limited information is available on how they stimulate innate immunity. This knowledge is critical for improving these vaccines for their potential use in humans. Dendritic cells (DCs) play a crucial role bridging innate and adaptive immunity. Therefore, enhancing the ability of rough vaccine strains to induce DC maturation and function could be critical for upregulating protective T-cell responses. Herein, we demonstrated that live vaccine strain RB51 induced significantly better (p≤0.05) DC maturation and function in vitro and upon intranasal inoculation in vivo compared to strain RB51SOD or strain 2308. Due to safety concerns of live vaccines, irradiated and heat killed vaccines were also tested; only live strain RB51 infected DCs induced significant (p≤0.05) DC function based on TNF-α and IL-12 secretion. DC activation occurs through Toll-like receptors (TLRs) 2, 4 and 9. Our study reported that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308, which was not dependent on a specific TLR. However, strain RB51 induced TNF – α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) higher number of strain RB51 colonies present at day 14 post infection. By unraveling the innate immune responses to Brucella, the ultimate goal of these studies is to develop a protective vaccine for animals and people against respiratory challenge. As such, we tested several vaccination strategies. Despite enhanced DC activation and function achieved by vaccine strains, they failed to protect mice against intranasal challenge with strain 2308. Future experiments will address host-pathogen interaction at the lung microenvironment and elucidate immune mechanisms that will enhance protection against aerosol exposure. / Ph. D.

Generation of reactive oxygen species (ROS) by mammalian host cells is a double-edged sword. ROS are clearly beneficial in directly killing pathogens and as a signaling molecule to alert macrophages and neutrophils to the site of infection. However, ROS are also capable of damaging host cells by destroying DNA, oxidizing proteins and lipids, inactivating enzymes, and eliciting apoptosis. Therefore the balance of ROS generation and clearance is essential for homeostasis. Although multiple mechanisms can contribute to the generation of ROS, NADPH oxidase (Nox) is a primary producer. In terms of clearance, several ROS scavenging enzymes are induced by Nrf2, a sensor of excessive ROS. The mechanisms behind the skewing of this balance toward prolonged accumulation of ROS under chronic inflammatory conditions are not well understood. Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria cell wall, is specifically recognized by Toll-like receptor 4 (TLR4). LPS triggers robust activation of Nox and ROS production through TLR4, while also activating Nrf2 and ROS clearance. Intracellular pathways regulating ROS generation and clearance mediated by TLR4 are not well defined. Since interleukin-1 receptor associated kinase 1 (IRAK-1) is a key downstream component of TLR4, we test the hypothesis that IRAK-1 may play a critical role in maintaining the balance of LPS triggered ROS generation and clearance. Using wild type and IRAK-1 deficient murine embryonic fibroblasts, we tested the dynamic induction of Nox1 (a key NADPH oxidase) and Nrf2 by varying dosages of LPS. Our data confirm that high dose LPS (as seen in acute bacterial infection) induced both Nox1 and Nrf2. The generation of Nox1 is IRAK-1 dependent. Low dose LPS (as seen in chronic metabolic endotoxemia) fails to induce Nrf2 and induces mild and prolonged expression of Nox1. Cells pre-challenged with low dose LPS are primed for more robust expression of ROS following a second LPS challenge. The conclusions and implications generated by this study are that chronic low dose endotoxemia (prevalent in adverse health conditions) may skew the balance of ROS generation and clearance to favor prolonged ROS accumulation, and that IRAK-1 represents a potential therapeutic target to treat chronic inflammatory diseases. / Ph. D.

