Orientador: Mary Marcondes / Banca:Wagner Luis Ferreira / Banca: Suely Regina Mogami Bomfim / Banca: Juliana Peloi Vides / Banca: Acácio Duarte Pacheco / Resumo: O presente estudo teve como objetivos investigar a presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp., e a presença do DNA de Leishmania spp. no sangue periférico de 284 indivíduos atendidos em um hospital público de área endêmica para leishmaniose visceral (LV). Baseando-se nos resultados de sorologia e da reação em cadeia da polimerase (PCR), 21,1% dos indivíduos foram considerados expostos. A presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp. foi observada em 20,8% e 37,7% dos indivíduos, respectivamente, observando-se uma associação significativa entre a presença dos dois anticorpos. Em 20,4% dos indivíduos verificou-se sororeatividade em ambos os testes, 17,3% apresentavam anticorpos anti-saliva de Lutzomyia spp. sem a presença de anticorpos anti-Leishmania spp. e um indivíduo apresentava somente anticorpos anti-Leishmania spp. sem a presença de anticorpos anti-saliva de flebotomíneo. Em apenas um indivíduo foi possível amplificar fragmento do DNA de Leishmania spp. no sangue periférico. Concluiu-se que a infecção por Leishmania spp. pode estar sendo subdiagnosticada em áreas endêmicas para LV e, portanto, sugere-se que indivíduos atendidos em hospitais sejam melhor avaliados quanto à possibilidade de infecção e posterior desenvolvimento da doença. Ainda, a presença de anticorpos anti-saliva de flebotomíneo pode ser um possível indicador de exposição ao vetor. / Abstract: The present study aimed to investigate the presence of anti-Leishmania spp. and anti-saliva of Lutzomyia spp. antibodies, and the presence of Leishmania spp. DNA in the peripheral blood of 284 people attended at a public hospital in an endemic area for visceral leishmaniasis (VL). Based on the results of serology and PCR, 21.1% of the people were considered exposed. The presence of antibodies anti-Leishmania spp. and anti-saliva of Lutzomyia spp. was observed in 20.8% and 37.7% of the individuals, respectively, with a significant association between the presence of both antibodies. In 20.4% of the people there was serum reactivity in both tests, 17.3% had antibodies anti-saliva of Lutzomyia spp. without the presence of anti-Leishmania spp. antibodies, while one individual had only anti-Leishmania spp. antibodies without the presence of antibodies against saliva of sand flies. In only one individual it was possible to amplify the DNA fragment of Leishmania spp. in peripheral blood. It was concluded that the infection by Leishmania spp. may be underdiagnosed in endemic areas for VL and, therefore, it is suggested that people referred to hospitals should be better evaluated for the possibility of infection and subsequent development of the disease. Furthermore, the presence of antibodies anti-sandfly saliva may be an indicator of exposure to vector. / Doutor

Soybean oil (SBO), menhaden fish oil (FO), chicken fat (CF) or hydrogenated soybean oil (HSBO) were incorporated at 5% of the total diet to study changes in the immunological status of both Japanese quail (JAP) and Bobwhite quail (BOB). The SBO diet, in which 66% of the total fatty acids were polyunsaturated fatty acids (PUFA), was rich in linoleic acid (LA 18:2 n-6), alpha-linolenic acid (ALA 18:3 n-3) and low in saturated fatty acid (SFA). The FO diet which contained about 50% PUFA, had only 40% n-6 fatty acids and 8% n-3 PUFA. The trans fatty acid isomers and other monounsaturated fatty acids (MUFA) were high in the HSBO diet. The diet containing CF provided a relatively balanced fatty acid composition with 18% SFA, 31% MUFA and 50% PUFA. Plasma fatty acid and hepatic fatty acid profiles consistently reflected their respective dietary lipid treatments. There were no differences in the fatty acid profile between blood and liver within respective dietary treatments in the two species. Dietary fatty acids had no effect on antibody titers against sheep red blood cells (SRBC) at 1, 2 and 8 months following the start of dietary lipid treatment in JAP. However, female JAP fed FO had a significantly (p< 0.05) higher antibody production compared to the other dietary lipid treatments at 4 months following the start of fatty acids supplementation. BOB fed either FO or SBO diets had a higher immunoglobulin G production compared to birds fed the CF diet. The total antibody titer was significantly higher in BOB fed SBO compared to CF. Dietary fatty acids had a significant effect on cell-mediated immunity (CMI) as accessed by toe web thickness 24 hours post intradermal injection of phytohemagglutinin-P (PHA) in both JAP and BOB. In general, birds fed a FO diet had a significantly higher CMI