Einfluss von ZNS-Verletzung auf die m-RNA-Expression der Protease-Aktivierenden Rezeptoren (PARs) und Indentifizierung eines Interaktionspartners für PAR-2 in Retina

Protease-activated receptor, optic nerve crush, focal ischemia, protein-protein interaction, alpha crystallin A

Cistatina C, PCR, Log TG/HDLc e Sindrome Metabolica estao Relacionados a Microalbuminuria na Hipertensao

Fundamento: Em pacientes com hipertensão arterial sistêmica, a microalbuminúria é um marcador de lesão endotelial e está associada a um risco aumentado de doença cardiovascular. Objetivo: O objetivo do presente estudo foi determinar os fatores que influenciam a ocorrência de microalbumiúria em pacientes hipertensos com creatinina sérica menor que 1,5 mg/dL. Métodos: Foram incluídos no estudo 133 pacientes brasileiros atendidos em um ambulatório multidisciplinar para hipertensos. Pacientes com creatinina sérica maior do que 1,5 mg/dL e aqueles com diabete mellitus foram excluídos do estudo. A pressão arterial sistólica e diastólica foi aferida. O índice de massa corporal (IMC) e a taxa de filtração glomerular estimada pela fórmula CKD-EPI foram calculados. Em um estudo transversal, creatinina, cistatina C, colesterol total, HDL colesterol, LDL colesterol, triglicerídeos, proteína C-reativa (PCR) e glicose foram mensurados em amostra de sangue. A microalbuminúria foi determinada na urina colhida em 24 horas. Os hipertensos foram classificados pela presença de um ou mais critérios para síndrome metabólica. Resultados: Em análise de regressão múltipla, os níveis séricos de cistatina C, PCR, o índice aterogênico log TG/HDLc e a presença de três ou mais critérios para síndrome metabólica foram positivamente correlacionados com a microalbuminuria (r2: 0,277; p < 0,05). Conclusão: Cistatina C, PCR, log TG/HDLc e presença de três ou mais critérios para síndrome metabólica, independentemente da creatinina sérica, foram associados com a microalbuminúria, um marcador precoce de lesão renal e de risco cardiovascular em pacientes com hipertensão arterial essencial.

Mitsuda's reactions: induced by BCG in the normal Rhesus ("Macacca mulatta")

The reversals of Mitsuda's reactions induced by BCG have been objected to based on the possiblem interference of other determination causes of the phenomenon: tuberculous primo-infections, communicants of unsuspected leprosy, revearsals due to other causes, such as anti-diphteric and anti-tetanic vaccination, etc. In order to study the problem, we have used Rhesus monkeys (Macaca mulatta), which were reared in isolation, in an attempt to avoid the referred to interferences. Prior to the experiments, all animals were tested and found negative to radiograph, tuberculin and lepromin tests and were then submitted to the application of BCG vaccine (from 1 to 3 days old), in different doses and by different via. At different times, after the application of BCG, they were again submitted to the radiographic, tuberculin and lepromin tests. In the tables I to IV the experiences were summarised. From the experiments, the following conclusions were reached: 1 - From 12 Rhesus that received BCG 11 showed reversals of the Mitsuda reaction (91.7%). 2 - These reverseals took place both in tests effected shortly after BCG (from 6 days to 2 months), and tests effected much later (from 7 to 12 months after BCG). 3 - Some differences were found in the results, according to the dosis and the application via of the BCG. a) - The testicular and peritonela via (0,02g) were the only that determined strong positive Mitsuda's reactions (+++). b) - By oral via, animals that received high dosis (0.6g and 1.2 g), there resulted uniform and regular reversals, even though of low intensity (+); but from those who got small doses (0.2 g.) one showed no reversals in all tests, and the other presented reversals in the 2nd and 3rd tests only, also with low positivity (+). 4) In the 2nd and 3rd Mitsuda's reactions in the same animals, positivity was always precocious (generally within 48 hours), one getting the impression that there occurs a sensibilization of the animal body by the antigen with the repetition of the tests, even though the intensity of the reaction always remains the same. This precocious reaction (Fernandez type) occurs both shortly and long time after the application of the BCG. Its precocity depends not of the antigen only because the first Mitsuda's reaction after the BCG application occurs after some time and seems not influenced by the control lepromin test effected on the Rhesus before the BCG. 5) On the control group, the animals which received a.a.f. bacilli suspensions (Mycobacterium sp.; M. avium, and M. smegmatis), did not show reverseals of the Mitsuda's reaction. Two Rhesus, however, which received dead BCG (120ºC autoclave 1 hour), one intradermically (0.006 g) and the other orally (1.2 g), did both present reversals of the Mitsuda's reaction, with weak positivity (+). In all animals of the control-group, the allergic reactions were found negative. 6) Strong local inflammatory reactions were observed in the Rhesus that had received living BCG by intradermal via, and in the one submitted to multipunctures, there occurred the formation of a large caseous abcess. 7) The allergic tuberculinic and infratuberculinic reactions appeared dissociated from the Mitsuda's reactions: sometimes they are more precocious, occurring before of the lepromin test; on other occasions they disappear, when the Mitsuda's reactions still persist; and finally, they may be absent, when the latter occur, especially after the oral application of the BCG. 8) In Rhesus which received BCG by testicular and peritonela via, in the infratuberculinic test (0.1 ml of total BCG extract), besides the classic answer, which occurs between 48 and 96 hours, one could observe a delayed answer (15 to 20 days), represented by a non-erythematous nodule, which persists for 11-14 days.