Brucella abortus is amongst the top 5 zoonotic diseases worldwide. The overall goal of this research is to generate a safe and effective vaccine for humans. Brucella abortus strain RB51, approved for use in cattle, provides protection by initiating a strong T-helper 1 (Th1) type response is a candidate vaccine. Based on a model for aerosol exposure mice were vaccinated intranasally (IN) with strain RB51 and challenged IN with B. abortus strain 2308, strain RB51 did not protect. Protection against Brucella is mediated through TLRs 2, 4 and 9. The addition of TLR 2 or TLR 4 and a trend with TLR9 agonists with intranasal RB51 vaccination significantly increased bacterial clearance in the lung after strain 2308 challenge. Therefore, we hypothesized that combining TLR agonists 2, 4, and 9 with strain RB51 IN would upregulate protection and clearance in the lung against strain 2308 challenge (IN), by upregulating the DC1 and CD4 Th1 and CD8 immune response. This study showed that protection is not upregulated by combining all TLR agonists. Overall the addition of TLR 2 and 4 vs. TLR 2, 4 and 9 agonists affects the immune response and impacts the level of clearance. Our data support the development of a DC1 Th1 CD8 response, based on serology, and both DC and T-cell activation and function by the group which received the TLR 2 and 4 agonists and to a lesser degree the group receiving TLR 2, 4, and 9 agonists. Additional studies are warranted to further define the differential mechanisms and endpoints of protection. / Master of Science

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: The communicable disease tuberculosis (TB) is responsible for millions of deaths each year, on a global scale. At present the contribution of host genetics in TB is generally accepted and, together with environmental aspects (e.g. nutrition and crowding) and the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis); it will possibly have a hand in the outcome of disease. Clearly, TB is a multifaceted disease and the repercussions for studying genetic susceptibility are that many genes will potentially be implicated. To date a variety of genes such as NRAMP1 and HLA have been implicated in influencing the host response to TB, albeit with varying effects in different populations. Some of the more recently implicated genes are the pattern recognition receptors, the Toll-like receptors (TLRs). Genetic variation in theses genes has been associated with a myriad of different diseases, including those of an infectious nature, such as TB. In the case of TB, TLR2 is the most prominent candidate with TLR8 and 9 more recently implicated. One of the more well known genes implicated with TB is the vitamin D receptor (VDR), as the antimicrobial gene cathelicidin (CAMP), one of the most important agents of mycobacterial killing, has a VDR response element in its promoter. TLR2, VDR and CAMP are all connected in a complex pathway essential for the host defence against M. tuberculosis. Nine single nucleotide polymorphisms (SNPs) in three TLR genes (TLR2,8 & 9) were investigated via a case-control approach to determine their potential role in human genetic susceptiblity to TB in the Coloured population of South Africa. The effect of the VDR polymorphism Cdx2 on the expression of cathelicidin mRNA and protein expression was also investigated. Three genes were found to contribute significantly to genetic host susceptibility in the Coloured population of South Africa. An allelic association (p = 0.031) was observed for the TLR8 (located on the X-chromosome) SNP rs3761624, with the A-allele being more prominent in females. Four haplotypes of TLR8 were found to be significantly linked to TB susceptibility with the three SNP haplotype rs3761624-rs3764879-rs3764880, specifically the allelic combination of G/C/A [p = 0.004, OR = 2.67(95% CI: 1.90-3.74)], showing a marked association (p = 0.001). The TLR9 introexon2 boundary SNP rs352139 was significantly associated with TB susceptiblity on a genotypic (P = 