Dissertationen zum Thema „Immune humoral response“

Abstract Heavy alcohol consumption places a substantial burden on health all over the world. Metabolites of alcohol evoke alterations that lead to tissue damage in many organs. Phosphatidylethanol (PEth) is a unique phospholipid formed in the cellular membranes during the metabolism of ethanol after alcohol consumption. PEth has attracted special attention as it is postulated to be a reliable marker of long term heavy alcohol consumption. The aims of present study were to investigate the immunogenicity of phosphatidylethanol in mice and to analyze the plasma antibodies binding to phosphatidylethanol in humans. In this study a clear immune response was generated in mice immunized with PEth in human low density lipoprotein (LDL) carrier. Mouse monoclonal IgM antibodies binding specifically to phosphoethyl head group of PEth were generated using hybridoma technology. Since PEth was shown to be immunogenic in mice, plasma was analyzed for the presence of antibodies also in humans. PEth-specific antibodies of IgG, IgA and IgM isotypes in plasma were detected in heavy drinkers of alcohol with or without pancreatitis as well as in the controls. The plasma levels of the antibodies binding to PEth were significantly lower in the study subjects with heavy alcohol use and in this present study sample the low IgA levels to PEth were better indicators of heavy alcohol consumption as compared to the some of the traditional markers of heavy alcohol use. The antibody levels to PEth associated significantly to plasma antibodies binding to malondialdehyde-acetaldehyde adducts that are known to be formed during alcohol metabolism but not to antibodies binding to phosphocholine which is generated by lipid oxidation in humans. In conclusion, this study demonstrates that phosphatidylethanol is immunogenic in mice when using carriers such as human LDL in the immunization process. The binding of the monoclonal antibodies specifically to the PEth head group suggests that it would be feasible to develop a diagnostic immunoassay to PEth. The presence of antibodies binding to PEth in plasma indicates that PEth may be a target of humoral immunity in humans
Tiivistelmä Runsas alkoholinkulutus aiheuttaa maailmanlaajuisesti merkittäviä terveydellisiä haittoja. Alkoholin aineenvaihduntatuotteet muuttavat kudoksien rakenteita ja aiheuttavat kudosvaurioita. Fosfatidyylietanoli on alkoholin aineenvaihdunnan tuloksena solukalvoilla syntyvä fosfolipidi, jota on tutkittu kahdenkymmenen vuoden ajan lupaavana alkoholin suurkulutuksen merkkiaineena. Tutkimuksen tavoitteena oli selvittää fosfatidyylietanolin immunisoinnin aiheuttamaa vasta-aineiden muodostumista koe-eläinmallina käytetyissä hiirissä sekä määrittää ihmisten plasmanäytteistä vasta-aineita, jotka sitoutuvat fosfatidyylietanoliin. Tutkimuksessa havaittiin immuunivasteen muodostuminen hiirissä, jotka immunisoitiin ihmisen LDL hiukkasiin liitetyllä fosfatidyylietanolilla. Hiiren monoklonaalisia fosfatidyylietanoliin sitoutuvia IgM-luokan vasta-aineita tuotettiin tutkimuksessa soluviljelyn avulla. Fosfatidyylietanolin aiheuttama vasta-aineiden muodostuminen hiirillä johdatti mittaamaan fosfatidyylietanoliin sitoutuvia vasta-aineita myös ihmisiltä. Tutkimuksessa havaittiin fosfatidyylietanoliin sitoutuvia IgG-, IgA- ja IgM-luokan vasta-aineita alkoholin suurkuluttajilla, alkoholihaimatulehdusta sairastavilla ja verrokkihenkilöillä. Vasta-aineiden pitoisuudet olivat alkoholia runsaasti käyttävillä koehenkilöillä merkitsevästi pienemmät kuin verrokkiryhmällä. Matalat IgA-vasta-ainepitoisuudet osoittautuivat aineistossa paremmaksi alkoholin suurkulutuksen osoittajiksi kuin eräät tavanomaisesti käytetyt alkoholinkäytön merkkiaineet. Plasman fosfatidyylietanoli-vasta-aineiden ja alkoholin aineenvaihdunnan seurauksena syntyvien malondialdehydi-asetaldehydi-addukteihin sitoutuvien vasta-aineiden määrän välillä havaittiin merkitsevä yhteys, jota ei havaittu rasvojen hapettumisen seurauksena syntyvien fosfokoliini-vasta-aineiden ja fosfatidyylietanoli-vasta-aineiden välillä. Tutkimus osoittaa, että hiirillä voidaan aikaansaada vasta-ainevälitteinen immuunivaste, kun ne rokotetaan ihmisen LDL-hiukkaseen liitetyllä fosfatidyylietanolilla. Fosfatidyylietanoliin spesifisesti sitoutuvien monoklonaalisten vasta-aineiden tuottaminen voi tulevaisuudessa johtaa immunologisen diagnostisen määritysmenetelmän kehittämiseen. Fosfatidyylietanoliin sitoutuvien plasman vasta-aineiden havaitseminen viittaa siihen, että fosfatidyylietanoli on vasta-ainevälitteisen immuunivasteen kohde myös ihmisillä

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

This thesis reports on investigations carried out to identify the important antigens inducing a humoral immune response in periodontal disease. A number of different techniques were used. The first study examined the systemic immune response, specifically the effects of treatment on the immune response of patients with chronic periodontitis against a panel of periodontal pathogens and a large panel of antigen preparations from these bacteria. The results of the study indicated that there was no difference in antibody titre following treatment against any of the putative periodontal antigenic targets tested. These results conflict with many published reports, however, differences in factors such as length of treatment and antigen preparation often make the comparison of different studies futile. An investigation to examine the phenomenon of cross-reactivity between antibodies induced against putative periodontal pathogens was the second part of this overall project. The results suggested a high proportion of cross-reactivity between the 4 periodontal pathogens P. gingivalis, A. actinomycetemcomitans, P. intermedia and B. forsythus. Results indicating such a high cross-reactivity between these micro-organisms prove that the issue of cross-reactivity is an important one that should be considered when carrying out any immunological and microbiological investigations in this field. Following the above investigations of the systemic immune response the remainder of this thesis is a report of three studies examining the local immune response in periodontal disease.

This study characterises various aspects of barramundi (Lates calcarifer) humoral immunity, including ontogeny, temperature modulation and kinetics following challenge with Streptococcus iniae. It was discovered that Staphylococcal protein A (SpA) was able to efficiently isolate antibody from serum, and that all barramundi Ig found in serum is tetrameric with a weight of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72 kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) of the tetrameric Ig which was consistent with those observed with other species. Polyclonal and monoclonal antibodies were produced against the protein A purified barramundi Ig, and various ELISA formats were developed. These serological tools were used to investigate aspects of barramundi humoral immunity. Examination of ontogeny of humoral immunity, revealed that barramundi possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be detected, which continues to increase over the following months. However, it is not until seven week post-hatch that barramundi fingerlings are able to mount a prolonged immune response following vaccination with S. iniae. Environmental temperature was also found to significantly impact the ability of barramundi to respond to vaccination with S. iniae. Barramundi maintained at low temperatures (<230C) displayed a diminished, delayed and highly variable humoral immune response following vaccination, with many of the experimental animals failing to respond to primary vaccination. These responses could be mediated by either administering a booster vaccine or by elevating the environmental temperature. This study also demonstrated that there was a relationship with specific serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with low titres being more susceptible to disease. Specific antibody in barramundi could be generated through natural exposure to the bacterium from the environment or through vaccination. Thus bath vaccination of fish (50,000) held at two facilities resulted in elevated systemic antibody levels and lower observed mortality, when compared to the unvaccinated control fish. Infections due to S. iniae were determined to be associated with elevated water temperatures. Laboratory trials and field data indicated that water temperatures between 24 and 280C resulted in the highest barramundi mortality. A weak association was also determined with low pH and mortality, with fish exposed to low pH’s (<6.0) being more susceptible to infection. No association was observed with mortality and salinity. Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa protein from cell wall of S. iniae. The Mab’s displayed a high level of specificity for S. iniae, including those from Australia, Israel and America, and minimal cross-reactivity with other bacterial species tested. The Mab’s were used in an immunohistochemical study that confirmed the neurotropic nature of S. iniae infections, as well as demonstrating the presence of the bacterium in the intestine of infected fish.

This study characterises various aspects of barramundi (Lates calcarifer) humoral immunity, including ontogeny, temperature modulation and kinetics following challenge with Streptococcus iniae. It was discovered that Staphylococcal protein A (SpA) was able to efficiently isolate antibody from serum, and that all barramundi Ig found in serum is tetrameric with a weight of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72 kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) of the tetrameric Ig which was consistent with those observed with other species. Polyclonal and monoclonal antibodies were produced against the protein A purified barramundi Ig, and various ELISA formats were developed. These serological tools were used to investigate aspects of barramundi humoral immunity. Examination of ontogeny of humoral immunity, revealed that barramundi possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be detected, which continues to increase over the following months. However, it is not until seven week post-hatch that barramundi fingerlings are able to mount a prolonged immune response following vaccination with S. iniae. Environmental temperature was also found to significantly impact the ability of barramundi to respond to vaccination with S. iniae. Barramundi maintained at low temperatures (<230C) displayed a diminished, delayed and highly variable humoral immune response following vaccination, with many of the experimental animals failing to respond to primary vaccination. These responses could be mediated by either administering a booster vaccine or by elevating the environmental temperature. This study also demonstrated that there was a relationship with specific serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with low titres being more susceptible to disease. Specific antibody in barramundi could be generated through natural exposure to the bacterium from the environment or through vaccination. Thus bath vaccination of fish (50,000) held at two facilities resulted in elevated systemic antibody levels and lower observed mortality, when compared to the unvaccinated control fish. Infections due to S. iniae were determined to be associated with elevated water temperatures. Laboratory trials and field data indicated that water temperatures between 24 and 280C resulted in the highest barramundi mortality. A weak association was also determined with low pH and mortality, with fish exposed to low pH’s (<6.0) being more susceptible to infection. No association was observed with mortality and salinity. Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa protein from cell wall of S. iniae. The Mab’s displayed a high level of specificity for S. iniae, including those from Australia, Israel and America, and minimal cross-reactivity with other bacterial species tested. The Mab’s were used in an immunohistochemical study that confirmed the neurotropic nature of S. iniae infections, as well as demonstrating the presence of the bacterium in the intestine of infected fish.

