Modulation of parasitemia and antibody responce to Trypanosoma cruzy by cyclophosphamide in Calomys callosus (Rodentia, Cricetidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MODULATION OF PARASITEMIA AND ANTIBODY-RESPONSE TO TRYPANOSOMA-CRUZI BY CYCLOPHOSPHAMIDE IN CALOMYS-CALLOSUS (RODENTIA, CRICETIDAE)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan.Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. callosus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10(3) T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callosus to T. cruzi.

Robust Control of Evolutionary Dynamics

The application of principles from evolutionary biology has long been used to gain new insights into the progression and clinical control of both infectious diseases and neoplasms. This iterative evolutionary process consists of expansion, diversification and selection within an adaptive landscape - species are subject to random genetic or epigenetic alterations that result in variations; genetic information is inherited through asexual reproduction and strong selective pressures such as therapeutic intervention can lead to the adaptation and expansion of resistant variants. These principles lie at the center of modern evolutionary synthesis and constitute the primary reasons for the development of resistance and therapeutic failure, but also provide a framework that allows for more effective control.

A model system for studying the evolution of resistance and control of therapeutic failure is the treatment of chronic HIV-1 infection by broadly neutralizing antibody (bNAb) therapy. A relatively recent discovery is that a minority of HIV-infected individuals can produce broadly neutralizing antibodies, that is, antibodies that inhibit infection by many strains of HIV. Passive transfer of human antibodies for the prevention and treatment of HIV-1 infection is increasingly being considered as an alternative to a conventional vaccine. However, recent evolution studies have uncovered that antibody treatment can exert selective pressure on virus that results in the rapid evolution of resistance. In certain cases, complete resistance to an antibody is conferred with a single amino acid substitution on the viral envelope of HIV.

The challenges in uncovering resistance mechanisms and designing effective combination strategies to control evolutionary processes and prevent therapeutic failure apply more broadly. We are motivated by two questions: Can we predict the evolution to resistance by characterizing genetic alterations that contribute to modified phenotypic fitness? Given an evolutionary landscape and a set of candidate therapies, can we computationally synthesize treatment strategies that control evolution to resistance?

To address the first question, we propose a mathematical framework to reason about evolutionary dynamics of HIV from computationally derived Gibbs energy fitness landscapes -- expanding the theoretical concept of an evolutionary landscape originally conceived by Sewall Wright to a computable, quantifiable, multidimensional, structurally defined fitness surface upon which to study complex HIV evolutionary outcomes.

To design combination treatment strategies that control evolution to resistance, we propose a methodology that solves for optimal combinations and concentrations of candidate therapies, and allows for the ability to quantifiably explore tradeoffs in treatment design, such as limiting the number of candidate therapies in the combination, dosage constraints and robustness to error. Our algorithm is based on the application of recent results in optimal control to an HIV evolutionary dynamics model and is constructed from experimentally derived antibody resistant phenotypes and their single antibody pharmacodynamics. This method represents a first step towards integrating principled engineering techniques with an experimentally based mathematical model in the rational design of combination treatment strategies and offers predictive understanding of the effects of combination therapies of evolutionary dynamics and resistance of HIV. Preliminary in vitro studies suggest that the combination antibody therapies predicted by our algorithm can neutralize heterogeneous viral populations despite containing resistant mutations.

16/6-idiotype expressing antibodies induce brain inflammation and cognitive impairment in mice: the mosaic of central nervous system involvement in lupus

Background: The 16/6-idiotype (16/6-Id) of the human anti-DNA antibody was found to induce experimental lupus in naive mice, manifested by production of autoantibodies, leukopenia and elevated inflammatory markers, as well as kidney and brain involvement. We assessed behavior and brain pathology of naive mice injected intracerebra-ventricularly (ICV) with the 16/6-Id antibody. Methods: C3H female mice were injected ICV to the right hemisphere with the human 16/6-Id antibody or commercial human IgG antibodies (control). The mice were tested for depression by the forced swimming test (FST), locomotor and explorative activity by the staircase test, and cognitive functions were examined by the novel object recognition and Y-maze tests. Brain slices were stained for inflammatory processes. Results: 16/6-Id injected mice were cognitively impaired as shown by significant differences in the preference for a new object in the novel object recognition test compared to controls (P = 0.012). Similarly, the preference for spatial novelty in the Y-maze test was significantly higher in the control group compared to the 16/6-Id-injected mice (42% vs. 9%, respectively, P = 0.065). Depression-like behavior and locomotor activity were not significantly different between the16/6-Id-injected and the control mice. Immunohistochemistry analysis revealed an increase in astrocytes and microglial activation in the hippocampus and amygdala, in the 16/6-Id injected group compared to the control. Conclusions: Passive transfer of 16/6-Id antibodies directly into mice brain resulted in cognitive impairments and histological evidence for brain inflammation. These findings shed additional light on the diverse mosaic pathophysiology of neuropsychiatric lupus.