0.02) and allelic scale [p = 0.05, OR=0.70; (95% CI: 0.55–0.90)], with the T allele more frequent in controls. The TLR9 two SNP haplotype consisting of rs5743836 and rs352139 was linked (p = 0.037) to TB susceptibility, specifically the combination of the alleles A/T [p = 0.013, OR=0.71; (95% CI: 0.55–0.92)]. No gene-gene interaction between TLR2, TLR8 and TLR9 was observed. No significant conclusions could be drawn from the analysis of the mRNA and protein expression of CAMP in samples harbouring the different genotypes of the VDR polymorphism Cdx2. Genetic variations in the TLR8 and 9 genes were identified as potential factors that influence genetic host susceptibility to tuberculosis in the Coloured population of South Africa. / AFRIKAANSE OPSOMMING: Die oordragbare siekte tuberkulose (TB) is elke jaar verantwoordelik vir miljoene sterftes wêreldwyd. Die invloed van die gasheer genoom op TB vatbaarheid word huidiglik aanvaar, tesame met die invloed van omgewingsfaktore (dieet, oorbevolking ens.) en die bakterium Mycobacterium tuberculosis (M. tuberculosis). Dit is duidelik dat TB ‘n veelvlakkigesiekte is wat beïnvloed sal word deur ‘n menigte verskillende gene. ‘n Verskeidenheid gene is al betrek by TB genetiese vatbaarheid, onder andere NRAMP1 en HLA, hoewel hul effekte in uiteenlopende bevolkings verskil. Sommige van die onlangse gene wat betrek is in TB genetiese vatbaarheid is die Toll-like reseptore (TLRs). Genetiese variasie in hierdie gene is geassosieer met ‘n wye verskeidenheid van siektes, insluitend aansteeklik van aard, onder andere TB. In die geval van TB speel TLR2 ‘n prominente rol, terwyl TLR8 en TLR9 meer onlangs geïmpliseer is. Een van die meer bestudeerde TB vatbaarheidsgene is die vitamiene D reseptor geen (VDR). VDR is direk betrokke in die uitdrukking van die anti-mikrobiale geen cathelicidin (CAMP),’n integrale komponent in die vernietiging van mikobakterieë. Die CAMP geen het ‘n VDR respons-element in sy promotor. Die TLR2, VDR en CAMP gene word verbind deur ‘n komplekse netwerk wat integraal is tot die liggaam se vermoë om TB af te weer. Nege enkel nukleotied polimorfismes (ENPs) in drie gene (TLR2,8 & 9) is vir hierdie studie ondersoek, deur gebruik te maak van ‘n pasiënt-kontrole assosiasiestudies, om te bepaal watter rol hul speel in genetiese vatbaarheid vir TB in die Kleurling bevoling van Suid-Afrika, al dan nie. Die invloed van die VDR polimorfisme Cdx2 op die uitdrukking van die mRNS (boodskapper ribonukleïensuur) en proteïen van die geen CAMP is ook ondersoek. Ons het gevind dat drie gene beduidend bygedra het tot genetiese vatbaarheid vir TB in die Kleurling populasie. ‘n Alleel verwante assosiasie (p = 0.031) was gevind vir die TLR8 SNP rs3761624, waar die A-alleel meer algemeen was in vroue. Vier haplotipes vir TLR8 het beduidende assosiasies met TB vatbaarheid getoon. Die drie SNP haplotipe rs3761624-rs3764879- rs3764880, spesifiek die alleel kombinasie C/G/A [p = 0.004, OR = 2.67(95% CI: 1.90-3.74)] het sterk assosiasie (p = 0.001) met TB getoon. Die TLR9 intron-ekson2 grens SNP, rs352139 het beduidende assosiasie met TB getoon op ‘n genotipiese (p = 0.02) sowel as alleliese skaal [p = 0.05, OR=0.70; (95% CI: 0.55–0.90)], met die T alleel meer algemeen in kontroles. Die twee SNP haplotipe bestaande uit rs5743836 en rs352139 het TB vatbaarheid beïnvloed (p = 0.037), spesifiek die alleliese kombinasie van A/T [p = 0.013, OR=0.71; (95% CI: 0.55–0.92)]. Geen noemenswaardige interaksies tussen TLR2, 8 en 9 is gevind nie. So ook is geen beduidende resultate gevind vir die effek van die VDR SNP Cdx2 op die uitdrukking van CAMP mRNS en proteïen nie. Genetiese variasie in die TLR8 en 9 gene is geïdentifiseer as moontlike faktore wat gasheer genetiese vatbaarheid vir TB in die Kleurlingbevolking van Suid-Afrika beïnvloed. / WW Roome Trust