Group A streptococcus (GAS) is a strictly human pathogen that causes infections ranging from asymptomatic carriage to the highly lethal streptococcal toxic shock syndrome (STSS). GAS are classified according to the sequence of the variable 5’ end of the emm-gene that encodes the surface associated M-protein. In the late 1980s, outbreaks of GAS infections with high rates of STSS were reported in several parts of the world, including Sweden. Superantigens (SAgs), a group of exotoxins, have been described as key mediators of STSS due to their capacity to polyclonally activate T-cells and induce a massive release of inflammatory cytokines. Previous reports have revealed that sera from STSS patients have lower capacity to neutralize this SAg-mediated immune stimulation and a higher prevalence of GAS isolates with specific emm-genotypes during disease outbreaks. The aims of this thesis were to analyse the protective antibody response mounted by the host against SAgs produced by the infecting GAS isolate and to characterise the isolates emm-genotypes and SAg gene profiles. The clinical material examined was collected from patients with STSS, sepsis, erysipelas, or tonsillitis in Sweden between 1986 and 2001. Both acute- and convalescence-phase sera were analyzed, along with the infecting GAS isolates. The 92 clinical GAS isolates examined were found to exhibit a high degree of genetic diversity in terms of the number and identity of their SAg genes. Isolates with a given emm-genotype could be divided into subgroups on the basis of their SAg gene profiles. Ten different SAg gene profiles were identified in the 45 emm1 isolates examined; one of these ten was highly persistent, being observed in 22 isolates collected over 14 years. Two of the 11 known SAg genes in GAS, smeZ-1 and speA, were more prevalent in the emm1 associated profiles than in the SAg gene profiles of isolates with other emm-genotypes. Patients infected by GAS with the emm1-genotype were less likely to produce acute-phase sera that could effectively neutralize the T-cell mitogenicity induced by the infecting isolate’s extracellular products (EP). Sepsis patients whose sera exhibited this lack of neutralizing ability were more prone to developing STSS. Most patients whose acute-phase sera did not effectively neutralize the EP from the infecting isolate lacked protective antibodies in their convalescent-phase sera despite having elevated ELISA titers. The results reported herein show that combining SAg gene profiling with emm-genotyping may be useful for tracking the spread of GAS clones in the community. It was also shown that a lack of neutralizing activity in convalescence-phase sera might be due to an inability of those patients to mount a protective immune response against SAgs produced by the infecting GAS isolate.

Some 42 million individuals worldwide are infected by the Human Immunodeficiency Virus (HIV) and no cure or vaccine is available. This thesis addresses approaches to humoral immunity to HIV-1. In primary infection, the cytotoxic T lymphocyte (CTL) response is detected early and is thought to play a role in the viral decline. Neutralising antibodies (NAbs) are detected much later. However, non-neutralising anti-HIV-1 Env glycoprotein Abs (non-NAbs) are present concomitantly with the CTL response. The possible role of non-NAbs with complement was investigated using sequential sera and viruses expressing gpl20 Env (gpl20) glycoproteins amplified from blood samples from a cohort of newly HIV-1 infected patients. Autologous gpl20 sequences were cloned and expressed into a replication-competent HIV-1 backbone. The autologous Ab pattern was studied. In the presence of complement, inactivation of autologous and heterologous HIV could be detected as early as day 9 post-onset of symptoms (POS). IgG were partly responsible for triggering the classical complement cascade. In parallel, a new approach was investigated to generate a recombinant vaccine to HIV-1. Camelids synthesise IgG devoid of light chains. These IgG fragments (Vhh) share the same characteristics as classical IgG but have unusually long CDR H3 regions that can adopt more flexible conformations. The possibility of generating Vhh fragments that mimic the neutralising CD4 binding site (CD4BS) of HIV-1 was investigated. A llama was immunised with IgGl bl2 (bl2), a potent cross-neutralising human NAb overlapping the CD4BS of HIV-1. The non-classical Vhh repertoire was cloned, the resulting libraries were panned against bl2 by phage display and five specific anti-bl2 Vhh fragments were isolated. Each of the five fragments was tested in animals for the induction of an anti-HIV-1 NAb response. These studies are discussed with reference to the control of HIV-1 infection by drugs and vaccines.

The immune system is critical for the maintenance of homeostasis. Due to the fact that a coordinated effort between organ systems is required for internal stability, it has been postulated that the immune system interacts with the neuroendocrine system. Clinical, anatomical, and receptor studies have provided evidence for a bi-directional communication between the nervous and immune systems. The goal of the present studies was to determine the potential influence of the sympathetic nervous system (SNS) on the primary antibody forming cell (AFC) response. The adrenergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been utilized extensively by researchers to explore the possible relationship between SNS and the antibody response. However, the literature describing the humoral effects observed following 6-OHDA treatment is irresolute. In an attempt to provide insight into these apparent discrepancies, studies were conducted comparing the effects of 6-OHDA and its non-neurotoxic congener, 5-hydroxydopamine (5-OHDA). Both chemicals, when added directly into cultures containing naive splenocytes, suppressed the in vitro AFC response. Analysis following in vivo treatment with 6-OHDA or 5-OHDA revealed that while both chemical treatments resulted in suppression of the AFC response following in viva sensitization, only the splenocytes from 6-OHDA treated mice were suppressed when subsequently sensitized in vitro. Pretreatment with desmethylimipramine (DMI) blocked the observed 6-OHDA- and 5-OHDA-induced immunosuppression displayed in vivo, indicating that the uptake of the chemicals into adrenergic neurons was required. As an alternative method for removing the sympathetic influence in the spleen, studies were conducted with chlorisondamine, a non-competitive ganglionic blocker. While direct addition of chlorisondamine was without effect in the in vitro-in vitro AFC response, the in viva AFC response following chlorisondamine treatment was significantly suppressed. In addition to the suppression of the primary AFC response, 6-OHDA and chlorisondamine treatment resulted in a time dependent increase in the level of DNA fragmentation in the thymus. Analysis of serum corticosterone levels in 6-OHDA- and chlorisondamine-treated mice revealed that both treatments elevated levels of serum corticosterone. Given the potential role of corticosterone in the 6-OHDA- and chlorisondamine-induced immunosuppression, studies were conducted to determine if the glucocorticoid receptor antagonist, RU-486, was able to block the DNA fragmentation in the thymus and the suppression of the AFC response demonstrated after 6-OHDA or chlorisondamine treatment. While RU-486 was effective at blocking the 6-OHDA- and chlorisondamine-induced thymic effects, it was unable to block the suppression of the primary AFC response. Collectively, these studies reveal that removal of the peripheral SNS by either sympathectomy with 6-OHDA or ganglionic blockade with chlorisondamine can result in 1) thymic alterations which are mediated by increased levels of corticosterone and 2) suppression of the primary AFC response which is independent of the elevated corticosterone levels. Importantly, these results provide evidence for positive modulation of the humoral immune response by the sympathetic nervous system.

Measles virus belongs to the Morbillivirus genus, Paramixoviridae family. It is the causative agent of a highly contagious acute infective disease, typical of infancy, characterized by fever, skin rash, cough and conjunctivitis, and a generalized immune suppression (Griffin, 2013). The virus is transmitted through the respiratory tract, multiplicates in its upper part and in regional lymph nodes, thus resulting in lymphatic and hematic dissemination with appearance of first clinical signs after 9-19 days (de Vries et al., 2015). In 30% of the cases, complications in the lower respiratory tract or the central nervous system (CNS) can occur. The first sign of infection is represented by an early immune depression, due to the loss of B and T immune memory cells (Mina et al., 2015), resulting in an increased susceptibility to opportunistic infections and to life-threatening complications such as pneumonia and/or gastro-intestinal disease (de Vries et al., 2015). However, this type of disease is paradoxically associated with the induction of a strong and specific immune response to the virus, which is usually permanent (Laksono et al., 2016). There is no specific treatment against measles, and this is the reason why vaccination is considered the best strategy against the virus. Furthermore, the monotypic nature of the virus and the lack of an animal reservoir, make measles a considerable candidate for eradication (Rota et al., 2016). Although a combined vaccine, called MMR (measles, mumps and rubella) is used in routinely vaccination schedule, measles remains a significative cause of morbidity and mortality, particularly during infancy (Moss & Griffin, 2012; Wolfson et al., 2009; Nandy et al., 2003). MMR live attenuated vaccine is very efficacious in protecting people against measles, mumps, and rubella, and preventing the complications caused by these diseases. The measles virus contained in the vaccine is represented by the live attenuated Edmoston B strain. The World Health Organization recommends two doses of vaccine for all children and adults; the first dose should be given at 13-15 months of age. The second dose is often done at 5 - 6 years, in Italy. About 3 out of 100 people who get two doses of MMR vaccine will get measles if exposed to the virus. However, they are more likely to have a milder illness, and are also less likely to spread the disease to other people (Centers for Disease Control and Prevention, 2018). Epidemiologic studies have shown that the level of neutralizing antibodies at the time of exposure to wild type (WT) virus in the community is a good indicator of protection from infection, with higher titers necessary to prevent infection than to prevent disease (rash) (Chen et al., 1990). High avidity antibodies are required to neutralize CD150-mediated WT MeV infection of lymphoid cells (Polack et al., 2003). However, levels of circulating anti-measles neutralizing antibody tend to reduce or even to fade during lifetime, especially among vaccinated subjects (Kennedy et al., 2019; Davidkin et al., 2008; Carryn et al., 2019; Seagle et al., 2018; Gonçalves et al., 2015; Le Baron et al., 2007). Because CD4+ T cell help is required for isotype and affinity maturation of antibody-secreting cells, B cell memory and maturation of CD8+ T cell memory, cellular immune response is also important for the induction of protective immunity (Laksono et al., 2018). All these things highlight the necessity to invest on studies focused on the correlates of protection against Measles virus. Recent evaluation systems for vaccines point towards the measurement of Tcell quality with regards to cytokine secretion as a protective correlate in addition to antibody titers in serum during the course of an immune response. Although the generation of immune memory supports the concept of vaccine efficacy, direct assessment of immune memory cells and their precursors has not yet been established as a correlate of protection. With the growing knowledge on the phenotype, function and localization of the immune memory cells in the body, researchers think that these cells may provide a novel correlate of protection for evaluation of more efficacious vaccines. Finally, transcriptome-level characterization (mRNA-Seq data) of responses to measles virus stimulation in antibody responders (either vaccinated or naturally infected) and those who have not responded to the vaccine, could help to identify plausible regulators (genes/pathways) that drive the observed differences among these subjects. Such study may help to develop a panel of biomarkers to monitor, besides the antibody response, the immune response to measles vaccine with the aim to protect, in case of outbreaks, not only the fragile subjects, but also the vaccinated subjects who eventually become seronegative along the time, with a booster composed of specific, immunogenic MeV proteins.