Morbidity-mortality and performance evaluation of Brahman calves from in vitro embryo production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transferência de imunidade passiva em bezerros das raças Nelore e Limousin e proteinograma sérico nos primeiros quatro meses de vida

Com o objetivo de investigar alguns aspectos relacionados à transferência de imunidade passiva em bovinos de corte, foram selecionados 90 bezerros aparentemente sadios, 45 da raça Nelore e 45 da raça Limousin, distribuídos em 3 grupos (com 15 bezerros cada) de acordo com o número de parições de suas mães: primeira cria; segunda cria; e terceira ou mais crias. Amostras de sangue foram colhidas de cada bezerro entre 24 e 36 horas de vida e com 15, 30, 60, 90 e 120 dias. Determinaram-se as concentrações de proteína total no soro (PT) e no plasma (PPT), a atividade sérica da gamaglutamiltransferase (GGT), e as concentrações séricas de albumina, alfa, beta e gamaglobulinas por eletroforese em gel de agarose e de IgG estimada por meio do método de turvação pelo sulfato de zinco. Empregou-se a análise de variância bifatorial para as variáveis mensuradas na primeira colheita. O comportamento das variáveis em função da idade foi estudado por meio da análise de variância de medidas repetidas. Correlações foram estabelecidas entre as variáveis. A transferência de imunidade passiva foi bem sucedida nos bezerros de ambas as raças e o número de parições das mães não interferiu no processo. As concentrações mais elevadas de gamaglobulinas ao término do primeiro dia de vida declinaram até valores mínimos aos 60 dias. A partir dessa idade, a elevação conseqüente à produção ativa de anticorpos foi mais precoce nos bezerros taurinos e mais lenta nos zebuínos. A gamaglobulina, ao término do primeiro dia de vida, correlacionou-se com as seguintes variáveis: IgG (r=0,859), PPT (r=0,807), PT (r=0,811) e GGT (r=0,399). O fator etário exerceu efeito sobre todas as variáveis mensuradas. As variações das proteínas séricas obedeceram a um padrão de comportamento fisiológico ao longo dos quatro primeiros meses de vida, de forma geral, não distinto em taurinos e zebuínos.

Período de absorção intestinal de macromoléculas em cabritos recém-nascidos após a ingestão de colostro bovino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação clínica, morbi-mortalidade e desenvolvimento de bezerros da raça Brahman produzidos por fertilização in vitro e inseminação artificial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteinograma sérico de bezerros recém-nascidos alimentados com colostro de vacas com mastite

The aim of this study was to evaluate the serum protein concentration in newborns fed with colostrum derived from healthy cows (n = 10), cows with subclinical mastitis (n = 10) and cows with clinical mastitis (n = 10). 30 Holstein cows were assigned to their respective groups according to macroscopic examination of colostral secretion, somatic cell count, CMT and presence of bacteria in colostrum samples. Blood samples of the calves were collected immediately after birth, at 24 and 48 hours after ingestion of colostrum. The total protein was measured by the biuret method and the concentrations of immunoglobulin A (IgA), immunoglobulin G (IgG), transferrin, albumin and haptoglobin was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). No differences were obser- ved amongst groups in the concentrations of albumin, total protein and IgA. In animals from cows with subclinical and clinical mastitis haptoglobin concentrations were higher than those of healthy animals. The concentrations of IgG and transferrin were significantly lower in calves from cows with mastitis. We concluded that the ingestion of colostrum from infected and uninfected glands from cows with mastitis (GII e GIII) is unlikely to be an important contributor to the high rate of failure of passive transfer of immunoglobulins in calves.

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.

