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This thesis was focused on the pro-inflammatory cytokines IL-1beta and IFN-gamma. Absolute and differential leukocyte count was also monitored. The experiment was conducted at 8 clinically healthy heifers, hybrids of Holstein and Czech Pied that have been housed by tethering in stalls and fed with a standard diet. The inflammatory reaction was induced by lipopolysaccharide (LPS; 5 ug in 20 ml PBS), as a control a phosphate buff-ered saline (PBS) was used. Results were measured at 1, 2, 3 and 7 days after stimulation of mammary gland by above-mentioned factors. Concentration of each cytokine was detected by a sandwich ELISA using commercially available kits. At 1 day after stimulation of mammary gland by LPS and PBS an average number of leukocytes, which was statistically significantly higher in the case of stimulation by LPS (P <0.01), was detected. After 7 days there was a significant decrease in the total number of leukocytes. There has also been a shift in the differential leukocyte count. Most abundant cell type were neutrophils, whose number was higher in the case of stimulation by LPS. Between day 1 and day 7 after challenge, there was a gradual reduction in the proportion of neutrophils. In the same period an increase in the proportion of macrophages and lymphocytes was detected. Concentration of IL-1beta also increased, 1 day after the activation a striking increase has been detected. In following days there was gradual decline of IL-1beta concentration almost to the level prior to treatment of the mammary gland. In the case of IFN-gamma similar pattern in the form of strong growth and a subsequent gradual decline in concentration to the original values was detected. There was found positive correlation between the increase in IL-1beta and IFN-gamma concentration and a shift in the differential leukocyte count in favor of neutrophils, which confirmed the important role of these pro-inflammatory cytokines in the establishment of inflammatory response and the mobilization of the components of natural and specific immunity.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-05-19T23:58:33Z No. of bitstreams: 1 TESE - Marilda - 2016 F.pdf: 1951206 bytes, checksum: f0d79f0e2b09130f482d4c5c8d7f00d3 (MD5) / Made available in DSpace on 2017-05-19T23:58:34Z (GMT). No. of bitstreams: 1 TESE - Marilda - 2016 F.pdf: 1951206 bytes, checksum: f0d79f0e2b09130f482d4c5c8d7f00d3 (MD5) / Introdução: A avaliação de risco para tuberculose (TB) em profissionais da saúde baseia-se no número de indivíduos doentes atendidos nas instituições, na evidência de sua transmissão entre pessoas dentro das instituições, ou cálculo de taxas de conversão do teste cutâneo tuberculínico (TCT). Sugere-se que os resultados negativos para este teste sejam repetidos periodicamente, de acordo com o risco que a instituição apresente e, ou, após exposições ocupacionais. O fato de apenas 10% das pessoas infectadas com Mγcobacterium tuberculosis desenvolverem a doença clínica sugere que diversos mecanismos podem desempenhar um papel importante na imunopatogênese da doença. Estudos genéticos estão sendo desenvolvidos com o objetivo de identificar possíveis marcadores de predisposição ou proteção ao desenvolvimento desta doença e sugerem que o desequilíbrio na produção de citocinas pró e anti-inflamatórias tem um importante papel na TB. Objetivo: Avaliar a liberação de interferon gama (IFN-γ) e o polimorfismo de citocinas nos PS com TCT de repetição, negativo e positivo, que trabalham em uma unidade de referência secundária e terciária em TB. Métodos: A população do estudo foi constituída por 48 profissionais TCT de repetição negativo, com resultado < 5 mm e 45 TCT positivo > 10 mm. O DNA genômico foi extraído a partir de sangue total, utilizando o kit de extração de DNA mini spin Kasvi. Foi realizada a genotipagem das citocinas (IL6, IL10, TNF, IFN-γ, TGFB1) e a associação entre o polimorfismo +874T/A do geneIFN- γ ,o resultado do ensaio de liberação de interferon gama IGRA (QFT®) e o TCT. Resultados: Não foi observada diferença estatística no polimorfismo do gene IFN-γ +874 T/A e o resultado do IGRA e TCT. A concordância entre o TCT e IGRA foi feita utilizando o índice Kappa que mostrou κ=0,24. Os resultados relativos aos fenótipos previstos de alto (TT), intermediário (TA) e baixo produtor (AA) de IFN-γ, a partir do polimorfismo na posição +874T/A, em ambos os grupos mostrou que a maior frequência foi o de baixo produtor. Ao analisar o grupo TCT+ / IGRA +, os dados mostram correlação dos testes in vivo e in vitro para reatividade em 24 indivíduos. O polimorfismo desses indivíduos tem o perfil dos fenótipos previstos de baixo produtor, sendo apenas 6 de intermediário e 3 de alto produtor. Conclusão: A concordância entre o TCT e o IGRA na população estudada é mediana e não foi observada diferença no polimorfismo do gene IFN-γ+874T/A para frequência de fenótipo previsto de baixo produtor, comparando os grupos do estudo e seus respectivos IGRA. Os polimorfismos dos genes IFN γ +874T/A, TNF -308G/A, IL6 -174G/C IL10 - 1082G/A, -819C/T e -592C/A e TGFβ1 -509 C/T não parecem ser marcadores de predisposição ou proteção ao desenvolvimento da TBIL. Foi encontrada diferença estatística no gene TGF1 +869 T/C.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-04-24T19:07:37Z No. of bitstreams: 1 Dissertação_Med_ Tatiana Moura.pdf: 1314903 bytes, checksum: d553217eb6f2d6d979a75a8179ee209b (MD5) / Approved for entry into archive by Flávia Ferreira(flaviaccf@yahoo.com.br) on 2013-04-26T16:49:27Z (GMT) No. of bitstreams: 1 Dissertação_Med_ Tatiana Moura.pdf: 1314903 bytes, checksum: d553217eb6f2d6d979a75a8179ee209b (MD5) / Made available in DSpace on 2013-04-26T16:49:27Z (GMT). No. of bitstreams: 1 Dissertação_Med_ Tatiana Moura.pdf: 1314903 bytes, checksum: d553217eb6f2d6d979a75a8179ee209b (MD5) / A Leishmaniose Tegumentar Americana (LTA) é uma doença endêmica no Brasil. No entanto, não existe um bom modelo experimental para o estudo da doença. Nosso objetivo foi desenvolver um modelo de infecção com L. braziliensis, o principal agente etiológico da LTA em nosso país, levando em consideração o inoculo de parasitas e o sítio de infecção. [MATERIAL E MÉTODOS] Camundongos BALB/c foram infectados com 105 Leishmania braziliensis (MHOM/BR/01/BA788), na derme da orelha. Os animais foram acompanhados durante 10 semanas para a avaliação do desenvolvimento da lesão e para a avaliação da resposta imune. [RESULTADOS] Observamos que a expansão parasitária foi acompanhada pelo desenvolvimento de uma lesão na derme da orelha, similar à observada em pacientes com LTA (lesão nodular e ulcerada no centro), a qual regrediu espontaneamente, como evidenciado pela presença de uma cicatriz. A análise histopatológica da orelha infectada mostrou a presença de, inicialmente, um infiltrado focal constituído por células mononucleares (linfócitos e monócitos), neutrófilos e poucos parasitas. No auge do desenvolvimento da lesão, havia predominância de macrófagos infectados os quais foram, em seguida, substituídos por um infiltrado inflamatório constituído por histiócitos, plasmócitos, neutrófilos e fibroblastos e pela ausência de parasitas. Os parasitas podem ser detectados no linfonodo regional, durante toda a infecção. A análise da expressão de quimiocinas no linfonodo regional mostra um aumento na expressão de quimiocinas recrutadoras de monócitos/macrófagos e neutrófilos Observamos também um aumento na expressão de IFN-γ, IL-4, IL-5 e IL-10, tanto por células T CD4+ quanto por células T CD8+. Com a regressão da lesão, a expressão destas citocinas diminuiu. [CONCLUSÃO] A inoculação de L. braziliensis na derme da orelha de camundongos constitui um modelo de resistência devido ao desenvolvimento de uma resposta imune do tipo Th1. Contudo, nesse modelo, os parasitas são capazes de sobreviver no linfonodo regional de camundongos infectados apesar do desenvolvimento de uma resposta imune capaz de curar a lesão / Salvador

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Introduction: Visceral leishmaniasis is a serious public health problem in several parts of the developing world. In leishmaniasis, protection and healing correlate with the development of Th1 immune response, and IFN-&#947; is considered a key molecule in this response. In contrast, the Th2 response results in disease progression. It is noteworthy that after contact with Leishmania the production of IFN-&#947; differs between healthy individuals. Some of them are high IFN-&#947; producer (HIFN-&#947;P), while others respond with low IFN-&#947; producer (LIFN-&#947;P). This has been observed in the in vitro response to several species of Leishmania, as well as in the early phase of the disease in patients with leishmaniasis. The activation of T lymphocytes can be modulated by immunomodulatory receptors present on the surface of lymphocytes and antigen presenting cells (APCs). Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein that promotes Th2 differentiation. Evidences support the involvement of this molecule in the immune response against parasites. Objective: To evaluate the role of SLAM signaling pathway in the modulation of immune response of peripheral blood mononuclear cells (PBMC) from individuals low IFN-&#947; producers (LP) against Leishmania chagasi. Methodology: Healthy individualsâ PBMC were stimulated in vitro with L. chagasi and monoclonal anti-SLAM (&#945;-SLAM), in the presence or not of inflammatory cytokines (rIFN-&#947; or rIL-12). The supernatants of the cultures were analyzed by ELISA for the determination of IFN-&#947; and interleukin 10 (IL-10) concentrations. Results: Upon stimulation of PBMC with L. chagasi, the blocking of the SLAM signaling pathway with &#945;-SLAM did not affect the synthesis of IFN-&#947; and IL-10, regardless of treatment with rIL-12. However, after rIFN-&#947; treatment of antigen stimulated cells it occurred a download of IL-10 synthesis and upload IFN-&#947; secretion, regardless of the blockade of SLAM signaling pathway. Conclusions: The blocking of SLAM signaling pathway with &#945;-SLAM at the concentration of 10&#956;g/mL does not interfere significantly in the IFN-&#947; and IL-10 production of PBMC from individuals LP stimulated with Leishmania chagasi promastigotes. Treatment of PBMC with rIFN-&#947; is able to induce a reduction of IL-10 and an increment of IFN-&#947; in the supernates cultures, whereas treatment with rIL-12 enhanced IFN-&#947; production, but does not interfered with IL-10. It is necessary to make further studies to better understand the role of the SLAM signaling pathway in the immune response of LP against Leishmania chagasi. / IntroduÃÃo: Nas leishmanioses, proteÃÃo e cura se correlacionam com o desenvolvimento de resposta imune tipo Th1, e IFN-&#947; à considerada uma molÃcula chave nesta resposta. Ao contrÃrio, a resposta Th2, resulta na progressÃo da doenÃa. Vale ressaltar que a produÃÃo de IFN-&#947;, apÃs o contato com a Leishmania, difere entre indivÃduos sadios, alguns apresentam alta produÃÃo de IFN-&#947; (AP), enquanto outros respondem com baixa produÃÃo (BP). Isto tem sido observado com diversas espÃcies de Leishmania, assim como na infecÃÃo natural, na fase inicial da doenÃa. A ativaÃÃo do linfÃcito T pode ser modulada por coestimuladores, presentes na superfÃcie de linfÃcitos e nas APCs, a exemplo da molÃcula sinalizadora na ativaÃÃo de linfÃcitos T (SLAM). EvidÃncias apontam para o envolvimento dessas molÃculas na regulaÃÃo da resposta imunolÃgica. Objetivo: Avaliar o papel da via de sinalizaÃÃo de SLAM na modulaÃÃo da resposta imune em cÃlulas mononucleares de sangue perifÃrico (CMSP) de indivÃduos BP, frente à estimulaÃÃo in vitro por Leishmania chagasi. Metodologia: CMSP de indivÃduos sadios BP foram estimuladas in vitro com L. chagasi na ausÃncia ou na presenÃa do anticorpo monoclonal anti-SLAM (&#945;-SLAM), com ou sem tratamento por citocinas proinflamatÃrias (rIFN-&#947; ou rIL-12). Os sobrenadantes das culturas foram analisados por ELISA para IFN-&#947; e interleucina (IL)-10. Resultados: Sob estimulaÃÃo de L. chagasi, o bloqueio da via SLAM nÃo modificou a sÃntese de IFN-&#947; e IL-10, independente do tratamento com rIL-12. No entanto, o tratamento com rIFN-&#947; reduziu a sÃntese de IL-10 e elevou a secreÃÃo de IFN-&#947; endÃgeno, independente do bloqueio da via SLAM. ConclusÃes: O bloqueio da via SLAM, com adiÃÃo de &#945;-SLAM (10Âg/mL), nÃo interfere significativamente na produÃÃo de IFN-&#947; e IL-10. O tratamento das CMSP com rIFN-&#947; à capaz de induzir a reduÃÃo da produÃÃo de IL-10 e o aumento de IFN-&#947; de forma significativa, enquanto que o tratamento com rIL-12 aumenta a produÃÃo de IFN-&#947;, mas nÃo interfere na produÃÃo de IL-10 dos indivÃduos baixos produtores. Faz-se necessÃrio ampliar o estudo da aÃÃo imunomoduladora da SLAM frente à Leishmania chagasi, para um melhor entendimento do papel desta via de sinalizaÃÃo na resposta imunolÃgica dos BP.