Orientador: Margareth Castro Ozelo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Os principais problemas relacionados ao tratamento de pacientes portadores de hemofilia A estão relacionados ao uso terapêutico de fator VIII (FVIII), sendo estes o desenvolvimento de anticorpos neutralizantes anti-FVIII (inibidores), e o desenvolvimento de reações anafiláticas, que são eventos raros, no entanto potencialmente graves. As informações quanto aos isotipos de imunoglobulinas associados a estas duas situações clínicas ainda é limitado. O objetivo deste projeto foi avaliar as características da resposta imune humoral em pacientes com hemofilia A que apresentam inibidor e/ou em condição de reação alérgica ao FVIII. Para estas análises três metodologias foram aplicadas, (1) determinação de anticorpos inibitórios por método de Bethesda-Nijmegen, (2) determinação do isotipo de imunoglobulinas envolvidas subclasses da IgG, IgM e IgE anti-FVIII, por método de ELISA e (3) determinação de citocinas por método multiplex BDTM CBA® (cytometric bead array). Esse projeto foi dividido em três estudos. No primeiro estudo, foram analisadas amostras de 25 pacientes brasileiros com hemofilia A, sendo 44% destes afrodescendentes. Todos os pacientes receberam exclusivamente terapia de reposição com concentrado de FVIII derivado de plasma (pdFVIII) e produtos bypass após o desenvolvimento de inibidor. Cinco pacientes deste grupo foram acompanhados por uma análise longitudinal no período de até três anos. No segundo estudo, 4 pacientes com hemofilia A com inibidor foram avaliados no período em que foram tratados através do protocolo de indução de imunotolerância (ITI) para erradicação do inibidor, também em análise longitudinal. O terceiro consistiu da avaliação de incidência de reação alérgica em pacientes com hemofilia A. Três de 322 pacientes (0,9%) apresentaram reação alérgica após a exposição exclusivamente para pdFVIII durante os últimos quinze anos em nosso centro. Os resultados evidenciaram que a subclasse IgG4 é a principal na modulação em presença de anticorpos inibitórios, enquanto a IgG1 na maior parte das análises estava presente junto a baixos títulos de inibidor.Durante o tratamento de ITI os níveis das interleucinas anti-inflamatórias IL-4 e IL-6 acompanharam o decaimento dos títulos de inibidor e IgG4 nos pacientes que obtiveram sucesso ao tratamento. Além disso, no decorrer do protocolo observou-se uma resposta polarizada para o tipo Th1 como padrão de resposta na conquista da tolerância completa ao FVIII. No contexto da reação alérgica, apenas um dos três pacientes apresentou reatividade da IgE que foi exclusiva ao pdFVIII, sendo negativa no ensaio do IgE anti-rFVIII (anti-fator VIII recombinante), demonstrando que a reatividade não foi específica ao FVIII. O entendimento resposta imune humoral em pacientes com hemofilia A, incluindo a participação da IgG4 e IgG1 no mecanismos envolvendo a presença e erradicação dos inibidores e da IgE na reação alérgica, possibilita ampliar conceitos estabelecidos dos mecanismos envolvidos nessas duas situações. Isso poderá auxiliar no desenvolvimento de novos produtos menos imunogênicos e de novas estratégias para a indução de tolerância ao FVIII, que tenham maior eficiência e melhor custo benefício
Abstract: The main problems related to the treatment of hemophilia A patients are linked to the use of therapeutic factor VIII (FVIII). First, the development of neutralizing antibodies against FVIII (inhibitors), and second development of anaphylactic reactions, which are rare, however potencialy severe. The knowledge about the immunoglobulin isotypes associated with these two clinical situations is still limited.The aim of this project was to evaluate the characteristics of the humoral immune response in patients with hemophilia A who have inhibitors and/or allergic reaction to FVIII. For these analyzes three methods were used (1) inhibitory anti-FVIII antibodies assay by Bethesda-Nijmegen (2) immunoglobulins isotype ELISA assay for anti-FVIII IgG subclasses, IgM and IgE and (3) cytokines assay by BDTM Cytometric bead array (BD CBA®) multiplex method. This project was divided in three studies. In the first study, we analyzed samples from 25 Brazilian hemophilia A patients with 44% African-descents. All patients received exclusively replacement therapy with plasma-derived (pdFVIII) concentrates, and bypassing agents after the development of inhibitors. Five patients from this group were followed for a longitudinal analysis in a period up to three years. In the second study, 4 hemophilia A patients with inhibitor were evaluated also in longitudinal analyses, during the induction of immunotolerance (ITI) treatment for the eradication of the inhibitor. The third study included the evaluation of the incidence of allergic reaction among hemophilia A patients. Three out of 322 patients (0.9%) had allergic reaction after exclusively exposure to pdFVIII during the last fifteen years in our center. The results of these studies demonstrated that IgG4 subclass is the main immunoglobulin involved in the modulation of the inhibitory antibodies, while IgG1 is associated with low-titer inhibitors. During the ITI protocol, the anti- inflammatory interleukins, IL-4 and IL-6 decreased following the IgG4 reduction among the patients that achieved success in the ITI treatment. In addition, during the ITI protocol it was observed a polarized Th1 immune response after the complete success achievement. In the context of allergic reaction, only one out of three patients presented IgE reactivity that was exclusively to pdFVIII, and the assay IgE anti-rFVIII (anti-recombinant FVIII) was negative, confirming that the reativity was not specific to FVIII. The understanding of the humoral immune response in hemophilia A patients, including the role of IgG4 and IgG1 in the mechanisms involving the presence and eradication of inhibitors, and the participation of IgE in allergic reaction, allows to better understand the established concepts of the mechanisms involved in these two situations. This may help the development of less immunogenic new products and new strategies for induction of tolerance to FVIII, with higher efficiency and best value
Mestrado
Clinica Medica
Mestra em Clínica Médica

Melanoma, a potentially lethal form of skin cancer, is widely thought to be immunogenic in nature. While numerous studies have examined T cell-mediated immune responses to melanoma and their therapeutic potential, there has been less focus on B cell-mediated immune responses and the tumor-reactive antibodies they produce. The aim of this work was three-fold: (1) to develop a methodology to detect antibodies secreted by human B cells that recognize melanoma cell surface proteins; (2) to evaluate the mature B cell repertoire of individuals with melanoma for antibody subclass composition and the presence and prevalence of anti-tumor antibodies; and (3) to study patient-derived antibodies and two engineered antibodies recognizing melanoma cells for their propensity to activate immune effector cells and their capacity to kill or restrict the growth of tumor cells. As part of this thesis, a novel tumor cell-based ELISA was developed for the detection of tumor-reactive antibodies. Utilizing this new assay, the presence and prevalence of melanoma-reactive IgG antibodies derived from ex vivo cultured peripheral blood B cells from a cohort of 21 patients with melanoma (Stage I, n=1; Stage II, n=8; Stage III, n=6; Stage IV, n=6) were compared to those from healthy volunteers (n=10). While B cells from melanoma patients secreted IgG antibody concentrations comparable to those from healthy volunteer B cell cultures, a significantly increased reactivity of antibodies derived from patients to primary and metastatic melanoma cells was measured compared to healthy volunteers (P<0.001). Interestingly, there was a significant reduction in antibody responses to melanoma with advancing disease stage that was not found to be solely due to a -ii-reduction in the B cell memory compartment size. Comparing IgG antibody subclass distribution among cutaneous tumors, patient lymph nodes and peripheral blood B cells all isolated from individuals with metastatic disease, elevated proportions of IgG4 subclass antibodies were observed in cutaneous tumors which present a novel finding of this thesis. These findings point to differentially polarized humoral immune responses in cutaneous tumor microenvironments. Lastly, an antibody derived from a patient was then selected and preliminary evaluations of reactivity and specificity to a range of melanoma cell lines and primary human melanocytes were conducted. Using a live cell imaging cytotoxicity assay, this patient-derived melanoma-specific antibody was observed to kill melanoma cells via antibody-mediated cellular cytotoxicity. Additionally, two engineered monoclonal antibodies recognizing a melanoma associated antigen were found to partially restrict tumor cell migration and adhesion and to kill melanoma cells via antibody-dependent cellular cytotoxicity or phagocytosis. In summary, examining the humoral immune response to melanoma and the effector function of antibodies targeting melanoma cells provides insight into the discovery of new therapeutic strategies for the treatment of melanoma.

It is now well established that gluten sensitivity comprises a spectrum of disorders, which affect different target organs. The small-intestine (coeliac disease), skin (dermatitis herpetiformis) and peripheral and central nervous systems are most frequently affected. These manifestations may occur alone or in combination with one another. Neurological complications affect approximately 6-10% of patients with gluten sensitivity with ataxia being the most frequent disorder seen in these patients. It has recently been established that previously undiagnosed gluten sensitivity may present solely with ataxia (gluten ataxia) and this disease entity may account for a large number of patients with sporadic idiopathic ataxia. Preliminary findings suggest an immune pathogenesis for gluten ataxia, in common with other manifestations of gluten sensitivity. The studies in this thesis are concerned with investigation of the humoral immune response in the pathogenesis of gluten ataxia. Assessment using enzyme-linked immunosorbent assay, immunohistochemistry and western blotting has shown the presence of elevated levels of circulating gluten sensitivity associated antibodies (anti-gliadin and anti-tissue transglutaminase antibodies) in patients with gluten ataxia. Studies have also shown the cross-reaction of anti-gliadin antibodies with epitopes on cerebellar Purkinje cells. In addition, patients with gluten ataxia possess circulating antibodies directed against cerebellar Purkinje cells, which are distinct from anti-gliadin antibodies although the target antigen remains unknown. Finally, studies have also shown that patients with gluten ataxia have elevated levels of circulating antibodies directed against glutamic acid decarboxylase. These studies suggest a role for the humoral immune response in gluten ataxia. However, preliminary evidence is also suggestive of a T-cell mediated response and the relative contributions of each in the pathogenesis of gluten ataxia remains to be elucidated.