Les récepteurs Toll-like (TLRs) détectent des structures conservées exprimées par différentes classes de microorganismes, jouant ainsi un rôle majeur dans la réponse immunitaire. Les TLRs localisés dans les endosomes (TLR3, 7, 8 et 9) reconnaissent principalement des acides nucléiques dérivés de microbes. Cependant, ils peuvent également être responsables de la reconnaissance d’acides nucléiques endogènes et contribuer au développement d’auto immunité. A la différence du TLR8humain, le TLR8 murin n’induit pas de réponse à l’ARN simple brin et a ainsi été considéré comme non fonctionnel. Le but de cette étude est d’étudier le rôle du TLR8 murin dans l’immunité. Nous avons montré que les cellules dendritiques déficientes en TLR8 surexpriment le TLR7 et présentent une réponse accrue à une stimulation de TLR7. Chez la souris, la déficience en TLR8 entraine une augmentation des taux d’anticorps circulant (IgM, IgG, IgG2a), des autoanticorps, et au niveau rénal la présence de dépôts de complexes immuns. A l’inverse des souris TLR8-/-, les souris TLR7/8-/- sont protégées de tout symptôme. Nos résultats indiquent donc que chez la souris le TLR8 joue un rôle primordial dans la modulation de l’expression de TLR7, et cette régulation est cruciale dans le contrôle du développement d’autoimmunité spontanée. / Toll-like receptors (TLRs) detect conserved molecular products of microorganisms and play anessential role in the induction of immune responses. Endosomal TLRs (TLR3, 7, 8 and 9) sensenucleic acids derived from microbes. However they can also recognize self nucleic acids and thus beinvolved in the development of autoimmunity. Unlike human TLR8, murin TLR8 does not respond tosingle-stranded RNA suggesting that it could be not functional. In the current study, we investigatedthe role of murine TLR8 signaling in immunity. We found that TLR8-/- dendritic cells overexpressTLR7 and are hyperesponsive to various TLR7 ligands. In mice, TLR8 deficiency leads to increasedlevels of IgM, IgG, IgG2a circulating antibodies, autoantibodies and in the kidney to higher depositionof immunocomplexes while double TLR7/8-/- mice are protected from autoimmune features. Thesedata provide evidence for a pivotal role of murine TLR8 in the regulation of murine TLR7 expressionand this control is critical for the prevention of spontaneous autoimmunity development.

Hepatitis C virus (HCV) establishes a chronic infection in about 70% of infected individuals that is associated with the development of liver cirrhosis and hepatocellular carcinoma. The mechanisms by which HCV avoids clearance by the host immune response are not fully understood. The first aim of this project was to determine whether immune cell subsets could become infected by HCV in vitro. None of the haematopoietic subsets analysed expressed all of the required entry factors, CD81, SR-BI, claudin-1 and occludin. Also, PBMCs were not susceptible to infection with HCVpp and HCVcc expressing glycoproteins of hepatotropic strains. Infection by a supposedly lymphotropic strain (SB) was found to be inefficient. The second aim was to identify in vitro immunomodulatory effects of HCV on innate immune cells that may impact on the immune response activated in acute infection. Crosslinking of CD81 on NK cells by antibody was found to have a minor inhibitory effect on their activation via CD16, but CD81 crosslinking by viral particles had no detectable effect. In contrast to other viruses, HCVcc elicited very little interferon-α production by pDC. HCVcc also did not affect pDC or mDC responses to TLR ligation. Systemic cytokine and chemokine responses were analysed in subjects with primary acute HCV infection and in HCV-infected patients undergoing liver transplantation (LT). Interestingly, induction of systemic type I and type III interferon was not observed in either group. Marked perturbations in systemic cytokine and chemokine levels were detected in uninfected LT patients, precluding use of HCV-infected LT patients to study the innate immune response activated in response to acute viral replication. Together, these results suggest that HCV may principally evade innate immune cell responses by avoidance rather than impairment strategies.