response than those fed HSBO. A diet high in n-3 PUFA increased the index of cutaneous basophil hypersensitivity (CBH), while the high trans fatty acid isomers suppressed the CBH response. By observing a CBH response over a 72-hour period in JAP, it was concluded that quail fed CF or SBO had a different peak response time (12 hours post PHA challenge) and amplitude compared with those fed FO or HSBO (24 hours post PHA challenge). Phagocytic ability was not affected by dietary lipid treatments in BOB while the quail fed FO diet had a faster carbon clearance rate. The FO fed JAP had a significantly higher response (p< 0.05) to concanavalin A ensiformis (CONA) compared to HSBO fed birds. There was no difference in B lymphocyte proliferation stimulated by lipopolysacchride (LPS) in female JAP, whereas it was significantly higher in male JAP fed SBO compared to those fed FO and HSBO. Phorbol 12-myristate 13-acetate/ionomycin calcium salt (PMA/ION) was used to nonspecifically stimulate cell proliferation by increasing chromosome mitosis. Dietary FO or HSBO suppressed cell proliferation stimulated by PMA/ION. However, JAP fed SBO or CF had a significantly higher PMA/ION stimulated lymphocyte proliferation compared those fed FO or HSBO. In male BOB, the FO fed birds had the highest response to all mitogens. In contrast, female BOB did not show any dietary effects by lymphocyte proliferation. Consistent with JAP, BOB fed HSBO had depressed lymphocytes proliferation in response to various mitogens stimulation. In general, female birds had a higher plasma total protein (PTP) and lower pack cell volume (PCV) compared to their males counterparts in both BOB and JAP. In summary, in in vivo experiments, feeding a diet high in menhaden fish oil that is rich in n-3 PUFA enhanced the CMI. There was a minimal effect on antibody production caused by feeding n-3 PUFA in JAP since a significant treatment effect was only found at one sampling period, while BOB were more sensitive to dietary lipid manipulation and had a higher antibody production with SBO or FO treatments. Dietary lipids exerted different effects in the two species in in vitro experiments. While both BOB and JAP fed FO had higher lymphocyte proliferation to CON A mitogen compared to those fed HSBO, only male BOB showed a higher proliferation to LPS. Feeding HSBO that contained a higher content of trans fatty acid isomers, MUFA, but lower PUFA content resulted in the lowest lymphocyte proliferation to various mitogens in both BOB and JAP. / Ph. D.

Abstract Carbamylation of proteins in vivo occurs by cyanate, non-enzymatically when urea is dissociated, and by myeloperoxidase-catalyzed oxidation from thiocyanate. Carbamylation of low-density lipoprotein is suggested to enhance atherogenesis in patients with with chronic kidney disease and uremia. This thesis study assessed the questions of whether healthy humans or uremic patients under enhanced carbamylation have antibodies recognizing carbamyl-epitopes in plasma, and what is their role in vivo. Also, humoral immune response to carbamyl-LDL immunization and its impact on atherogenesis in LDLR-/- mice was investigated. In this thesis study, plasma antibodies to carbamylated proteins were detected in humans, and IgG antibodies to carbamylated proteins were associated with uremia and smoking, conditions with enhanced carbamylation. The human IgG and IgM antibodies binding to carbamyl-epitopes were associated with oxidation-specific epitopes in plasma. Monoclonal Fab antibodies with characteristics of a natural antibody and ability to bind both carbamyl- and malondialdehyde-derived epitopes were cloned from healthy humans. An investigated Fab antibody was able to bind epitopes found in atherosclerotic lesions and inhibit the uptake of modified LDL by macrophages. Human plasma antibodies and the monoclonal Fab bound to epitopes found on apoptotic cells. Human B-cells secreted antibodies with similar cross-reactive binding properties between carbamyl- and malondialdehyde adducts and apoptotic cells in vitro. Immunization with mouse carbamyl-LDL without adjuvant resulted in specific IgG immune response in LDLR-/- mice, but also a cross-reaction with malondialdehyde-adducts was observed. Carbamyl-LDL immunized mice had enhanced plasma antibody binding to apoptotic cells. Carbamyl-LDL immunization did not affect atherogenesis in mice. This thesis demonstrates that IgG antibodies to carbamyl-epitope might serve as a novel indicator of carbamylation in vivo in uremic patients or smokers. The cross-reactivity between antibodies binding to carbamylated and oxidation-specific epitopes, and apoptotic cells may have a role in explaining the link between enhanced atherogenesis and kidney disease. / Tiivistelmä