RESUMO:Aterosclerose é uma das principais causas de morbilidade e mortalidade no mundo ocidental. É responsável, direta ou indiretamente, pela maior percentagem de gastos com a saúde na maioria dos países europeus. A “teoria lipídica” da aterosclerose, que se baseia na dislipidemia como causa primária para a doença vascular tem algumas implicações práticas importantes: permite a definição de linhas de orientação e protocolos simples e ainda estabelece alvos terapêuticos que podem ser atingidos na maior parte dos casos com a atual intervenção farmacológica. A associação da aterosclerose com o sistema imunológico (a “teoria imunológica”), forneceu por sua vez novas formas de explorar os mecanismos envolvidos e abriu novas perspetivas para um conhecimento mais completo da doença. No entanto, levanta dificuldades evidentes no que diz respeito às possibilidades terapêuticas. De todos os intervenientes no processo aterosclerótico (bioquímicos, imunológicos e anatómicos), as lipoproteínas de elevada densidade (HDL) são atualmente reconhecidas como um dos fatores mais importantes na aterogénese. Isto é baseado no reconhecimento das múltiplas propriedades anti-aterogénicas das HDL como por exemplo: a anti-oxidante, a anti-inflamatória e a antitrombótica, bem como o seu importante papel na melhoraria da função endotelial. Atualmente, é consensual que as funções anti-aterogénicas das HDL vão além do seu papel no transporte reverso do colesterol (RCT) e a importância das HDL no processo aterosclerótico baseia-se não apenas no seu papel protetor impedindo a formação da placa de ateroma, mas também na estabilização destas, prevenindo a sua ruptura e, consequentemente o evento trombótico. Como fundamentais no processo aterosclerótico estão reconhecidos dois principais conjuntos de eventos: um caracterizado por alterações no metabolismo das lipoproteínas que resultam em lipoproteínas pró-inflamatórias e pró-oxidantes que interagem com os componentes celulares da parede arterial e que conduzem à formação da placa de ateroma; o outro evento é a resposta imunológica desencadeada contra um novo conjunto de antigénios que por sua vez leva à produção de citoquinas pró-inflamatórias. Dada a complexidade da HDL e das suas múltiplas funções estas lipoproteínas tornaram-se um potencial alvo para a resposta auto-imune, e cujas consequências podem explicar algumas das associações identificados em estudos clínicos e epidemiológicos. Contudo esta interação entre o sistema imunológico e HDL nunca foi exaustivamente estudada. Portanto, pomos a hipótese de que em condições oxidativas e pró-inflamatórias, um aumento do antigénio (HDL) conduz a um consequente acréscimo na produção de anticorpos anti-HDL (aHDL) responsáveis pela alteração quantitativa e / ou qualitativa das HDL. O conceito de que estes anticorpos podem contribuir tanto para a evolução a longo prazo do processo aterosclerótico, como para o desencadeamento de eventos clínicos pode também explicar a heterogeneidade encontrada em cada doente e nos grandes estudos clínicos, no que diz respeito aos fatores de risco e outcomes clínicos. Para além disso, a confirmação desta hipótese pode permitir explicar porque é que as intervenções terapêuticas atualmente em desenvolvimento para aumentar os níveis de HDL, não conseguem mostrar a tão esperada redução do risco vascular. O objetivo geral desta tese foi identificar e caracterizar a resposta humoral contra os componentes da HDL, e avaliar possíveis mecanismos que possam contribuir para a modificação das propriedades anti-aterogénicas das HDL. Para alcançar este objetivo investigou-se: 1) A presença de anticorpos aHDL em doentes com lúpus eritematoso sistémico (SLE) e em doentes com manifestações clínicas de aterosclerose, como os doentes com doença arterial coronária (CAD), acidente vascular cerebral isquémico (IS) e diabetes tipo 2; 2) Os principais alvos antigénicos dentro do complexo das HDL e a associação entre os títulos de anticorpos aHDL e diferentes características clínicas destas doenças; 3) As modificações das funções normais associadas às HDL, em particular da função anti-oxidante e anti-inflamatória; 4) A atividade biológica dos anticorpos aHDL isolados do soro de doentes através de um conjunto de experiências in vitro de inibição da atividade da paraoxonase 1 (PON1) e da expressão de moléculas de adesão em culturas de células endoteliais. Para tal foi necessário estabelecer um método de isolamento dos anticorpos. Os anticorpos aHDL isolados do soro de doentes foram utilizados de forma a identificar as potenciais alterações dos sistemas celulares utilizados; 5) O efeito de fármacos usados no tratamento das dislipidemias, em particular o ácido nicotínico e as estatinas, na variação dos títulos de anticorpos aHDL através de ensaios clínicos randomizados, controlados com placebo e em dupla ocultação. Os métodos utilizados neste trabalho incluíram: técnicas imunológicas (como por exemplo, enzyme-linked immunoabsorbent assay - ELISA, ensaio imunoturbidimetrico e cromatografia de imuno-afinidade) técnicas bioquímicas (tais como a quantificação de atividade enzimática por espectrofotometria e por luminescência), experiências com cultura de células e citometria de fluxo. Os nossos resultados mostram que: 1) A presença de anticorpos aHDL, e mais especificamente anticorpos contra alguns do seus principais componentes como a apolipoproteína A-I (ApoA-I, principal apolipoproteína presente nas HDL) e a PON1 (o enzima que mais contribui para a propriedade anti-oxidante das HDL), quer em doentes com doenças auto-imunes, como o SLE, quer em doentes com manifestações clínicas de aterosclerose, como CAD, IS e diabetes tipo 2. Os doentes apresentaram títulos de anticorpos IgG aHDL, aApoA-I e aPON1 significativamente mais elevados do que controlos saudáveis com a mesma idade e sexo. 2) A correlação positiva estatisticamente significativa entre os títulos de aHDL e aApoA-I e aPON1 sugere que estes sejam dois dos principais alvos antigénicos dentro do complexo das HDL. Os anticorpos encontrados nestes doentes estão associados com a diminuição da atividade da PON1 e a uma redução da capacidade anti-oxidante total (TAC) do soro, um aumento dos biomarcadores de disfunção endotelial (como por exemplo dos metabolitos do óxido nítrico - NO2- e NO3-, as moléculas de adesão vascular e intracelular - VCAM-1 e ICAM-1 e os níveis de 3-nitrotirosina). Nos doentes com SLE os títulos destes estão associados a um aumento do dano cardiovascular e à atividade global da doença avaliados pelas escalas SLICC/ACR DI e BILAG score, respetivamente. Enquanto que nos doentes com diabetes tipo 2 estes anticorpos estão associados com um aumento dos níveis de glicemia em jejum (FGP) e hemoglobina glicada (HbA1c). 3) Após se ter estabelecido um método de isolamento dos anticorpos que permite isolar quantidades significativas de anticorpos do soro de doentes sem perder a sua especificidade, foi identificada a capacidade dos anticorpos isolados do soro de doentes inibirem de uma forma dependente da concentração a atividade da PON1 até um máximo de 70% no caso dos doentes com SLE e ente 7-52% no caso dos anticorpos isolados de doentes com CAD e IS. 4) O efeito anti-inflamatório das HDL na inibição da produção de VCAM-1 induzida por citoquinas (como o TNF-) foi revertido em mais de 80% pelos anticorpos aHDL isolados do soro de doentes. 5) A angiogenesis induzida por HDL através do aumento do fator de crescimento do endotélio vascular (VEGF) foi anulada em 65% pelos anticorpos aHDL isolados do soro de doentes. 6) Os atuais agentes farmacológicos disponíveis para aumentar as concentrações de HDL-C estão associados a um aumento dos títulos de anticorpos.-------- ABSTRACTAtherosclerosis is the major cause of morbidity and mortality in the western world. It is also responsible, directly or indirectly, for the highest percentage of health costs in most European countries. Despite the use of new technologies for the diagnosis of vascular disease and regardless of the major advances in treatment, the atherosclerosis-related clinical burden is still raising. The “lipid theory” of atherogenesis, which identifies dyslipidemia as the primary cause of this vascular disease has some important practical implications: it allows the definition of simple guidelines and establishes therapeutic targets which can be generally met with current pharmacologic intervention. The association between atherosclerosis an the immune system (the immune concept) has in turn provided new ways of exploring the mechanisms involved in this condition and has opened new perspectives in the understanding of the disease. However, it raises obvious difficulties when it comes to treatment options. Of all the players (biochemical, immunological and anatomical) involved in this matter, high-density lipoproteins (HDL) are currently recognised as one of the most important factors in atherogenesis. This is based on the recognition of HDL's multiple anti-atherogenic properties: anti-oxidant, anti-inflammatory and antithrombotic, as well as its capacity to improve endothelial function. Nowadays, it is widely recognized that the anti-atherogenic functions of HDL go beyond reverse cholesterol transport (RCT), and the importance of HDL is based not just on its ability to reduce atheroma formation but also on its ability to stabilise plaques, therefore preventing their rupture and ultimately thrombosis. Two main set of events have been recognised as fundamental in atherogenesis: one, characterized by lipoprotein metabolism alterations, resulting in pro-inflammatory and pro-oxidative lipoproteins, which interact with the normal cellular elements of the arterial wall leading to atheroma formation; the other, the immune cellular response towards new sets of antigens which lead to the production of pro-inflammatory cytokines. Given to HDL complexity and multiple functions this lipoprotein has became a potential target for an auto-immune response, the consequences of which may explain some of the association identified in epidemiological and clinical studies, though the interaction between the immune system and HDL has never been thoroughly addressed. Therefore, we hypothesized that under oxidative and pro-inflammatory conditions, the increase in the antigen (HDL) would lead to a consequent increase in the production of anti-HDL (aHDL) antibodies be responsible for quantitative and/or qualitative changes of HDL. The concept that these antibodies may contribute either to the long-term evolution of atherosclerosis or to the triggering of clinical events may also explain the heterogeneity found in individual patients and in large cohorts regarding risk factors and clinical outcomes. Moreover this may be a major breakthrough in understanding why therapeutic interventions that increase HDL levels, failed to show the anticipated reduction in vascular risk. The overall aims of this thesis were to identified and characterize the humoral response towards HDL components and to evaluate the possible mechanisms that may contribute to the modifications of the anti-atherogenic properties of HDL. To achieve this objective we investigated: 1) the presence of aHDL antibodies in patients with systemic lupus erythematosus (SLE) and in patients with atherosclerosis-related clinical events, such as coronary artery disease (CAD), ischemic stroke (IS) and type 2 diabetes; 2) the association between the titres of aHDL antibodies and different clinical features of these diseases; 3) the modifications of the anti-atherogenic properties of HDL; 4) the biologic effect of aHDL antibodies isolated from serum of patients on the anti-oxidant and anti-inflammatory properties of HDL; 5) the effect of different pharmacologic treatments for dyslipidemia on the prevalence and activity of aHDL antibodies. The methodologies used in this work included immunologic-related techniques (e.g. enzyme-linked immunoabsorbent assay – ELISA, immunoturbidimetric immunoassay and immunoaffinity chromatography), biochemical techniques (enzymatic assays with quantification by spectrophotometry and luminescence methods), cell culture experiments and flow cytometry. Our results indicate that: 1) The titres of IgG aHDL, anti-apolipoprotein A-I (aApoA-I) and anti-paraoxonase 1 (aPON1) antibodies were higher in patients with SLE, CAD, IS and type 2 diabetes when compared with age and sex matched healthy controls. 2) The antibodies found in these patients were associated with decreased PON1 activity, (the enzyme responsible for most of the anti-oxidant effect of HDL), reduced total anti-oxidant capacity (TAC) of serum and increased biomarkers of endothelial dysfunction (nitric oxide metabolites, adhesion molecules, nitrotyrosine). In patients with SLE the antibody titres were associated with an increase in disease-related cardiovascular damage and activity whereas in patients with type 2 diabetes they were directly related with the fasting glucose plasma (FGP) levels and the glycosylated haemoglobin (HbA1c). 3) The antibodies isolated from serum of our patients, directly inhibited HDL-associated PON1 activity in a dose dependent way ranging from 7 to 52%. 4) The anti-inflammatory effect of HDL, measured by the percentage of inhibition of the cytokine-induced production of vascular adhesion molecules (VCAM-1), was reduced in more than 80% by aHDL antibodies isolated from our patients. 5) The HDL-induced angiogenesis by increasing vascular endothelial growth factor (VEGF) levels was abrogated in 65% by the antibodies isolated from serum of patients. 6) The current available pharmacologic agents for increasing HDL-C concentrations were associated with an increase in the titres of IgG aApoA-I antibodies. This increase was higher in the extended release niacin when compared to statins probably due to their dampening effect on oxidative stress. In conclusion, aHDL antibodies are present in different pathologic conditions. aHDL antibodies represent a family of self-reacting immunoglobulins, of which ApoA-I and PON1 might be the most relevant targets. These antibodies are biologically active, interfering with the HDL anti-oxidant and anti-inflammatory properties and, consequently, with the atherosclerotic process. The pathogenic potential of these antibodies may lead to the identification of a new biomarker for vascular disease, whilst presenting itself as a novel target for a different treatment approach which may redefine the treatment strategies and clinical trials design for HDL interventions in the future.

Thesis (M.S.)--University of Missouri-Columbia, 2006.
"August 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

I investigated the humoral immune response to streptococcal cell walls (SCW) in arthritis susceptible Lewis and resistant Fisher rats. All rats were given a single intraperitoneal injection of either SCW or saline (controls). Rats were sacrificed, three rats per time point, over an eleven week period and serum was collected for ELISA. SCW injected Lewis rats produced anti-SCW antibody, whereas control rats did not. Anti-SCW antibody was significantly elevated over controls between days 14-28 (post injection). Both saline and SCW injected Fisher rats produced anti-SCW antibody, but with different kinetics. Anti-SCW antibody increased by day 7 and remained elevated over controls till day 21, after which there was no difference. ELISA were designed to determine the SCW epitope(s) recognized by anti-SCW antibody. Formamide extracts of SCW, peptidoglycan and polysaccharide, were investigated along with the terminal epitope of polysaccharide, N-acetyl-D-glucosamine, and the peptidoglycan precursor peptide. The data revealed that anti-SCW antibody was directed against a combined SCW epitope, given the lack of significant binding to any of the SCW epitopes tested. Isotype analysis of anti-SCW antibody revealed that the Lewis response was composed primarily of IgG2a whereas the Fisher response was composed primarily of IgM. Binding of rat IgG isotypes to whole streptococcus, SCW, peptidoglycan, and polysaccharide was investigated, given the possibility of background binding by the streptococcal Fc-receptor. Streptococcal binding of rat IgG was specific for IgG2c and the polysaccharide portion of SCW was necessary for binding. Passive immunization of naive Lewis rats with antibody from rats with active arthritis was ineffective at transferring the disease. However, subcutaneous injection of affinity purified anti-SCW antibody or IgG into Lewis rats, followed twenty-four hours later by a single intraperitoneal injection of SCW, suppressed the acute phase and inhibited the chronic disease. IgM rheumatoid factor (RF) was present in the serum of both saline and SCW injected Lewis and Fisher rats. However, SCW injection only induced a significant increase in IgM RF (between days 3-7) in Lewis rats. Passive immunization of Fisher rats with affinity purified IgM RF (from Lewis serum), three days post SCW injection, was ineffective at inducing arthritis.