The tripartite motif (TRIM) family comprises at least 80 members in humans, with most having ubiquitin or SUMO E3 ligase activity conferred by their N-terminal RING domain. TRIMs regulate a wide range of processes in ubiquitination-or sumoylation-dependent manners in most cases, and fewer as adaptors. Their roles in the regulation of viral infections, autophagy, cell cycle progression, DNA damage and other stress responses, and carcinogenesis are being increasingly appreciated, and their E3 ligase activities are attractive targets for developing specific immunother-apeutic strategies for immune diseases and cancers. Given their importance in antiviral immune response, viruses have evolved sophisticated immune escape strategies to subvert TRIM-mediated mechanisms. In this review, we focus on their regulation of IFN-I-mediated innate immune response, which plays key roles in antiviral and antitumor defense.

Background: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. Objective: We have examined the effects of IFN-α on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. Methods: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. Results: IFN-α induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-α was not significantly different (P = .6). IFN-α induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-α at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-α on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-α. IL-13 mRNA stability was unaffected by IFN-α, suggesting transcriptional regulation. Conclusion: IFN-α differentially regulates antigen-stimulated IL-4 and IL-13 generation.

Background: Diminished cytokine production in infancy has been associated with an increased risk for allergen sensitization and early-life wheeze. Objective: We sought to assess the effect of low cytokine production in the first year of life on the development of wheeze through age 13 years. Methods: Cytokine production (IFN-γ and IL-2) by mitogen-stimulated mononuclear cells was determined from peripheral blood samples (9.4 months, n = 118) in a subset of healthy infants enrolled in the Tucson Children's Respiratory Study. The occurrence of wheeze during the previous year was ascertained at ages 2, 3, 6, 8, 11, and 13 years by means of questionnaire. Relative risk for wheeze was computed with generalized estimating equations. Results: The risk of wheezing between 2 and 13 years was significantly higher for subjects with low 9-month IFN-γ production (relative risk, 2.29; 95% CI, 1.35-3.89) and borderline significant for those with intermediate IFN-γ production (relative risk, 1.59; 95% CI, 0.95-2.68) compared with those who produced high levels of IFN-γ (P value for linear association = .002). Nine-month IL-2 production was unrelated to wheeze. In relation to complex wheezing phenotypes, 9-month IFN-γ production was inversely related to toddler wheeze (occurring only before age 6 years, P = .03) and chronic wheeze (occurring before and after age 6 years, P = .007) but not school-age wheeze (occurring only after age 6 years, P = .06). Conclusion: The results suggest that characteristics of the immune system present during the first year of life can anticipate the likelihood of development of episodes of airway obstruction characterized by wheezing. Clinical implications: Immune susceptibility to asthma is established very early during postnatal life.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are recognized by different cellular pathogen recognition receptors (PRRs), which are expressed on cell membrane or in the cytoplasm of cells of the innate immune system. Nucleic acids derived from pathogens or from certain cellular conditions represent a large category of PAMPs/DAMPs that trigger production of type I interferons (IFN-I) in addition to pro-inflammatory cytokines, by specifically binding to intracellular Toll-like receptors or cytosolic receptors. These cytosolic receptors, which are not related to TLRs and we call them "Toll-free" receptors, include the RNA-sensing RIG-I like receptors (RLRs), the DNA-sensing HIN200 family, and cGAS, amongst others. Viruses have evolved myriad strategies to evoke both host cellular and viral factors to evade IFN-I-mediated innate immune responses, to facilitate their infection, replication, and establishment of latency. This review outlines these "Toll-free" innate immune pathways and recent updates on their regulation, with focus on cellular and viral factors with enzyme activities.