Proteiinien karbamylaatiota tapahtuu syanaatin vaikutuksesta. Sitä muodostuu urean hajotessa tai myeloperoksidaasin katalysoimana tiosyanaatin hapettuessa. Low-density lipoproteiinin eli LDL:n karbamylaation on esitetty edistävän valtimonkovettumataudin eli ateroskleroosin kehittymistä munuaisten vajaatoimintaa sairastavilla ureemisilla potilailla. Väitöskirjatyössä tutkittiin, onko terveillä ihmisillä ja ureemisilla potilailla karbamyyli-epitooppeja tunnistavia vasta-aineita, ja mikä niiden merkitys on elimistössä. Humoraalista immuunivastetta karbamyyli-LDL-immunisaation jälkeen sekä sen vaikutusta ateroskleroosin kehittymiseen tutkittiin LDL-reseptoripuutteellisilla hiirillä. Tutkimuksessa osoitettiin, että ihmisillä on plasmassa karbamyloituja proteiineja tunnistavia vasta-aineita. IgG-luokan vasta-aineet ovat yhteydessä uremiaan ja tupakointiin, joissa karbamylaatio on lisääntynyt. Karbamyyli- ja hapettuneita epitooppeja tunnistavien plasman IgG- ja IgM-vasta-aineiden välillä havaittiin olevan yhteys. Työssä kloonattiin terveistä ihmisistä monoklonaalisia Fab-vasta-aineita, joilla on luonnollisten vasta-aineiden kaltaisia ominaisuuksia ja kyky sitoutua sekä karbamyyli- että malonidialdehydi-epitooppeihin. Yksi tutkittu Fab-vasta-aine sitoutui valtimonkovettumataudin ateroomissa oleviin epitooppeihin ja esti muuntuneen LDL:n sisäänoton makrofagi-soluihin. Ihmisen plasman vasta-aineet ja monoklonaalinen Fab-vasta-aine sitoutuivat apoptoottisten solujen pinnalla oleviin rakenteisiin. Soluviljelyolosuhteissa ihmisen B-solut tuottivat vasta-aineita, joilla oli samanlaisia ristireaktio-ominaisuuksia karbamyyli- ja malonidialdehydi-epitooppeja sekä apoptoottisia soluja kohtaan. Karbamyyli-LDL-immunisaatio sai aikaan IgG-immuunivasteen hiirillä karbamyyli-LDL:a kohtaan, mutta myös ristireaktio malonidialdehydi-rakenteita sekä apoptoottisia soluja kohtaan havaittiin. Karbamyyli-LDL-immunisaatio ei vaikuttanut ateroskleroosin kehittymiseen hiirillä. Tutkimus osoittaa, että IgG-vasta-aineet karbamyyli-epitooppeja kohtaan voivat olla uudenlainen karbamylaation merkkiaine elimistössä ureemisilla potilailla ja tupakoitsijoilla. Karbamyloituneiden ja hapettuneiden epitooppien sekä apoptoottisten solujen välillä havaituilla vasta-aineiden ristireaktioilla voi olla merkitystä valtimonkovettumataudin etenemiseen munuaisten vajaatoiminnassa.

Follicular dendritic cells (FDCs) reside in primary B-cell follicles and in the light zones of germinal centers (GCs) in secondary follicles, where their dendrites interdigitate forming extensive networks intimately interacting with B-cells. In GCs, FDCs can be found at the edges attached to the supporting reticular fibers. They trap and arrange immune complexes (ICs) in vivo and in vitro in a periodic manner with 200–500Å spacing and provide both antigen-specific and non-specific accessory signals to B-cells. FDCs exist in resting and activated states, with two characteristically different phenotypes. In their activated state, FDCs upregulate the expression of accessory molecules and cytokines important in the FDC-B cell interaction in GCs. We sought to determine the mechanisms influencing the transition of FDCs from a resting to an activated state in GCs and their impact on T-cell dependent (TD) and independent (TI)-GC reactions (GCRs). We found that IC-FDC interactions via FDC-FcgammaRIIB induce the upregulation of FDC-FcgammaRIIB, -ICAM-1, and -VCAM-1, at both the protein and mRNA levels. We also reported for the first time the expression of TLR-4 on FDCs. Moreover, engagement of FDC-TLR4 with LPS activated NF-kappaB, up-regulated expression of important FDC-accessory molecules, including FcgammaRIIB, ICAM-1, and VCAM-1, and enhanced FDC accessory activity in promoting recall IgG responses. Moreover, IC-activated FDCs produced IL-6 and FDC-IL-6 promoted GCRs, somatic hypermutation (SHM) and IgG production. Further, we reported that binding of FDCs to collagen coated surfaces induced restoration of their dendritic processes and networks in vitro. In addition, we designed an FDC-supported in vitro model capable of induction and assessment of primary human antibody responses to protein antigens characterized by class-switching and affinity maturation. Uniquely, we generated TI immune responses to TD protein Ags in the complete absence of T cell help in vivo and in vitro. In the presence of FDC-associated second signals such as BAFF and C4BP, FDC- FcgammaRIIB-periodically trapped-ICs induced the production of Ag-specific IgM, GC-development and plasmablast-differentiation in anti-Thy-1-pretreated nude mice. Purified murine and human B cells cultured in vitro with IC-bearing FDCs also showed the production of antigen–specific IgM within just 48 h.