Acute and chronic HBV infection and the resulting liver damage including hepatocellular carcinoma (HCC) are major health problems placing large demands on health resources. In high prevalence areas transmission occurs principally perinatally or in early childhood resulting in a high incidence of chronic life-long carriers. Progress in the study of HBV infection, replication and pathogenesis has depended to a large extent on the development of animal models such as the duck/DHBV model. Although less related to HBV than the mammalian models DHBV was used to demonstrate the unique replication strategy of hepadnaviruses and is now providing an insight into the immunological mechanisms of viral clearance or persistence. In this thesis the duck model was manipulated to provide models of both chronic carriage and acute hepatitis with viral clearance by varying the time and titre of DHBV inoculated. In addition a percutaneous needle liver biopsy which permitted multiple biopsies over a short time-span was developed. The lack of systematic studies of the avian immune response to DHBV has limited our understanding of the epidemiology, natural history and pathogenesis of DHBV infection. There is insufficient cross reaction between avian and mammalian viral antigens to permit the use of human reagents. The development of virus-specific assays has been restricted by the lack of duck-specific reagents. A radioimmunoassay to detect anti-DHBs activity in serum was established and the humoral immune response in ducks which were either experimentally inoculated or naturally exposed to DHBV infection was studied. Anti-DHBs activity was detected in a significant number of ducks from DHBV infected flocks. The anti-DHBs activity was found to pass passively from dam to offspring and neutralised DHBV infectivity in vivo. Acquired immunity may explain the variable infectivity of DHBV when injected into day-old ducklings from different flocks from similar genetic stock. Despite the importance of CMI in the pathogenesis of HBV infection the role of viral antigens in determining the outcome of infection is not clear. In order to determine the nature and timing of the specific immune response involved in clearance of infection the CMI of the duck/DHBV model was studied. Initially, the conditions were defined for optimum splenocyte responses to commonly used non-specific mitogens PHA, ConA and LPS and found to differ from those reported for peripheral blood mononuclear cells. The optimal conditions for antigen-specific mononuclear transformation were determined. The duck model was then used to study the CMI of uninfected. transiently or chronically infected and vaccinated ducks to antigenic stimulation and related these findings to humoral response and outcome of infection. The rate of clearance of viral particles from the circulation is related to the dosage given rather than the age of the duck. The infection rate of adult ducks also depends on the virus dose. In individual adult ducks with slow removal of viral inoculum or uptake of virus by blood mononuclear cells is unrelated to the development of infection. In baby ducks appearance of progeny virus occurs by 24 hours after inoculation. The lack of replication within this time frame in adult ducks or in tissue culture supernatant may reflect a higher percentage of cells refractory to infection. Adult ducks demonstrated a viraemia of limited duration and low titre while the viraemia in baby ducks was of high titre and constant. The IDso endpoint of 26-day—old ducks was found to be 1000 times the 11?0 dose of day-old ducks. A Chi-square analysis comparing livers with or without inflammation showed that DHBV positive livers had significantly (P<0.01) more inflammation than DHBV negative livers. The repeated inoculation of adult ducks with small doses of DHBV resulted in the production of neutralising antibody and anti-DI-[Bs activity. The presence of anti-DHBs activity was determined in 168 day-old duckling, 77 two month old, 20 six month old and 99 twelve month old ducks from 3 commercial farms. Significant anti-DHBs activity was detected in 1/53 ducklings from non exposed flocks compared with 28/117 ducklings hatched from DHBV infected flocks (p<0.01). Significant anti-DHBs activity was detected in only 1/40 non-exposed ducks compared with 42/ 1 56 ducks from infected flocks (p<0.01). However, naturally occurring anti-DI-[Bs levels are lower than levels obtained in the vaccinated ducks and showed a positive correlation to DHBV exposure but a negative correlation to infection. The anti-DHBS activity in day-old ducks fiom DHBV endemic flocks suggests that immunity is transferred from dam to offspring via the egg. This passively acquired anti-DHBs activity was able to neutralise DHBV infectivity in vivo in 75% of cases (p<0.02).. Optimisation of mitogen concentration for stimulation of duck splenic mononuclear cell cultures was 5x105cells/well. Once the cell concentration increased to le0 cells/well the unstimulated cultures exhibited blastogenesis and uptake of 3H thymidine, so that the high counts obtained for the mitogen stimulated cultures were not significantly different from those obtained for unstimulated controls. Cell numbers below 5x105cells/well resulted in decreased 3I-I thymidine uptake and lower SI. The response to each mitogen was different and there was also substantial duck to duck variation in response to mitogen stimulation. PHA at concentrations between 5 and 20pg/ml, produced good transformation (SI>3 00) from day l to 4 of culture. Peak transformation was achieved with Con A concentrations of between 10 to 20ug/ml for all four ducks tested. The transformation of SMC in response to LPS was poor when compared with that obtained with PHA or Con A in the three ducks tested. The concentration of LPS required to give maximal transformation responses varied between 10 and 40ug/ml and resulted in SI of up to 20.The optimum cell concentration for peripheral blood mononuclear cells (PBMC) varied between ducks and in order to maximise antigen or mitogen, cell concentrations needed to be optimised on an individual basis. Maximum antigen-specific transformation varied from duck to duck but occurred between days 4 and 7. The optimum antigen concentration varied between ducks from 1 to lOOOng/ml for DHBsAg and between 10 and lOOng/ml for DHBcAg. As expected the level of transformation (SI) was not as great as that obtained with stimulation with PHA which would be expected to stimulate a greater number of cells non-specifically. However, the antigen specific transformation response was similar to that obtained by stimulation with LPS. In acute DHBV infection PBMC from 2/8 and 3/8 responded to stimulation core or surface antigen respectively with one duck responding to both antigens. Both ducks that responded to core antigen cleared DHBV from the serum and the liver, while the ducks that responded to surface antigen cleared DHBV DNA from the serum , albeit only temporary in R89.In ducks that developed chronic DHBV infection 3/5 and 2/5 ducks responded to core and surface antigen respectively. The two ducks with the greatest liver pathology responding to DHBsAg. Vaccination with the 17kDa S protein produced a poor immunological response in the majority of ducks with only 2/4 responding with significant DHBsAg specific proliferation and one with concurrent anti-DHBs production. In the majority of immune ducks whether vaccinated or convalescent from infection the PBMC responded to stimulation by both antigens following a challenge with DHBV. However, this CMI response was temporary falling to non-significant levels quickly. The two sample Wilcoxon's rank sum test was applied to test for any significant difference between the above groups. The PBMC transformation response to both surface and core antigen in the immune ducks following challenge was significantly different from the transformation responses obtained from non-exposed ducks and ducks acutely infected with DHBV (P<0.05).

The purpose of the present study was to determine if psychological stress, defined as academic examination stress, would systematically produce changes in immune parameters (immunoglobulin concentration) and psychological functioning. It was hypothesized that as examination stress occurred there would be an effect on immunological function consistent with heightened psychological activity/stress. Subjects were 23 master's and doctoral students in psychology who volunteered for the research project. All subjects were administered a series of psychological tests to measure stress, personality factors, emotional states, and anxiety levels. All tests were administered and.blood samples drawn over a period of 15 months across two lowstress and two high-stress periods. Immunological tests included white blood cell (WBC) differential count and radial immunodiffusion (RID) for the determination of concentration of different immunoglobulin classes (IgA, IgG, IgM) in serum. Data were treated to a one-way analysis of variance (ANOVA) with repeated measures, t /test for correlated samples correlational matrix between variables across assessments and discriminant function analysis. Results showed (1) increased immunoglobulin levels during periods of stress; (2) immunoglobulin G most consistently related to stress and probably most indicative of the stressed condition and biological resistance to stress; (3) anxiety related to external events; (4) increase in anxiety under stress; and (5) anxiety inversely correlated with emotional stability and coping skills while positively related to tension, increased number of somatic complaints, and obsessive-compulsive trends. Firm support was provided for the hypothesis that as stress occurred, there would be consistent changes in immunological functioning associated with heightened psychological activity/stress. It was concluded that a response pattern to stress was adaptive along both psychological- and immunological dimensions and that the concept of bodymind interaction was the most realistic approach to understanding the total response patterns.

While Rhodococcus equi (R. equi) remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study this bacterium has proven difficult. As a result, the reasons for the underlying foal’s susceptibility to this disease are not well understood. Furthermore, data regarding the immune response of foals to R. equi infection remains controversial. We hypothesized that foals are susceptible to R. equi early in life and that this susceptibility decreases with age. Also, we hypothesized that specific subclasses of IgG antibodies to the virulence-associated protein of R. equi, VapA, predict the outcome of exposure. The objectives of this study were: (1) to develop an R. equi challenge model that resulted in slow progressive disease in some foals as well as spontaneous regression of lesions in others, (2) using the developed model, to investigate the age-related susceptibility of young foals to R. equi, (3) to describe the humoral immune response of foals following experimental challenge and natural infection. The use of a low dose of R. equi to challenge neonatal foals resulted in slow, progressive disease characterized by pulmonary abscessation and spontaneous regression in approximately 50% of the foals. When this low dose was used in 1, 2 or 3-week-old foals, a marked decrease in disease susceptibility was observed as the foals aged. The immunological responses seen after experimental challenge reflect those observed after natural infection. While there was a significant increase of VapA-specific IgG and IgG subclasses over time in both pneumonic and healthy foals, use of VapA-specific IgG(T) showed good sensitivity and specificity when used as a diagnostic tool for R. equi pneumonia. In summary, this study shows that foal susceptibility to R. equi occurs early in life and decreases with age. Whereas all foals developed VapA-specific IgG antibodies post-exposure, IgG(T) appeared to be predictive of infection.