Gammaglobulin and selenium status in healthy neonatal dairy calves in Switzerland

Serum samples from 142 calves and their dams were analyzed for gammaglobulins (gammaG, calves) and selenium concentrations (Se, calves and dams). A questionnaire provided information about birth and colostrum management. The calves and their dams were distributed into two groups according the calves' gammaG concentration (< 10 and >= 10 g/L), Se concentrations were compared between groups. The correlation between gammaG and Se concentrations in the calves and their dams was analyzed. Risk factors for failure of passive transfer and Se deficiency were assessed based on the questionnaire. The gammaG concentration of 42.9 % of the calves was < 10 g/L (median: 10.9). Calves showed significantly higher gammaG values after optimized colostrum administration than calves with suboptimal colostrum administration (p < 0.004). The median Se concentration was 26.8 and 36.5 microg/L for the calves and dams, respectively. A high correlation was observed between the Se concentration of the dam and her calf (r = 0.72, p < 0.001). The calves' Se and gammaG concentrations were not significantly correlated. These results demonstrate that further efforts toward better information of farmers regarding colostrum management and Se supply are warranted.

Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.

Vigor, variação na concentração de metabólitos sanguíneos e avaliação da transferência de imunidade passiva em bezerros leiteiros neonatos

O objetivo deste trabalho foi identificar o padrão de vigor e status metabólico através da determinação da frequência dos sinais objetivos e metabólitos sanguíneos após o nascimento, possivelmente associados à transferência de imunidade passiva, além de determinar o melhor horário para o uso do refratômetro de Brix na avaliação da transferência de imunidade passiva em bezerros neonatos após a ingestão de colostro. No primeiro experimento foram utilizados 46 bezerros neonatos de ambos os sexos, nascidos entre setembro de 2013 e julho de 2014. A avaliação do vigor dos animais foi realizada entre 15 e 30 minutos após o nascimento com o auxílio da escala APGAR modificada pela Universidade de Guelph. A colheita de sangue para análise de metabólitos seguiu os horários de 0h (antes da ingestão), 1h, 2h, 4h, 6h, 12h, 24h, 48h contadas a partir da ingestão do colostro. Os parâmetros sanguíneos foram determinados por kit enzimático específico para cada parâmetro em Sistema Automático para Bioquímica. Todos os animais utilizados neste estudo foram oriundos de partos eutócicos e apresentaram, de acordo com a escala APGAR, vigor satisfatório. As concentrações de metabólicos sanguíneos dos neonatos mostraram que o consumo de colostro aumentou a disponibilidade de energia e a concentração de frações proteicas, comumente utilizadas como indicativos da transferência de imunidade passiva. No segundo experimento foram utilizados 47 bezerros neonatos de ambos os sexos, nascidos entre março e julho de 2014. O colostro foi ordenhado após o parto e a qualidade determinada com auxílio do colostrômetro e do refratômetro digital. A colheita do sangue para a análise dos parâmetros sanguíneos seguiu os horários de 0h (antes da ingestão), 1h, 2h, 4h, 6h, 12h, 24h, 48h, 72h, 96 e 120h, contadas a partir da ingestão do colostro. A concentração de proteína total foi determinada por refratômetro de Brix e por kit enzimático em Sistema Automático para Bioquímica. Os demais parâmetros sanguíneos foram determinados por kit enzimático específico para cada parâmetro em Sistema Automático para Bioquímica. A contagem global e diferenciada das células sanguíneas foi realizada nos tempos 0, 12 e 24h após o fornecimento do colostro. O colostro se manteve na faixa de boa qualidade, segundo os critérios de classificação de medidas pelo colostrômetro e refratômetro digital de Brix. O consumo de colostro foi o principal fator de variação dos parâmetros estudados neste trabalho. A avaliação da transferência de imunidade passiva em bezerros recém-nascidos deve ser realizada por volta das 24 horas após a ingestão do colostro, pois neste momento a absorção de macromoléculas colostrais já está encerrada e a PT alcança estabilidade. A partir deste período, não é possível determinar se as frações proteicas avaliadas são de origem colostral ou endógena, o que não garante avaliação segura sobre a transferência de imunidade passiva.

Estudo da transferência de imunidade passiva, em vitelos, no concelho de Guimarães

Dissertação de Mestrado Integrado em Medicina Veterinária