Estudio de los mecanismos moleculares involucrados en la inmunosupresión observada durante la infección con Trypanosoma cruzi: Rol de la vía PD-1/PD1-L y E3 ubiquitina ligasas. Molecular mechanismns involved in the immunosupression during Tryapanosoma cruzi infection. Role of PD1-PD1-L pathway and E3 ubiquitin ligases.

Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, un problema de salud importante en América Latina, así como también en América Central, ya que causa infección crónica afectando a millones de personas [1]. Durante esta enfermedad se han descripto varias alteraciones de la respuesta inmune, entre ellas una severa inmunosupresión durante la etapa aguda de la infección, tanto en humanos como en ratones. Células T provenientes de ratones infectados activadas in vitro, muestran reducción en la respuesta proliferativa a mitógenos, característica de un estado de inmunosupresión [2-4]. La falla del sistema inmune durante estadios tempranos de la infección probablemente colabore con la diseminación y el establecimiento del parásito. Un gran número de estudios se han focalizado en la identificación de mecanismos moleculares responsables del fenómeno de inmunosupresión, entre los mecanismos citados se ha demostrado presencia de células supresoras [5-9], factores inmunosupresores presentes en el parásito [2, 3, 10-13], producción excesiva de óxido nítrico [14], disminuida producción de IL-2 y reducida expresión del receptor de IL2 en células de bazo de animales infectados [9, 15-17]. Muchos de estos mecanismos han sido exhaustivamente investigados, sin embargo no está del todo claro si existen mecanismos adicionales involucrados en la inmunosupresión de la célula T. Adicionalmente, en los últimos años nuevas moléculas que median la regulación negativa de la célula T, entre las cuales están PD-1/PD1-L [18], arginasa [19] y E3 ubiquitina ligasas [20-22], han sido reportadas durante inmunosupresión en diversas infecciones. Trypanosoma cruzi, the etiological agent of Chagas’ disease, is parasite causing chronic infections in human and other mammalian species. There is an important immunosupresion during the acute phase of the infection that contribute to the dissemination and installation of the parasite. Several studies have been focused on identifying the mechanisms involved in the immunosupresion; however it is not clear if there are additional mechanisms implicated. In addition, during the last years new molecules involved in the negative T cell regulation such as PD-1/PD1-L pathway and E3 ubiquitin ligases (E3-Ub-Lig) have been reported. It has been demonstrated, that E3-Ub-Lig control the amount and localization of intracellular signal mediators, limiting T cell activation. Moreover, these mechanisms mediate the immunosupresion observed during several infections leading to the persistence of the pathogen in the host. In this project the role of E3-Ub-Lig on the T cell immunosupresion and hipo-response mechanisms observed during T. cruzi infection will be studied. On the other hand, it has been reported that some pathogens release proteins with E3-Ub-Lig activity modifying the ubiquitination process to promote their survival and replication in the host. Recently, a protein with E3-Ub-Lig activity was identified in T. cruzi, however its target molecule has not been discovered yet. Therefore, one of the aims of this project consists on studying different potential target molecules for this novel E3-Ub-Lig. In addition, during the last years, important progress has been done about the biological rol of PD-1/PD1-L pathway on the regulation of the immune response in several infections. However, it is not well known how PD-1/PD1-L pathway transduces signals at intracelular level to block T cell response. Because of this, it is interesting to study if there is any relation between the PD-1/PD1-L pathway and E3-Ub-Lig on the mechanism of T cell immunosupression during T. cruzi infection.

Participación de las células supresoras mieloides (CSM) y T regulatorias (Treg) en el control de la infección con Trypanosoma cruzi y el desarrollo de la patología inflamatoria asociada a la infeccion. Role of Myeloid-Derived Suppressor Cells and regulatory T cells in the control of T. cruzi infection and the infection-associated pathology.