Saccharomyces cerevisiae (bakers'/brewers' yeast) is a ubiquitous dietary constituent in the developed world. Previous studies, using semi-quantitative ELISA techniques, suggested that patients with Crohn's disease have higher titres of IgG and IgA isotypespecific antibodies to this yeast than are found in normal control subjects or patients with ulcerative colitis. For this study, in order to allow more stringent assay standardisation and more meaningful numerical comparison of the relative antigen-binding capacities of different sera, a quantitative ELISA was developed for measurement of anti-yeast antibodies, using a soluble extract of yeast (sacc) as the antigen. The finding of raised levels of yeast antibodies in Crohn's disease was confirmed, and the data suggest that this may be related to the presence of disease in the small bowel, although this latter observation did not reach statistical significance. Patients with chronic liver disease also had higher antibody levels than controls, but less markedly so than in Crohn's disease. When sera were tested in a similar assay for antibodies to bovine casein, no difference was found between controls and the Crohn's or liver disease group. The response of peripheral blood mononuclear cells (PBMC) to sacc was examined using a proliferation assay measuring uptake of tritiated thymidine. Cells from normal controls demonstrated dose-dependent proliferation, the time-course of which resembled that obtained with known recall antigens. Following separation of cell populations by rosetting with sheep erythrocytes, the responding cells were shown to be T-lymphocytes and the magnitude of the response was sensitive to the number of antigen-presenting cells present in the culture. When positive selection with immunomagnetic beads was used to further separate T-cells into highly purified CD4+ and CD8+ populations, responsiveness to yeast co-separated with the CD4+ subset. Following negative selection of cells expressing CD45RO or CD45RA, responsiveness was largely, but not exclusively, confined to the CD45RO+ population. Limiting dilution analysis of peripheral blood T-cells gave estimates of the sacc-specific precursor cell frequency in keeping with values previously reported for recall antigens, although the experimental data could not be shown to conform to single-hit kinetics. By sequential stimulation in long term culture, it was possible to obtain populations of cells which were uniquely responsive to sacc but unresponsive to other recall antigens. At some concentrations of sacc, proliferation responses of PBMC from Crohn's disease patients were higher than those in normal subjects, but the difference was not convincing overall. Digestion of sacc with pronase abolished the T-cell response but left specific antibody-binding intact, supporting the suggestion that antibody recognition is dependent on carbohydrate epitopes. Yeast cell wall mannan is implicated as the likely site of B-cell epitopes; evidence pertaining to T-cell epitopes is less conclusive. Thus, this study provides evidence that immune sensitisation to a common dietary constituent frequently occurs in the normal population, leading to detectable humoral and cellular immune responses. The T-cell response appears to be genuinely antigen-specific, and not due to non-specific lymphocyte activation. The gastrointestinal lymphoid system may be the site at which primary sensitisation occurs. In patients with Crohn's disease, the humoral response is enhanced, possibly as a consequence of inflammatory processes in the small bowel.

It has been suggested that ankylosing spondylitis (AS) is a form of reactive arthritis similar to that observed among patients with genitourinary or intestinal infections. This hypothesis was supported by several studies which found an increased isolation rate of Klebsiella species from the stools of AS patients. The strongest known association of a human leucocyte group A antigen (HLA) with disease is observed with HLA-B27 and AS. One hypothesis proposed to explain this association is molecular mimicry, in which antigenic similarities between HLA-B27 and arthritogenic bacteria lead to a cross-reacting immune response and inflammation. Non-secretion of ABO blood group substances, an autosomal recessive characteristic, is another factor associated with susceptibility to disease, particularly to infection of mucosal surfaces. In a study in 1987, non-secretors were found to be over-represented in patients with AS, evidence which supported the hypothesis of an infectious aetiology in AS. The aims of this work were to reassess the association of non-secretion with AS; to study the faecal flora in a cohort of patients with spondylarthropathy; to determine the prevalence of bacteria expressing antigens cross-reactive with the HLA-B27 antigen; to examine the humoral responses of patients and controls to their own faecal flora; and to correlate these findings with the disease activity of the patients as assessed clinically and by the erythrocyte sedimentation rate and C-reactive protein as laboratory parameters of inflammation. The proportion of non-secretors in this AS population was identical to that in the control population when determined by haemagglutination inhibition assay and confirmed by an enzyme-linked immunosorbent assay (ELISA) for Lewis antigens. Careful scrutiny of the secretor status in a group of patients examined in both present and initial studies revealed that 27% of non-secretors in the previous survey had been wrongly typed and were indeed secretors.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A leishmaniose visceral pode ser incluída como uma das causas de miopatia inflamatória em cães, entretanto, pouco se sabe sobre a patogênese da doença no sistema muscular, sendo incriminada muitas vezes apenas à natureza catabólica da enfermidade. O objetivo deste estudo foi avaliar, por meio de imunoistoquímica, a presença de formas amastigotas de Leishmania sp, linfócitos T (CD3+), macrófagos e IgG nos músculos tríceps braquial, extensor carpo radial, bíceps femoral e gastrocnêmio de 23 cães naturalmente acometidos por leishmaniose visceral. Dentre os 92 músculos avaliados,11 (12%) apresentaram marcação antigênica para formas amastigotas de Leishmania sp, 35 (38,1%) para linfócitos T (CD3+), 29 (31,5%) para macrófagos e 14 (12%) para IgG. Os resultados obtidos permitiram concluir que em cães com leishmaniose visceral apresentam imunomarcação para formas amastigotas de Leishmania sp., linfócitos T CD3+, macrófagos e IgG, sugerindo a participação direta do parasito e de uma resposta imune celular e humoral na fisiopatogenia da lesão muscular.
Visceral leishmaniasis may be included as a cause of inflammatory myophathy in dogs, however, little is known about the pathogenesis of the disease in the muscular system, which is frequently associated with the catabolic nature of the illness. The purpose of this study was investigate, through immunohistochemistry, the presence of amastigote forms of Leishmania sp, T lymphocytes (CD3+), macrophages and IgG in the muscle triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius of 23 dogs with visceral leishmaniasis. Among 92 evaluated muscles, 11 (12%) presented antigenic marking for amastigote forms of Leishmania sp., 35 (38,1%) for T lymphocites (CD3+), 29 (31,5%) for macrophages and 14 (12%) for IgG. The results of the present experiment led to the conclusion that in dogs with visceral leishmaniasis there may be a straight participation of the parasite and of cellular and humoral immune response in the ethiopatogeny of the muscular injury.

Orientador: Rosangela Zacarias Machado
Banca: Solange Maria Gennari
Banca: Aramis Augusto Pinto
Banca: Deise Aparecida de Oliveira Silva
Banca: Ana Patricia Yatsuda Natsui
Resumo: Neospora caninum é um protozoário do Filo Apicomplexa, que foi primeiramente descrito como causa de encefalomielite em filhotes caninos sorologicamente negativos para Toxoplasma gondii. Estudos anteriores neste importante hospedeiro da cadeia epidemiológica de N. caninum demonstram que as respostas de anticorpos IgG são tardiamente detectadas e que a infecção clínica é de difícil indução. Desta forma, este trabalho objetivou o estudo da imunidade de cães frente à infecção oral por N. caninum. Os resultados obtidos a partir de análises de diversos animais experimentalmente infectados indicam que os cães apresentam uma prolongada fase aguda da infecção, com eliminação de oocistos associado à queda nos níveis de linfócitos T CD4+ e CD8+ e diminuição de MHC de classe II por células apresentadoras de antígeno. Adicionalmente, os animais apresentam soroconversão instável durante o mesmo período, sendo que somente IgG1 e IgG3 foram detectados em adultos e filhotes, respectivamente, entre o 2o e 3o mês de infecção. De forma concomitante, observa-se uma predominância da expressão de citocinas imunomoduladoras como TGF 1, IL-4 e IL-10. Após dois meses de infecção, o perfil da resposta se inverte, sendo observado picos de produção dos marcadores CD4 e CD8 de linfócitos T e citocinas próinflamatórias como IFN , IL-6 e IL-12, além do aumento nos títulos de anticorpos, principalmente IgG1 e IgG4 nos cães jovens. Com base nestes resultados, conclui-se que os cães apresentam uma relação de equilíbrio com N. caninum, a qual induz nesta espécie uma modulação da resposta imunológica durante a fase de merogonia.
Abstract: Neospora caninum is an Apicomplexan parasite firstly described as the cause of encephalomyelitis in puppies serologically negative to Toxoplasma gondii. Previous reports on the parasite’s definitive host indicate a late IgG antibody response and that clinical disease is difficult to be induced. The aim of this study was to investigate canine immunity during N. caninum oral infection. The results obtained from the analysis of infected animal’s samples indicate that dogs present a protracted acute phase, with oocyst shedding correlated to a drop in CD4+ and CD8+ T cell levels, and low MHC class II expression by antigen presenting cells. Additionally, the dogs presented an unstable seroconversion pattern in the same period, with only IgG1 and IgG3 being detected in adult dogs and puppies, respectively, between the second and third months of infection. Concomitantly, dominant Th2 cytokine expression was observed, with peak expression levels of TGF 1, IL-4 and IL-10. After 2 months of infection, the immunity profile shifts towards a Th1 response, with high levels of CD4 and CD8 lymphocytary marker production and pro-inflammatory cytokine expression (IFN , IL-6 and IL-12), besides of the raise in antibody levels, especially IgG1 and IgG4 in puppies. Based in the results presented herein, we may conclude that dogs present a balanced host-parasite relationship, modulating the host immune response during N. caninum merogony.
Doutor