Diversos estudos tem demonstrado que as células-tronco mesenquimais (CTM) são imbuídas de uma forte atividade imunossupressora in vitro, no entanto, os resultados de imunoterapias utilizando CTM têm sido variáveis até o momento. Nossa hipótese para tal variação são interações que devem ocorrer entre as CTM e fragmentos de patógenos circulantes nos pacientes, resultando na modulação da atividade imunossupressora. Para avaliar a ocorrência deste fenômeno em CTM de medula óssea, inicialmente foi avaliado a presença de diversos TLR através da marcação com anticorpos e posterior quantificação por citometria de fluxo, sendo observada a presença dos TLR2, TLR3, TLR4 e TLR9. No intuito de avaliar alterações no potencial imunossupressor, linfócitos T ativados e marcados a nível intracelular foram co-cultivados com CTM estimuladas com LPS, POLY IC e oligonucleotídeos com motivos CpG: DSP30, CpG-A e CpG-B, sendo sua proliferação quantificada por citometria de fluxo. Como resultados, foi observado que a estimulação com LPS e DSP30 levaram a perda e acentuação da capacidade supressora, respectivamente, enquanto o estímulo simultâneo com LPS e DSP30 resultou em sua manutenção. Tais modulações na imunossupressão foram corroboradas ao serem avaliadas modulações na expressão gênica, tendo em vista que o estímulo por LPS e DSP30 induziram no aumento da expressão de IL1 e TGF, respectivamente. Em seguida, foi avaliado o efeito das mesmas condições experimentais na indução a proliferação das CTM, ao ser mensurada alterações na quantidade de células em um equipamento de High content Screening (HCS). Como resultados, foi possível observar que somente o tratamento com DSP30 foi capaz de aumentar significativamente a quantidade de células, fenômeno corroborado ao ser mensurada a síntese de DNA, através da utilização de um produto comercial, seguido de análise por citometria de fluxo. No intuito de avaliar possíveis modulações na via NF-B, CTM estimuladas com LPS ou DSP30 foram sujeitas ao ensaio de imunoprecipitação de cromatina, utilizando anticorpos específicos a subunidades de RelA e RelB, sendo o DNA imunoprecipitado sujeito a PCR quantitativo com primers específicos para a regiões promotoras do gene VCAM-1. Como resultados, foi observado que o estímulo com LPS aumentou a atividade do RelA, enquanto não foram observados efeitos após o estímulo com DSP30. No entanto, o estímulo simultâneo com ambos os ligantes levou ao aumento de atividade de RelA e RelB. Ao serem avaliadas estas condições em um ensaio de imunofluorescência analisado em HCS, foi possível observar maiores níveis da proteína RelB no citoplasma das células tratadas com DSP30, sugerindo um aumento da sua formação. Apesar dos mecanismos moleculares subjacentes aos resultados observados ainda necessitarem de maior elucidação, nosso trabalho indica que a estimulação das CTM com DSP30 pode trazer benefícios no sentido de potencializar a imunossupressão e proliferação celular, além de impedir a perda da imunossupressão, decorrente da interação com LPS. Tais resultados poderão servir como diretrizes para o aprimoramento de imunoterapias utilizando CTM de medula óssea, principalmente em casos de pacientes com infecções por patógenos. / Several studies have shown that mesenchymal stem cells (MSCs ) are imbued with a strong immunosuppressive activity in vitro , however , the results of immunotherapies using CTM has been mixed so far. Our hypothesis for this variation are interactions that must occur between the CTM and fragments of circulating pathogens in patients , resulting in the modulation of the immunosuppressive activity. To evaluate the occurrence of this phenomenon in bone marrow MSCs was initially evaluated the presence of various TLR by staining with antibodies and subsequent quantification by flow cytometry , the presence of TLR2 , TLR3 , TLR4 and TLR9 was observed . To assess changes in the immunosuppressive , activated T lymphocytes and labeled intracellularly potential were co-cultured with MSC stimulated with LPS , poly IC and oligonucleotides with CpG motifs : DSP30 , CpG - A and CpG - B , and their proliferation measured by flow cytometry. As a result , it was observed that stimulation with LPS and DSP30 led to loss of stress and suppressing ability , respectively, while simultaneous stimulation with LPS and DSP30 resulted in maintenance. Such modulations in immunosuppression were corroborated when assessing modulations in gene expression , given that the stimulus induced by LPS and DSP30 in increased expression of TGFb and IL1 , respectively. Then , the effect of the same experimental conditions inducing proliferation of MTC to be measured changes in the amount of cells in a High Content Screening equipment (HCS ) was measured . As a result , it was observed that only the DSP30 treatment was able to significantly increase the amount of cells, phenomenon to be measured supported DNA synthesis through the use of a commercial product followed by analysis by flow cytometry . In order to evaluate possible modulations in NF-kB pathway , CTM stimulated with LPS or DSP30 were subjected to chromatin immunoprecipitation assay , using antibodies specific to subunits RelA and RelB , and the immunoprecipitated DNA subjected to quantitative PCR with primers specific to the promoter regions of VCAM-1 gene. As a result , it was observed that stimulation with LPS increased the activity of RelA , while effects were not observed after stimulation with DSP30 . However , simultaneous stimulation with both ligands led to increased activity of RelA and RelB . When these conditions are evaluated in an assay in HCS immunofluorescence analysis , we observed higher levels of RelB protein in the cytoplasm of cells treated with DSP30 , suggesting an increase in their formation. Although the molecular mechanisms underlying the observed results still require further elucidation , our work indicates that stimulation of MSC with DSP30 can bring benefits in terms of enhancing immunosuppression and cell proliferation , and prevent loss of immunosuppression resulting from the interaction with LPS . These results can serve as guidelines for the improvement of immunotherapies using CTM bone marrow , especially in cases of patients with infections caused by pathogens.