La infección de mamíferos con el T. cruzi resulta en diferentes alteraciones inmunológicas que permiten la persistencia crónica del parásito y destrucción inflamatoria progresiva del tejido cardiaco, nervioso y hepático. Los mecanismos responsables de la patología de la enfermedad de Chagas han sido materia de intensa investigación habiéndose propuesto que el daño producido en esta enfermedad puede ser consecuencia de la respuesta inflamatoria del individuo infectado y/o de una acción directa del parásito sobre los tejidos del hospedador. El propósito del presente proyecto es estudiar comparativamente, en dos cepas de ratones con diferente susceptibilidad a la infección y desarrollo de patología, la participación y los mecanismos efectores de las células supresoras mieloides (CSM) y las celulas T regulatorias inducidas por la infección experimental con Trypanosoma cruzi en el control de la infección con este protozoario y en el desarrollo de la patología hepática siendo los objetivos especificos desarrolar: - Investigar la generación y/o reclutamiento de células de CSM en bazo e hígado de ratones infectados con Trypanosoma cruzi y su contribución a la desigual susceptibilidad a la infección y respuesta inmune desarrollada en las cepas de ratones BALB/c y C57BL/6; - Investigar la capacidad de las CSM inducidas por la infección con T. cruzi en bazo e hígado de ratones de ambas cepas para suprimir la respuesta de células T in vitro e indagar sobre los mecanismos de supresión utilizados; - Investigar la generación y/o reclutamiento de células Treg durante la infección experimental con Trypanosoma cruzi, su participación en la desigual susceptibilidad a la infección y respuesta inmune desarrollada en ambas cepas de ratones y los mecanismos de supresión utilizados. - Analizar en tejido hepático o leucocitos infiltrantes la presencia de COX2, PGE2, MMP2 y 9, IL1b, IL6, IDO, IL10 y GM-CSF capaces de inducir la expansión de las CSM; - Dilucidar si la administración del ligando para TLR2 (Pam3CyS) previo a la infección de ratones C57BL/6 (en los cuales se detecta un menor número de CSM) es capaz de modular la respuesta inflamatoria y el daño hepático a través de la inducción de CSM y/o T reg en hígado y bazo. La comprension de los eventos celulares y moleculares que regulan la producción de citoquinas pro- y anti-inflamatorias y otros mediadores, así como el papel de los receptores de la inmunidad innata durante la infección con T. cruzi contribuirá a responder interrogantes que son claves para el diseño de nuevas estrategias de intervención inmune tendientes a preservar los mecanismos de defensa del huésped. Two nonexclusive mechanisms have been proposed to explain the Chagas’s disease pathology: 1) The pathology of the disease seems to be consequence of the inflammatory response triggered for the parasite; or 2) The damage is produced by the parasite direct effect. Recently, we reported that TLR2, TLR4 and TLR9 (innate immune response receptors) are differentially modulated in injured livers from BALB/c (lesser liver pathology) and C57BL/6 (elevated liver pathology) mice during Trypanosoma cruzi infection. The aim of our proposal is the study of role of Myeloid-Derived Suppressor Cells (MDSC) and regulatory T cells in the control of T. cruzi infection and the infection-associated pathology. Our specific aims are: -To study the induction or recruitment of MDSC in splenn and liver of BALB/c and C57BL/6 mice and their relationship with the differential susceptibility and immune response observed in these both mice strains; - To determine the ability and the mechanisms used by the T. cruzi-induced MDSC to suppress the T cell proliferative response; -To study the induction or recruitment of Treg in liver of BALB/c and C57BL/6 mice and their relationship with the differential susceptibility and immune response observed in these both mice strains; -To analize in liver tissue or tissue infiltrating lymphocytes the activation of COX2, PGE2, MMP2 y 9, IL1b, IL6, IDO, IL10 y GM-CSF known to promote the development of MDSC; -To determine whether the treatment with Pam3CyS (TLR2 ligand) is able to modulate the liver inflammatory respose and damage througth the induction of MDSC or Treg.