Thesis (PhD)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: New agents for the diagnosis, prevention and treatment of tuberculosis are urgently required. Yet, despite extensive tuberculosis research over recent years, no new drugs, vaccines or diagnostics have been identified to date. It is widely speculated that the major obstacle to the identification of new therapies is the lack of understanding of the hostpathogen interaction. This study has investigated whether patterns of antigen expression correlate with molecular epidemiological data and strain virulence through the analysis of protein expression and antigen recognition profiles of different M tuberculosis clinical isolates. Using polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and Western blotting, protein expression and antigen recognition by two genotypically different clinical strains that differed in their frequency in the study population have been compared. In addition to differences in protein expression and antigen recognition between the clinical strains and the reference strain H37Rv, protein expression differences between the clinical strains themselves were observed which may relate to strain frequency and virulence. Differential protein expression by M tuberculosis strains, may explain the heterogeneous host humoral immune response and why no fully effective serodiagnostic test has been developed to date. To explore this hypothesis, the potential of serodiagnosis in this community, where patients are infected with a wide variety of genotypically distinct strains, was investigated. IgG levels to three mycobacterial antigens showed that serodiagnosis of TB is possible in this community, despite infection by a wide variety of genotypically different M tuberculosis strains. Disease episode affected antibody levels, suggesting that care should be taken when evaluating serological diagnosis for repeat episode patients. This study has shown that M tuberculosis protein expression is dynamic and that the bacillus presents a hypervariabie array of antigens to the host immune system. It is likely that different antigens become immunodominant as antituberculosis chemotherapy progresses, and that these differentially expressed antigens may be tracked as predictors of treatment outcome. This hypothesis was tested by correlating Ag85-specific IgG with treatment response, as assessed by sputum smear conversion after two months of antimycobacterial chemotherapy. No significant correlation between antibody levels and treatment responses was observed, suggesting that antibodies may not be useful surrogate markers or that the incorrect antibody type or mycobacterial antigen were selected. Results were consistent with previous findings where patient-to-patient variation dictated the host humoral response. The results obtained in this study have demonstrated that although bacteriological factors may influence strain prevalence due to antigen variation and immune evasion, both bacteriological and host factors affect humoral immunity. Differential protein expression by M tuberculosis strains has potentially important implications for serodiagnosis and the development of subunit or DNA vaccines, by suggesting that multi-antigen cocktails should be used. Differential protein expression may also explain why patients do not develop adequate protective immunity and are susceptible to reinfection.
AFRIKAANSE OPSOMMING: Daar is 'n dringende behoefte vir nuwe middels vir die diagnosering, voorkoming en behandeling van tuberkulose. Ondanks intense tuberkulose navorsing gedurende die afgelope paar jaar, is daar geen nuwe tuberkulose medikasie, vaksines of diagnostiese metodes geïdentifiseer nie. Daar word gespekuleer dat die hoof struikelblok vir die identifisering van nuwe medikasie die onkunde oor die tuberkulose patogeen is. Deur die analise van proteien-uitdrukking en antigeen-erkenning profiele van verskillende M. tuberculosis kliniese isolate is daar tydens hierdie studie ondersoek ingestel of die patroon van antigeen uitdrukking korreleer met molekulêre epidemiologiese data and stam-virulensie. Proteien-uitdrukking en antigeen-erkenning deur twee genotipies verskillende kliniese stamme wat verskil in hul frekwensie in die bestudeerde populasie, is vergelyk deur middel van poli-akrielamied gel elektroforese, ensiem-gekoppelde immuunabsorberende analise en Westelike oordrag. Addisoneel tot die verskille in proteienuitdrukking en antigeen-ekenning tussen kliniese stamme en die verwysingstam H37Rv, is daar ook verskille aangedui tussen die kliniese stamme self wat kan dui op stam frekwensie en virulensie. Differensiële proteien-uitdrukking deur M. tuberculosis stamme, kan moontlik die heterogene gasheer se humorale immuunreaksie verduidelik en daarmee saam die rede waarom daar nie tot op hede 'n effektiewe sero-diagnostiese toets ontwikkel is nie. Daar is dus ondersoek ingestel na die potensiaal van sero-diagnose in 'n gemeenskap waar pasiënte geïnfekteer is met 'n wye verskeidenheid genotipiese stamme. Die IgG vlakke van drie mikobakteriële antigene het aangedui dat sero-diagnose van tuberkulose moontlik is in hierdie gemeenskap, ten spyte van infektering deur 'n wye verskeidenheid genotipies-verskillende M. tuberculosis stamme. Die tussenspel van die siekte het teenliggaampie-vlakke beïnvloed wat daarop dui dat daar versigtig moet gelet word tydens die evaluering van serologiese diagnose van geïnfekteerde pasiënte wat voorheen siek was. Hierdie studie toon dat M. tuberculosis proteïen-uitdrukking dinamies is en dat die bacillus 'n groot variëteit van antigene tot die immuun sisteem bied. Dit is moontlik dat verskillende antigene immuun dominant kan word soos wat antituberkulose chemoterapie toeneem, en dat hierdie verskillend-uitgedrukte antigene as 'n gevolg daarvan gebruik kan word as voorspellers vir behandeling. Hierdie hipotese is getoets deur die korrelering van Ag85-spesifieke IgG met die reaksie op behandeling soos geëvalueer deur speeksel-monster verandering na twee maande se anti-mikobakteriële chemoterapie. Daar was geen noemenswaardige korrelasie tussen teenliggaampie vlakke en die reaksie op behandeling nie, wat daarop dui dat die teenliggaampies nie toepaslike surrogaat merkers is nie of dat die verkeerde teenliggaampie-tipe of mikobakteriële antigeen geselekteer is. Hierdie resultate bevestig vorige bevindinge waar pasiënt-tot-pasiënt verskille die gasheer se humorale immuunreaksie gedikteer het. Die resultate wat uit hierdie studie volg dui dat alhoewel bakteriologiese faktore die stam-frekwensie kan beïnvloed as gevolg van antigeen-variasie en immuun-ontduiking, kan beide bakteriologiese en gasheer faktore die humorale immuunreaksie beïnvloed. Differensiële proteiën uitdrukking deur 'n verskeidenheid M. tuberculosis stamme het potensieël belangrike toepassings vir sero-diagnose en die ontwikkeling van subeenheid of DNS vaksines wat impliseer dat multi-antigeen mengsels gebruik moet word. Differensiële proteiën uitdrukking mag ook verduidelik waarom pasiënte nie 'n voldoende beskermende immuniteit opbou nie en sodoende ontvanklik is vir her-infeksie.

Aggregatibacter actinomycetemcomitans (Aa) is a powerful pathogen associated with a periodontal disease, and heat-shock protein 60 (HSP60) is considered to be a major virulence factor of the Aa. Periodontitis is associated with a higher risk of atherosclerosis. One of the possible mechanisms by which atherosclerosis is developed could be the humoral immune cross-reactivity between bacterial products and oxidized epitopes of LDL (low-density lipoprotein). Oxidization of LDL is known to be a major atherosclerosis promoting factor. The objectives of the study were to investigate whether mouse immune system can be induced to produce immunoglobulins against Aa HSP60, what type of immunoglobulins are produced, and do they show cross-reactivity with oxidized forms of LDL. Mice (C57BL/6J) were immunized with purified recombinant Aa HSP60 protein, blood samples were collected before and after immunization, and measurements were performed using enzyme linked immunosorbent assay (ELISA). Levels and types of immunoglobulins specific to Aa HSP60 were measured with ELISA, as well as possible cross-reactivity with oxidized forms of LDL. Specificity of immunoglobulins to Aa HSP60 was investigated with a liquid phase competitive ELISA method. The immunization with Aa HSP60 triggered IgG and IgM class humoral immune response, but not IgA class response. Both IgG and IgM antibodies were able to not only bind to Aa HSP60 but also crossreact with malondialdehyde-acetaldehyde (MAA) epitopes of LDL. Specificity testing revealed that most of the IgG class antibodies that bound to Aa HSP60 could be competed out with Aa HSP60, implicating that the adaptive immune responses are specific. Cross-reactivity testing demonstrated that a significant portion of IgG antibodies that bound to the fixed Aa HSP60 could be competed out with MAA-LDL. This implies that MAA-LDL possess epitopes sharing molecular mimicry with Aa HSP60 which can be recognized by the adaptive IgG antibodies
Aggregatibacter actinomycetemcomitans (Aa) on parodontiittin liittyvä voimakas patogeeni, jonka tärkeä virulenssitekijä on lämpöshokkiproteiini 60 (HSP60). Parodontiitti on liitetty kohonneeseen ateroskleroosiriskiin. Yhden ateroskleroosin syntyyn vaikuttavista mekanismeista uskotaan olevan vasta-ainevälitteisen immuunipuolustuksen ristireagoinnin bakteerituotteiden ja hapettuneen kolesterolin (LDL) välillä. LDL:n hapettumisen tiedetään olevan ateroskleroosille altistava tekijä. Tutkimuksessa selvitettiin, voidaanko hiiren immuunijärjestelmä aktivoida tuottamaan vasta-aineita Aa bakteerin HSP60 proteiinia vastaan, minkä tyyppisiä tuotetut vasta-aineet ovat, ja ristireagoivatko ne hapettuneiden LDL:n muotojen kanssa. Hiiret (C57BL/6J) immunisoitiin rekombinantti tekniikalla valmistetulla ja puhdistetulla Aa HSP60 proteiinilla. Verinäytteet kerättiin ennen ja jälkeen immunisaation, ja mittaukset suoritettiin entsyymivälitteisellä immunosorbenttimäärityksellä (ELISA). Aa HSP60 proteiinille spesifisten vasta-aineiden määrät ja tyypit, sekä niiden mahdolliset ristireaktiot hapettuneen LDL:n kanssa mitattiin ELISA-menetelmää käyttäen. Vasta-aineiden spesifisyys määritettiin käyttämällä inhiboivaa nestemäisen faasin ELISA-menetelmää. Immunisaatio Aa HSP60 proteiinilla aiheutti vasta-ainevälitteisen immuunivasteen. Tuotetut vasta-aineet olivat IgG ja IgM tyyppiä, IgA tyypin vasta-ainetta ei tuotettu. Sekä IgG että IgM tyypin vasta-aineilla havaittiin ristireaktio LDL:n malondialdehydi-asetaldehydi epitooppien (MAA-LDL) ja Aa HSP60 proteiinin välillä. Spesifiteettimäärityksessä kävi ilmi, että suurin osa IgG luokan vasta-aineista jotka sitoutuvat Aa HSP60 proteiiniin, voitiin inhiboida Aa HSP60 proteiinilla, joka viittaa vasta-aineiden olevan spesifisiä Aa HSP60 proteiinille. Ristireaktiotesteissä ilmeni, että merkittävä osa vasta-aineista jotka sitoutuvat Aa HSP60 proteiiniin, voidaan inhiboida myös MAA-LDL:n avulla. Tämä viittaa MAA-LDL:n sisältävän epitooppeja, jotka Aa HSP60:tä vastaan tuotetut IgG vasta-aineet tunnistavat rakenteellisen samankaltaisuuden vuoksi

Orientadora: Mary Marcondes
Banca: Raimundo Souza Lopes
Banca: Vera Lúcia Fonseca de Camargo Neves
Resumo: A leishmaniose visceral pode ser incluída como uma das causas de miopatia inflamatória em cães, entretanto, pouco se sabe sobre a patogênese da doença no sistema muscular, sendo incriminada muitas vezes apenas à natureza catabólica da enfermidade. O objetivo deste estudo foi avaliar, por meio de imunoistoquímica, a presença de formas amastigotas de Leishmania sp, linfócitos T (CD3+), macrófagos e IgG nos músculos tríceps braquial, extensor carpo radial, bíceps femoral e gastrocnêmio de 23 cães naturalmente acometidos por leishmaniose visceral. Dentre os 92 músculos avaliados,11 (12%) apresentaram marcação antigênica para formas amastigotas de Leishmania sp, 35 (38,1%) para linfócitos T (CD3+), 29 (31,5%) para macrófagos e 14 (12%) para IgG. Os resultados obtidos permitiram concluir que em cães com leishmaniose visceral apresentam imunomarcação para formas amastigotas de Leishmania sp., linfócitos T CD3+, macrófagos e IgG, sugerindo a participação direta do parasito e de uma resposta imune celular e humoral na fisiopatogenia da lesão muscular.
Abstract: Visceral leishmaniasis may be included as a cause of inflammatory myophathy in dogs, however, little is known about the pathogenesis of the disease in the muscular system, which is frequently associated with the catabolic nature of the illness. The purpose of this study was investigate, through immunohistochemistry, the presence of amastigote forms of Leishmania sp, T lymphocytes (CD3+), macrophages and IgG in the muscle triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius of 23 dogs with visceral leishmaniasis. Among 92 evaluated muscles, 11 (12%) presented antigenic marking for amastigote forms of Leishmania sp., 35 (38,1%) for T lymphocites (CD3+), 29 (31,5%) for macrophages and 14 (12%) for IgG. The results of the present experiment led to the conclusion that in dogs with visceral leishmaniasis there may be a straight participation of the parasite and of cellular and humoral immune response in the ethiopatogeny of the muscular injury.
Mestre

Feed costs are approximately 70% of total production cost for poultry producers. Poultry diets in the United States generally consist of 2 grains: corn and soybean meal. In recent years, the cost of these grains has dramatically increased. Due to these price increases, producers seek alternative feeds that provide adequate nutrition, and are also more affordable than â traditionalâ grains. Cottonseed meal is one alternative that is both affordable and an excellent source of crude protein. However, cottonseed meal contains gossypol, a pigment toxic to chickens. This study had two main objectives. The first objective was to determine if silymarin, an extract from milk thistle, could offset or prevent gossypol toxicosis. The second objective was to determine if divergent selection for humoral immune response would have an impact on the ability of the chicken to cope with gossypol toxicosis. Two preliminary studies were conducted. One determined basal activities of liver detoxification enzymes at various ages. The other determined concentrations of gossypol and silymarin that should be added to the diet to elicit a response. The information gathered from the second preliminary study was used to conduct the final experiment. In the final experiment, chickens from each of 2 lines selected for humoral immunity were exposed to diets containing gossypol, silymarin, gossypol and silymarin, and a control. Humoral immunity had no impact on the ability of the chicken to cope with gossypol toxicosis. Silymarin did not alleviate gossypol toxicosis. Future studies will focus on using a lower gossypol concentration in the diet.
Master of Science