O melanoma é o mais agressivo tumor da pele, cuja resistência aos tratamentos quimioterápicos tem promovido a crescente utilização de imunoquimioterapia, como é o caso da utilização de agonistas dos receptores Toll-Like (TLRs). Nesse contexto, os compostos abreviados por P-MAPA e seu sintético estrutural MRB-CFI-1 com reconhecidas propriedades antitumorais e imunológicas, são fortes candidatos na terapia e prevenção desse tipo de câncer. Esse estudo visa determinar o potencial anticâncer do P-MAPA e de MRB-CFI-1 contra o melanoma murino em consequência ao padrão de resposta microambiental semelhante ao de morte imunogênica, em regimes de tratamento terapêutico ou vacinal, na vigência de quimioterapia com cisplatina e/ou em associação com antígenos de células tumorais totais. Após avaliação In vivo do crescimento de tumores B16F10 implantados em modelos murinos selvagem e nocaute para o gene Myd88, na vigência ou não do tratamento com cisplatina e/ou P-MAPA, nossos resultados mostraram que o P-MAPA apresentou atividade pró-tumoral e antagonizou a ação da cisplatina em inibir o crescimento dos tumores, de forma dependente de Myd88. Além disso, através de análises qualitativa e quantitativa pelo software ImageJ em fotomicrografias de secções tumorais coradas histologicamente, observamos que o P-MAPA promoveu mudanças microambientais nos tumores que podem impactar negativamente em seu desempenho. Como monoterapia em esquema de vacinação com lisado tumoral total em combinação com quimioterapia, o P-MAPA em dose baixa falhou em suprimir o crescimento de tumores B16F10, mas o seu sintético MRB-CFI-1 foi capaz de prevenir o crescimento desse tipo de melanoma num regime de vacinação profilática. Apesar do sucesso terapêutico desse imunomodulador em diversos modelos de câncer e de doenças infecciosas, o P-MAPA não foi eficaz em produz respostas microambientais contra o melanoma murino, dados esses que limitam a aplicabilidade clínica do composto. De outro modo, o composto fosfato inorgânico MRB-CFI-1 foi protetivo em retardar o aparecimento desse tipo de doença. Assim, o presente estudo foi importante por ampliar o entendimento funcional do P-MAPA numa abordagem imunoquimioterápica em modelos biológicos de tumores de melanoma, e representa uma importante mudança na utilização de constituintes individuais similares ao P-MAPA que sejam mais eficazes, de fácil obtenção, e de produção controlada e garantida / Melanoma is the most aggressive skin cancer, whose resistance to chemotherapeutic treatments has promoted the increasing use of immunochemotherapy, as is the case for the use of Toll-Like receptor agonists (TLRs). In this context, the compounds abbreviated by P-MAPA and its structural synthetic MRB-CFI-1 with recognized antitumor and immunological properties are strong candidates in the therapy and prevention of this type of cancer. This study aims to determine the anti-cancer potential of P-MAPA and MRB-CFI-1 against murine melanoma as a consequence of the microenvironmental response pattern similar to that of immunogenic death in therapeutic or vaccine treatment regimens when using chemotherapy with cisplatin alone or in combination with whole tumor cell antigens. After In vivo evaluation of the growth of B16F10 tumors implanted in wild-type and Myd88 gene knockout mice, under treatment or not with cisplatin and / or P-MAPA, our results showed that P-MAPA showed pro-tumor activity and antagonized the action of cisplatin in inhibiting the growth of tumors in a Myd88-dependent manner. In addition, using qualitative and quantitative analysis by ImageJ software in histological images of tumor sections, we observed that P-MAPA promoted microenvironmental changes in tumors that may negatively impact its performance. As monotherapy in vaccination schedule with total tumor lysate in combination with chemotherapy, low dose P-MAPA failed to suppress the growth of B16F10 tumors, but its synthetic MRB-CFI-1 