CD95-induced apoptosis in health and disease dimers at the DISC and the effect of c-FLIP R on L. Monocytogenes infection

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013

Mathematical models of influenza A virus infection : from intracellular replication to virus growth in cell populations

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2015

Neuronal alterations in chronic cerebral Toxoplasma gondii infection

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Infection by microorganisms on soybean seeds obtained from plants treated with growth regulators

This research, deals with the effects of exogenous growth regulators on infection by microorganisms on soybean (Glycine max cv. Davis) seeds. To study the influence of the chemicals, soybean plants were sprayed with gibberellic acid (GA) 100 ppm, (2-chloroethyl) trimethylammonium chloride (CCC) 2,000 ppm, succinic acid-2,2-dimethy1hydrazide (SADH) 4,000 ppm, indolylacetic acid (IAA) 100 ppm, 2,3,5-triiodobenzoic acid (TIBA) 20 ppm (three applications), and Agrostemin (1g/10 ml/ 3 1). Application of growth regulators did not affect infect ion by microorganisms on soybean seeds. The prominent fungus isolated was Phomopsis sojae. Alternaria and Fusarium spp. were isolated from seeds. The presence of a bacterium on the seeds was observed. The delay in harvest and high humidity increased the number of seeds from which Phomopsis was recovered.

Investigações sobre doenças de Psittacideos

The present paper colligates the notions acquired in previous investigations, already published, and new observations upon diseases of the psittacidae, liable to be confused with psittacosis of parrots. The author calls attention to the indifference with regard to this question shown by investigators, even by those who dealt with the study of this disease on the occasion of the latest outbreak of psittacosis, in flagrant contrast with the researches upon the alterations induced by pathogenic agents of other diseases transmissible to man, when these agents pass through animals or when the latter are depositaries of the virus. This remark considerably enhances the importance of the presence paper from a hygienic and epidemiologic point of view, representing moreover a contribution to general knowledge and to veterinary medicine. The researches carried out since the appearance of the latest outbreak of psittacosis,-which occurred simultaneously with an epizooty in parrots lodged in aviary of the park of Agua Branca (Directory of Animal Industry of the State São Paulo)-led to the verification of the frequent existence in these animals of various diseases liable to be confused with psittacosis. These diseases are due to two kinds of pathogenic agents: virus and bacteria. In the first group there are to be found the diseases occasioned by the virus of human psittacosis, discovered by Western, Bedson and Simpson, and the disease me with in parrots coming from traders in S. Paulo. The infections by bacteria of the genus Salmonella and by those of other genera belong to the second group. As differential characters of the two infections due to virus, delineated on the strength of notions drawn from a detailed experimental study and from the literature on this subject, the following are given: ¹ Samples of our virus were sent, for comparison, to various investigators of psittacosis. Amongst them, Prof. M. Rivers acceded to our request; he found its nature to be different from that of the virus of psittacosis studiedby him. We are very much obliged to him for the attention he paid to this verification. Virus of psittacosis - Infectiousness: man, monkey, rabbit, mouse, hen, canary. Neurotropic affinity. Inclusions: small, protoplasmic. Exsiccation: the virus has good power of preservation. Symptoms: inactivity, drowsiness, frequent diarrhoea, oculo-nasal discharge and cough, coma. Duration: 4 to 5 days. Bodily lesions: congestion of intestines, splenomegaly. Virus of S. Paulo - Infects only psittacidae, particularly those of the genus Amazona. No localization in the nervous system. Large, nuclear. Is rapidly destroyed. Inactivity, inappetency, adynamia (drooping of the wings, indifference, leaning its beak against the bars of the cage in order not to fall down); profuse diarrhoea, of whitish stools, at times enterorrhagia; prolonged coma. 2 to 8 days. Foci of yellowish necrosis in liver, spleen and lung. At times, congestion of intestines. Characteristic features common to the two viruses.-They act in great dilutions, filter through tight candles though being partly retained, are preserved under glycerine or Bedson's solution, are stable at 55°C. heat and are destroyed by physical and chemical agents. Both virus diseases are very seldom met with in psittacidae: only once, amongst numberless sick parrots, the author met with a disease of the virus differring from that of psittacosis. This disease, greatly transmissible to man, ought to be more frequent, if it were common in parrots. On the contrary, bacteria cause diseases in these animals with great frequency, presenting variable characters, from a severe epizootic form, rapidly mortal, to ambulatory or silent forms, for the most part developing towards a cure or assuming a chronic character. Amongst the bacteria which cause the infection of this group the salmonellae predominate and amongst them the bacterium discovered by Nocard, as well as a species which in the course of this study is characterized under the name of Salmonella nocardi. The author believes that in the epizooty from which Nocard isolated his bacterium there was association of the virus-disease inducing the epizooty of that epoch in Paris with the bacterial disease, as must have happened in Argentina, where the disease was transmitted to man, and Santillan, according to Barros, isolated from the sick parrots bacteria of the genus Salmonella. The diseases of the two groups, that due to virus and that due to bacteria, are differentiated: Virus-diseases - Evolution: rapid, nearly always followed by death. Symptoms: sadness, profuse diarrhoea, of whitish stools, at times enterorrhagia, complete inappetency, adynamia, indifference, prolonged coma. Clinical forms: acute and subacute. Lesions: Foci of necrosis in liver and spleen without cellular reaction around the focus, yellow liver, multiple serositis. Presence of protoplasmic or nuclear granulations. Bacteriology: Complete lack or inconstant presence of bacteria in the organs and blood. Infectiousness of the organs and blood after filtration: positive. Bacterial diseases - Varies from one week to a month or more, not always fatal. Sadness, partial inappetency, tremblings, intensive thirst, mucous or mucosanguineous diarrhoea, lack of adynamia (reacts to stimulations and moves well at any time of the disease, though showing little disposition to locomotion), soiling of feathers. Frustrate, acute, subacute and chronic. Hepatic and intestinal cogestion, foci of necrosis in liver, spleen and lung with cellular reaction around the focus. Lack of granulations. Constant presence of bacteria in the organs and blood. Negative. The analysis of the litterature shows that the characteristic features of the diseases in parrots referred to parrot psittacosis, more frequently approach the bacterial diseases here described of these animals, a hypothesis which is reinforced by the observation of the greater frequency of infections...