Este estudo avaliou a imunidade humoral e celular em cães naturalmente infectados com L. (L.) chagasi correlacionando com a transmissibilidade para o vetor. Soros e biópsias de baço, linfonodo e pele foram coletados de 120 cães provenientes do Centro de Controle de Zoonoses do município de Araçatuba, São Paulo, Brasil. Os soros foram processados por ELISA para detecção de IgG, IgG1, IgG2 e IgE; e as biópsias foram processadas por técnicas histológicas usuais coradas pelo HE e imunoistoquímica para a detecção de parasito, macrófago e células T CD3. De acordo com os sinais clínicos, 65/120 (54%) cães foram classificados como sintomáticos e 55/120 (46%) como assintomáticos. O diagnóstico parasitológico foi confirmado em 71% dos sintomáticos e em 40% dos assintomáticos. A correlação dos sinais clínicos com parasitismo mostrou que a carga parasitária estava diretamente associada com cães sintomáticos (p<0.05). Em relação aos anticorpos específicos anti-L.(L.)chagasi, cães de área endêmica com diagnóstico parasitológico positivo mostraram maiores níveis de IgG total comparado com ambos os controles (p<0.05), sem diferença entre cães sintomáticos e assintomáticos. IgG1 esteve presente em baixos níveis e foi mais intensa no grupo sintomático parasito-positivo (p<0.05). Níveis mais elevados foram observados para IgG2 em cães de área endêmica (p<0.05), mas sem correlação com o parasitismo e sinais clínicos. IgE também esteve presente em baixos níveis, mas mostrou diferenças entre cães de área endêmica e cães de área não endêmica; e cães com diagnóstico parasitológico positivo mostrou níveis mais elevados que cães com diagnóstico parasitológico negativo (p<0.05). Histopatologicamente, linfonodos mostraram hiperplasia e hipertrofia de macrófagos na área medular e em muitos casos linfadenite granulomatosa. Na polpa branca do baço, hiperplasia folicular foi observada; e a polpa vermelha mostrou granulomas. As lesões de pele foram caracterizadas por infiltrado inflamatório crônico na derme formado por macrófagos, linfócitos e plasmócitos; variando de discreto a intenso, assim como de focal a difuso. Foi evidente a presença de granulomas epitelióides na pele de alguns animais. Imunoistoquímica mostrou presença de células marcadas pelo anticorpo anti-macrófago e anti-CD3 em 100% dos baços e linfonodos variando de intensidade entre discreto e intenso. Na pele, macrófagos foram positivos em 90% e células CD3 em 39% dos casos. Houve associação direta entre baixa expressão de células CD3 e alto parasitismo na pele. Animais assintomáticos mostraram baixa expressão de macrófagos junto com baixo parasitismo na pele. Em relação ao xenodiagnóstico, no 4o dia depois da alimentação, as fêmeas dos vetores foram dissecadas e examinadas para observação de parasitos no intestino. Formas promastigotas foram observadas em 27% das fêmeas que se alimentaram em cães sintomáticos e em 42% das fêmeas que se alimentaram em cães assintomáticos. A técnica da PCR foi também utilizada para avaliar as fêmeas positivas depois do xenodiagnóstico. DNA de Leishmania foi detectado em 24% das fêmeas que se alimentaram em cães sintomáticos e em 34% das fêmeas que se alimentaram em cães assintomáticos. Os dados mostraram que a imunidade humoral e celular não teve correlação direta com as formas clínicas de leishmaniose canina. A alta porcentagem de vetores infectados na alimentação em cães assintomáticos mostra a importância destes animais na transmissibilidade para o vetor.
These studies evaluate humoral and cellular immunity in dogs naturally infected with L. (L.) chagasi correlating to the transmissibility to the vector. Serum and biopsy from spleen, lymph node and skin were collected from 120 dogs referred to the Center of Zoonosis Control of Araçatuba city, São Paulo, Brazil. The sera were processed by ELISA for IgG, IgG1, IgG2 and IgE detection; and the biopsies were processed usual histological techniques stained by HE and immunohistochemistry for parasite, macrophage and T CD3 cells detection. According to the clinical signs, 65/120 (54%) dogs were classified as symptomatic and 55/120 (46%) as asymptomatic. Parasitological diagnosis was confirmed in 71% of symptomatic and in 40% of asymptomatic dogs. The correlation of clinical signs and parasitism showed that parasite burden was directly associated with symptomatic dogs (p<0.05). Concerning to L.(L.)chagasi-specific antibodies, dogs from the endemic area with positive parasitological diagnosis showed high levels of total IgG compared to both controls (p<0.05), without difference between symptomatic and asymptomatic dogs. IgG1 was present at low levels and was more intense in the parasite-positive symptomatic group (p<0.05). More elevated levels were observed for IgG2 in dogs from endemic area (p<0.05), but with no correlation to parasitism and clinical signs. IgE was also present at low levels, but showed differences between dogs from non-endemic and endemic areas; and dogs with positive parasitological diagnosis showed higher levels than dogs with negative parasitological diagnosis (p<0.05). Histopathologically, lymph nodes showed macrophage hyperplasia and hypertrophy in the medullary area and in many cases granulomatous lymphadenitis. In the white pulp of the spleen, follicular hyperplasia was observed; and the red pulp showed granulomas. The skin lesions were characterized by dermal chronic inflammatory infiltrate formed by macrophages, lymphocytes and plasma cells; it varied between descreet to intense, as well as focal to diffuse. The epithelioid granulomas were evident in the skin of some animals. Immunohistochemistry showed presence of labeled cells by anti-macrophage and anti-CD3+ antibodies in 100% of spleen and lymph nodes varying the intensity between mild to intense. Macrophage was positive in 90% of the skin and CD3 cells in 39%. There was a direct association between lower CD3 cells expression and higher parasite burden in the skin. Asymptomatic animals showed lower macrophage expression together with lower parasitism in the skin. Concerning to the xenodiagnosis, on the 4th day after the blood meal, female flies were dissected and examined for visible parasites in the gut. Promastigotes forms were observed in 27% of female which fed in symptomatic dogs and in 42% of female which fed in asymptomatic dogs. PCR technique was also used to evaluate the positive females after the xenodiagnosis. Leishmania DNA was detected in 24% of female which fed in symptomatic dogs and in 34% of female which fed in asymptomatic dogs. The data showed that the humoral and cellular immune response not has direct correlation to the clinical form of canine leishmaniasis. The high percentage of sand flies female infected by feeding in the asymptomatic dogs show the importance these animals on the parasite transmissibility to the vector.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Neospora caninum é um protozoário do Filo Apicomplexa, que foi primeiramente descrito como causa de encefalomielite em filhotes caninos sorologicamente negativos para Toxoplasma gondii. Estudos anteriores neste importante hospedeiro da cadeia epidemiológica de N. caninum demonstram que as respostas de anticorpos IgG são tardiamente detectadas e que a infecção clínica é de difícil indução. Desta forma, este trabalho objetivou o estudo da imunidade de cães frente à infecção oral por N. caninum. Os resultados obtidos a partir de análises de diversos animais experimentalmente infectados indicam que os cães apresentam uma prolongada fase aguda da infecção, com eliminação de oocistos associado à queda nos níveis de linfócitos T CD4+ e CD8+ e diminuição de MHC de classe II por células apresentadoras de antígeno. Adicionalmente, os animais apresentam soroconversão instável durante o mesmo período, sendo que somente IgG1 e IgG3 foram detectados em adultos e filhotes, respectivamente, entre o 2o e 3o mês de infecção. De forma concomitante, observa-se uma predominância da expressão de citocinas imunomoduladoras como TGF 1, IL-4 e IL-10. Após dois meses de infecção, o perfil da resposta se inverte, sendo observado picos de produção dos marcadores CD4 e CD8 de linfócitos T e citocinas próinflamatórias como IFN , IL-6 e IL-12, além do aumento nos títulos de anticorpos, principalmente IgG1 e IgG4 nos cães jovens. Com base nestes resultados, conclui-se que os cães apresentam uma relação de equilíbrio com N. caninum, a qual induz nesta espécie uma modulação da resposta imunológica durante a fase de merogonia.
Neospora caninum is an Apicomplexan parasite firstly described as the cause of encephalomyelitis in puppies serologically negative to Toxoplasma gondii. Previous reports on the parasite s definitive host indicate a late IgG antibody response and that clinical disease is difficult to be induced. The aim of this study was to investigate canine immunity during N. caninum oral infection. The results obtained from the analysis of infected animal s samples indicate that dogs present a protracted acute phase, with oocyst shedding correlated to a drop in CD4+ and CD8+ T cell levels, and low MHC class II expression by antigen presenting cells. Additionally, the dogs presented an unstable seroconversion pattern in the same period, with only IgG1 and IgG3 being detected in adult dogs and puppies, respectively, between the second and third months of infection. Concomitantly, dominant Th2 cytokine expression was observed, with peak expression levels of TGF 1, IL-4 and IL-10. After 2 months of infection, the immunity profile shifts towards a Th1 response, with high levels of CD4 and CD8 lymphocytary marker production and pro-inflammatory cytokine expression (IFN , IL-6 and IL-12), besides of the raise in antibody levels, especially IgG1 and IgG4 in puppies. Based in the results presented herein, we may conclude that dogs present a balanced host-parasite relationship, modulating the host immune response during N. caninum merogony.

The drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim was investigated using an in vitro culture system, an in vivo mouse model, and a clinical trial in HIV-infected patients. We hypothesized that combination exposure causes immune cell populations in the bone marrow to undergo apoptotic cell death, and that the toxicity would affect the host response to an infectious stimulus. Mice were dosed with zidovudine, sulfamethoxazole-trimethoprim, the combination of both drugs, or vehicle only control via oral gavage. Focusing on B-lineage cells in the bone marrow, we determined that cells of the rapidly cycling, early pre-B cell subset are targeted, as well as pro-B cells earlier in development. This toxicity was found to be cell cycle dependent, with an increase in percentage of cells in the S/G2/M phases of the cycle. In vitro experiments using the drugs in a bone marrow culture system demonstrated that the effect of cytotoxicity with combination exposure is synergistic and concentration-dependent. The mechanism of apoptosis that is induced appears to be caspase-independent. To measure host response in mice, animals treated with zidovudine plus sulfamethoxazole-trimethoprim were infected with Pneumocystis murina pneumonia, and the group that received the combination of agents had a blunted antigen-specific IgG response, possibly due to a decreased number of B cells and activated B cells in the draining lymph nodes of the lungs. A clinical trial was conducted in HIV-infected patients, dividing subjects into groups receiving zidovudine, sulfamethoxazole-trimethoprim, the combination of both, or neither agent. Upon vaccination with the influenza vaccine, the combination treatment group had a blunted humoral response, with reduced antigen-specific serum IgG titers as compared to the control group. We conclude that the drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim is clinically-significant, and clinicians must consider this toxicity when treating patients with these agents concurrently.

Thesis (Ph. D.)--West Virginia University, 2006.
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