was able to prevent the growth of this type of melanoma in prophylactic vaccination regimen. Despite the therapeutic success of this immunomodulator in various cancer models and infectious diseases, P-MAPA has not been effective in producing microenvironmental responses against murine melanoma, data that limit the clinical applicability of the compound. Otherwise, the inorganic phosphate compound MRB-CFI-1 was protective in delaying the onset of this type of disease. Thus, the present study was important because it broadened the functional understanding of P-MAPA in an immuno-chemotherapeutic approach in biological models of melanoma tumors and represents an important change in the use of individual constituents similar to P-MAPA that are more efficient, easily obtainable, and controlled and guaranteed production

Os mecanismos imunológicos que governam a interação entre o fungo Paracoccidioides brasiliensis e o hospedeiro têm sido pouco estudados. Tanto os componentes do fungo como os receptores dos fagócitos envolvidos nesta interação são pouco conhecidos. Baseados nestes fatos, nosso trabalho teve por objetivo caracterizar in vitro e in vivo o envolvimento do receptor Toll Like-2 (TLR-2) e da proteína adaptadora MyD88 (myeloid differentiation primary response gene 88) na infecção de camundongos pelo P. brasiliensis. O TLR-2 é um receptor da imunidade inata envolvido no reconhecimento de PAMPs (padrões moleculares associados aos patógenos), enquanto que MyD88 é uma molécula envolvida na sinalização celular induzida por muitos TLRs e que culmina com a ativação de vários fatores de transcrição, entre eles o NFB, envolvidos na ativação de genes ligados à resposta inflamatória. Para tanto, utilizamos camundongos C57Bl/6 deficientes e normais para TLR-2 e para MyD88. Demonstramos que, comparado ao grupo controle, animais TLR2-/- apresentavam uma infecção pulmonar menos grave associada com menor síntese de óxido nítrico (NO). Resultados equivalentes foram obtidos com macrófagos peritoneais e alveolares infectados in vitro. Inesperadamente, apesar das diferenças na carga fúngica, ambas as linhagens apresentavam tempo médio de sobrevida semelhante e lesões pulmonares de gravidade equivalente. Os estudos com leucócitos infiltrantes de pulmão revelaram um aumento de leucócitos polimorfonucleares neutrófilos (PMNs) nos animais TLR-2-/- associado com um menor número de linfócitos TCD4+ e TCD8+ ativados. Animais TLR-2-/- deficientes apresentaram uma discreta diferença quanto à síntese de citocinas pulmonares dos tipos Th1 e Th2, porém estes animais apresentaram maiores níveis de KC, uma quimiocina CXC envolvida na quimiotaxia de neutrófilos, assim com maiores níveis de citocinas Th17 (IL-6, IL-17, IL-23 e TGF-). Além disso, a resposta imune Th17 desenvolvida por animais TLR-2-/- esteve associada com menor expansão de células T regulatórias CD4+CD25+FoxP3+. Assim, o TLR-2 controla a imunidade inata e adaptativa frente ao P. brasiliensis e regula negativamente a resposta imune Th17 e a patologia pulmonar. Em relação aos estudos com animais deficientes para a proteína adaptadora MyD88 na paracoccidioimicose verificamos que sua ausência resultou numa produção deficiente in vitro e in vivo de NO, além de uma produção deficiente in vivo de citocinas do tipo Th1, Th2 e Th17. Animais MyD88-deficientes infectados desenvolveram uma resposta imune prejudicada, evidenciada pelo menor número de macrófagos ativados, assim como uma imunidade adaptativa menos eficiente, evidenciada pelo menor número de células T CD4 ativadas que afluíram aos pulmões. Este quadro culminou com uma carga fúngica maior nos pulmões dos animais MyD88- deficientes, como também permitiu uma exuberante disseminação do fungo para outros órgãos, como fígado e baço. Os pulmões e o fígado apresentaram graves lesões com a presença de granulomas coalescentes e ricos agregados fúngicos. Assim, camundongos MyD88-deficientes não foram capazes de