Capacidade receptora de Salmonella typhi A fatores R*1

Estudando a incidência de fatores infecciosos de resistência, em amostras de Salmonella, foi verificado apresentarem as amostras de S. typhi uma fraca habilidade receptora inicial a esses fatores.

Lisotipia Vi e classificação bioquímica de amostras de Salmonella typhi isoladas no Estado da Guanabara

Na presente investigação, os autores estudaram a distribuição dos lisótipos Ví, tipos fermentativos e tetrationato-redutase, em 110 amostras de Salmonella typhi, isoladas no período de 1966-1970, no Estado da Guanabara. Para estabelecer uma melhor configuração epidemiológica dos resultados, fez-se a divisão do Estado em seis regiões, respectivamente as zonas Sul, Centro, Norte, da E. F. Central, da E. F. Leopoldina e rural. Obteve-se no cômputo geral, a prevalência dos lisótipos A (59,09%), E 1a (19,09%), T (7,27%) e amostras Vi negativas (4,55%). Verificou-se ainda, que cerca de 70,90% das culturas foram classificadas no tipo fermentativo II, segundo esquema de Kristensen e 95,42% das amostras revelaram-se positivas na prova de detecção da tetrationato-redutase.

Avaliação de diferentes meios de enriquecimento para o isolamento de Salmonella, ocorentes em água de esgoto

Examinando-se 158 amostras de águas cloacais, provenientes de uma estação de tratamento e de uma elevatória de esgotos, localizadas na cidade do Rio de Janeiro, fêz-se uma avaliação da eficiência de quatro meios de enriquecimento para Salmonella. Os meios empregados foram o caldo tetrationato, segundo Kauffmann, o meio de Rappaport e duas modificações deste meio, previamente descritas. Os resultados encontrados, evidenciaram a superioridade de um dos meios modificados da Rappaport, em que se fez a substituição do verde malaquiata por outro corante bacteriostático (Metachromgelb II RD) e com a inclusão de tetrationato, à fórmula original. A maior eficiência assinalada para este meio se baseou nos resultados obtidos do enriquecimento dos diferentes grupos sorológicos e particularmente dos quinze sorotipos mais incidentes de Salmonella enteritidis, na presente investigação.

Pneumocystis carinii: its incidence in rodents and enhancement of the infection by corticosteroids

A survey was made on the incidence of Pneumocystis carinii in 361 rodents including sewer rats, albino rats, albino mice, guinea-pigs and rabbits. P. carinii was found in 4 of the 215 Rattus norvegicus examined (1,8%). These results accord with recent observations but disagree with investigations made by the researchers who first studied this parasite in the past when high indexes of infection were found. However, in 20 albino rats treated with corticosteroids (betamethazone) we found 8 positive (40%) and in 20 albino mice treated by the same way, 9 were positive for P. carinii (45%). These results confirm the opportunistic character of P. carinii in rodents already well demonstrated in man.

Considerações sobre a freqüência de sorotipos de Salmonella na cidade do Rio de Janeiro

Durante o período de 1962 a 1971, foram identificadas 164 amostras de salmonelas, isoladas a partir de fezes de crianças e de adultos, possuidores ou não, de problemas entéricos e residentes na cidade do Rio de Janeiro. Os resultados obtidos na caracterização sorológica dessas culturas, evidenciaram uma nítida predominância de Salmonella enteritidis, pertencentes ao grupo sorológico B. Dentre os sorotipos de Salmonella enteritidis, que se destacaram pela maior frequência de isolamentos, citam-se os seguintes: Typhimurium Newport, Anatum e Thompson.

Cross-infection experiments with "isospora" of cats and dogs

Feces of 34 dogs out of 251 (13.5%) from guanabara were positive for Isospora. From these 19 (7.5%) were i. rivolta, 13 (5.2%) were I. canis and 2 (0,7) were i. bigemina. "Free-sporocysts" of I. rivolta were eliminated by 9 dogs (3.5%). A "Caryospora-like" oocyst was seen once. Cross-infection experiments performed with Isospora from dogs and cats failed to produce infection while inoculations of these Isospora in their natural hosts succeeded. The results suggest that the species of Isospora occurring in cats are different from those of dogs.

Ocorrência de Salmonella em gânglios linfáticos de suínos aparentemente normais, abatidos no matadouro de Santa Cruz, cidade do Rio de Janeiro, Guanabara

Foi realizada uma investigação sobre a presença de Salmonella em gânglios linfáticos pré-escapulares, pré-crurais e mesentéricos, de 59 suínos aparentemente normais, abatidos no Matadouro de Sanata Cruz, Rio de Janeiro, GB. De um total de 177 gãnglios examinados isolaram-se 27 amostras de salmonelas, das quais 14 (51,84%) eram de gânglios mesentéricos: 5 (18.51%) de gânglios pré-escapulares e 8 (29,62%), de gânglios pré-crurais. A identificação sorológica das 27 Salmonella isoladas, revelou a existência de 4 sorotipos diferentes de Salmonella enteritidis distribuídos em dois grupos, com dominância do grupo somático B (soro tipo Typhimurium), para os gânglios mesentéricos, e 3 sorotipos distribuídos em 3 grupos, com dominância do grupo somático El (Sorotipo Anatum), para os gânglios pré-escapulares e pré-crurais. Considerando a presença de salmonellas em gãnglios pré-escapulares e pré-crurais, em 8,4% dos suínos examinados, os autores sugerem que estes gãnglios, sejam durante a fase de evisceração, retirados das carcaças dos animais, antes das mesmas serem enviadas para o consumo oúblico a fim de diminuir a contaminação pós-morte das carnes.