controlar a doença e morreram em um tempo mais curto que os animais MyD88-competentes, como evidenciado em experimentos de sobrevida. Assim, nossos achados demonstram que a sinalização intracelular mediada pela proteína MyD88 é importante para a ativação dos mecanismos fungicidas, assim como para a ativação das respostas imunes inata e adaptativa contra o P. brasiliensis. Em conjunto, nosso trabalho demonstra que tanto o TLR-2 quanto a molécula adaptadora MyD88 desempenham um papel relevante no controle da infecção, assim como na indução da resposta imune contra este patógeno fúngico primário. / The immunological mechanisms that govern the interaction between hosts and the dimorphic fungus Paracoccidioides brasiliensis have been scarcely studied. Both, fungal and phagocyte receptors involved in this interaction are poorly understood. Based on these facts, the aim of our study was to characterize in vitro and in vivo the role played by Toll Like Receptor-2 (TLR-2) and the adaptor protein MyD88 (myeloid differentiation primary response gene 88) in murine pulmonary paracoccidioidomycosis. The TLR-2 is a receptor of innate immunity involved in the recognition of PAMPs (pathogen associated molecular patterns), whereas MyD88 is a molecule involved in cell signaling induced by many TLRs . TLR-mediated activation results in the production of several nuclear transcription factors, including NFB, which activate important genes of the inflammatory response. Wild-type (WT) besides TLR- 2- and MyD88-deficient C57Bl/6 mice were used in our investigation. We showed that, compared to control animals, TLR2-/- mice developed a less severe pulmonary infection associated with reduced synthesis of nitric oxide (NO). Equivalent results were obtained with in vitro infected peritoneal and alveolar macrophages. Unexpectedly, despite the differences in fungal loads, TLR-2-/- and WT mice showed equivalent survival times and pulmonary lesions. Studies with lung infiltrating leukocytes revealed an increase of polymorphonuclear neutrophil leukocytes (PMNs) in TLR-2-/- mice associated with a low number of activated T CD4 and T CD8+ lymphocytes. Compared with WT mice, the TLR-2-deficient mice showed slight differences in the production of pulmonary Th1 and Th2 cytokines, but presented higher levels of KC, a CXC chemokine involved in neutrophil chemotaxis, besides increased levels of Th17 cytokines ( IL-6, IL-17, IL-23 and TGF-). Furthermore, the prevalent Th17 immune response developed by TLR-2-/- mice was associated with lower expansion of regulatory T cells CD4+CD25+FoxP3+. Thus, TLR-2 controls the innate and adaptive immunity against the P. brasiliensis infection and negatively regulates Th17 immune response and pulmonary pathology. Studies with MyD88-deficient mice showed an impaired production of NO in vivo and in vitro, and a deficient in vivo production of Th1, Th2 and Th17 cytokines. In addition, infected MyD88-deficient mice developed an impaired immune response, evidenced by poorly activated macrophages, as well as by an inefficient adaptive immunity mediated by a diminished influx of activated CD4+ T cells to the lungs. These events led to increased fungal loads in the lungs of MyD88-deficient mice and allowed a marked dissemination of the fungus to other organs such as liver and spleen, which presented severe lesions composed by coalescent granulomas containing high numbers of fungal cells. As consequence, MyD88-deficient mice were unable to control fungal growth and presented a decreased survival time. Our findings demonstrate that MyD88 signaling is important to the activation of fungicidal mechanisms and to the induction of the innate and adaptive immunity against P. brasiliensis. Altogether, our work shows that both TLR-2 and the adapter molecule MyD88, play an important role in controlling of P. brasiliensis infection, as well as in the induction of immune responses against this primary fungal pathogen.