Acquired resistance of mice against S. mansoni and lung granulomatous reaction induced by BCG

Studies carried out in Sw outbred mice showed that there is no correlation between the degree of lung granulomatous reaction and the level of acquired resistance against S. mansoni infection induced by BCG.

Influência do BCG no controle da imunodeficiência humoral induzida pelo mastocitoma P-815

O BCG, em solução lipídica, injetado por via intravenosa, foi capaz de reverter a imunosupressão humoral provocada pelo mastocitoma P-815, em camundongos singênicos DBA/2, aumentando tanto o número de células formadoras de placas hemolíticas quanto os títulos de anticorpos hemaglutinantes do soro. Não foram encontradas diferenças significativas nos títulos de anticorpos hemaglutinantes da classe IgG. Nenhum efeito bloqueador pôde ser notado na progressão normal do tumor.

Regulatory RNA binding molecules : implication for diabetes pathogenesis

Le glucose est notre principale source d'énergie. Après un repas, le taux de glucose dans le sang (glycémie) augmente, ce qui entraine la sécrétion d'insuline. L'insuline est une hormone synthétisée au niveau du pancréas par des cellules dites bêta. Elle agit sur différents organes tels que les muscles, le foie ou le tissu adipeux, induisant ainsi le stockage du glucose en vue d'une utilisation future.¦Le diabète est une maladie caractérisée par un taux élevé de glucose dans le sang (hyperglycémie), résultant d'une incapacité de notre corps à utiliser ou à produire suffisamment d'insuline. A long terme, cette hyperglycémie entraîne une détérioration du système cardio-vasculaire ainsi que de nombreuses complications. On distingue principalement deux type de diabète : le diabète de type 1 et le diabète de type 2, le plus fréquent (environ 90% des cas). Bien que ces deux maladies diffèrent sur beaucoup de points, elles partagent quelques similitudes. D'une part, on décèle une diminution de la quantité de cellules bêta. Cette diminution est cependant partielle dans le cas d'un diabète de type 2, et totale dans celui d'un diabète de type 1. D'autre part, la présence dans la circulation de médiateurs de l'inflammation nommés cytokines est décelée aussi bien chez les patients de type 1 que de type 2. Les cytokines sont sécrétées lors d'une inflammation. Elles servent de moyen de communication entre les différents acteurs de l'inflammation et ont pour certaines un effet néfaste sur la survie des cellules bêta.¦L'objectif principal de ma thèse a été d'étudier en détail l'effet de petites molécules régulatrices de l'expression génique, appelées microARNs. Basé sur le fait que de nombreuses publications ont démontré que les microARNs étaient impliqués dans différentes maladies telles que le cancer, j'ai émis l'hypothèse qu'ils pouvaient également jouer un rôle important dans le développement du diabète.¦Nous avons commencé par mettre des cellules bêta en culture en présence de cytokines, imitant ainsi un environnement inflammatoire. Nous avons pu de ce fait identifier les microARNs dont les niveaux d'expression étaient modifiés. A l'aide de méthodes biochimiques, nous avons ensuite observé que la modulation de certains microARNs par les cytokines avaient des effets néfastes sur la cellule bêta : sur sa production et sa sécrétion d'insuline, ainsi que sur sa mort (apoptose). Nous avons en conséquence pu démontrer que ces petites molécules avaient un rôle important à jouer dans le dysfonctionnement des cellules bêta induit par les cytokines, aboutissant au développement du diabète.¦-¦La cellule bêta pancréatique est une cellule endocrine présente dans les îlots de Langerhans, dans le pancréas. L'insuline, une hormone sécrétée par ces cellules, joue un rôle essentiel dans la régulation de la glycémie. Le diabète se développe si le taux d'insuline relâché par les cellules bêta n'est pas suffisant pour couvrir les besoins métaboliques corporels. Le diabète de type 1, qui représente environ 5 à 10% des cas, est une maladie auto-immune qui se caractérise par une réaction inflammatoire déclenchée par notre système immunitaire envers les cellules bêta. La conséquence de cette attaque est une disparition progressive des cellules bêta. Le diabète de type 2 est, quant à lui, largement plus répandu puisqu'il représente environ 90% des cas. Des facteurs à la fois génétiques et environnementaux sont responsables d'une diminution de la sensibilité des tissus métabolisant l'insuline, ainsi que d'une réduction de la sécrétion de l'insuline par les cellules bêta, ce qui a pour conséquence le développement de la maladie. Malgré les différences entre ces deux types de diabète, ils ont pour points communs la présence d'infiltrat immunitaire et la diminution de l'état fonctionnel des cellules bêta.¦Une meilleure compréhension des mécanismes aboutissant à l'altération de la cellule bêta est primordiale, avant de pouvoir développer de nouvelles stratégies thérapeutiques capables de guérir cette maladie. Durant ma thèse, j'ai donc étudié l'implication de petites molécules d'ARN, régulatrices de l'expression génique, appelées microARNs, dans les conditions physiopathologiques qui aboutissent au développement du diabète. J'ai débuté mon étude par l'identification de microARNs dont le niveau d'expression était modifié lorsque les cellules bêta étaient exposées à des conditions favorisant à la fois le développement du diabète de type 1 (cytokines) et celui du diabète de type 2 (palmitate). Nous avons découvert qu'une modification de l'expression des miR-21, -34a et -146a était commune aux deux traitements. Ces changements d'expressions ont également été confirmés dans deux modèles animaux : les souris NOD qui développent un diabète s'apparentant au diabète de type 1 et les souris db/db qui développent plutôt un diabète de type 2. Puis, à l'aide de puces à ADN, nous avons comparé l'expression de microARNs chez des souris NOD pré-diabétiques. Nous avons alors retrouvé des changements au niveau de l'expression des mêmes microARNs mais également au niveau d'une famille de microARNs : les miR-29a, -29b et -29c. De manière artificielle, nous avons ensuite surexprimé ou inhibé en conditions physiopathologiques l'expression de tous ces microARNs et nous nous sommes intéressés à l'impact d'un tel changement sur différentes fonctions de la cellule bêta comme la synthèse et la sécrétion d'insulinè ainsi que leur survie. Nous avons ainsi pu démontrer que les miR-21, -34a, -29a, -29b, -29c avaient un effet délétère sur la sécrétion d'insuline et que la surexpression de tous ces microARNs (excepté le miR-21) favorisait la mort. Finalement, nous avons démontré que la plupart de ces microARNs étaient impliqués dans la régulation d'importantes voies de signalisation responsables de l'apoptose des cellules bêta telles que les voies de NFKB, BCL2 ou encore JNK.¦Par conséquent, nos résultats démontrent que les microARNs ont un rôle important à jouer dans le dysfonctionnement des cellules bêta lors de la mise en place du diabète.

Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods.

The technique of sentinel lymph node (SLN) dissection is a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) is emerging as a highly sensitive technique to detect micrometastases in SLNs, but its specificity has been questioned. A prospective SLN study in melanoma patients was undertaken to compare in detail immunopathological versus molecular detection methods. Sentinel lymphadenectomy was performed on 57 patients, with a total of 71 SLNs analysed. SLNs were cut in slices, which were alternatively subjected to parallel multimarker analysis by microscopy (haematoxylin and eosin and immunohistochemistry for HMB-45, S100, tyrosinase and Melan-A/MART-1) and RT-PCR (for tyrosinase and Melan-A/MART-1). Metastases were detected by both methods in 23% of the SLNs (28% of the patients). The combined use of Melan-A/MART-1 and tyrosinase amplification increased the sensitivity of PCR detection of microscopically proven micrometastases. Of the 55 immunopathologically negative SLNs, 25 were found to be positive on RT-PCR. Notably, eight of these SLNs contained naevi, all of which were positive for tyrosinase and/or Melan-A/MART-1, as detected at both mRNA and protein level. The remaining 41% of the SLNs were negative on both immunohistochemistry and RT-PCR. Analysis of a series of adjacent non-SLNs by RT-PCR confirmed the concept of orderly progression of metastasis. Clinical follow-up showed disease recurrence in 12% of the RT-PCR-positive immunopathology-negative SLNs, indicating that even an extensive immunohistochemical analysis may underestimate the presence of micrometastases. However, molecular analyses, albeit more sensitive, need to be further improved in order to attain acceptable specificity before they can be applied diagnostically.

Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses.

The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.

Structured RNAs in the ENCODE selected regions of the human genome.

Functional RNA structures play an important role both in the context of noncoding RNA transcripts as well as regulatory elements in mRNAs. Here we present a computational study to detect functional RNA structures within the ENCODE regions of the human genome. Since structural RNAs in general lack characteristic signals in primary sequence, comparative approaches evaluating evolutionary conservation of structures are most promising. We have used three recently introduced programs based on either phylogenetic-stochastic context-free grammar (EvoFold) or energy directed folding (RNAz and AlifoldZ), yielding several thousand candidate structures (corresponding to approximately 2.7% of the ENCODE regions). EvoFold has its highest sensitivity in highly conserved and relatively AU-rich regions, while RNAz favors slightly GC-rich regions, resulting in a relatively small overlap between methods. Comparison with the GENCODE annotation points to functional RNAs in all genomic contexts, with a slightly increased density in 3'-UTRs. While we estimate a significant false discovery rate of approximately 50%-70% many of the predictions can be further substantiated by additional criteria: 248 loci are predicted by both RNAz and EvoFold, and an additional 239 RNAz or EvoFold predictions are supported by the (more stringent) AlifoldZ algorithm. Five hundred seventy RNAz structure predictions fall into regions that show signs of selection pressure also on the sequence level (i.e., conserved elements). More than 700 predictions overlap with noncoding transcripts detected by oligonucleotide tiling arrays. One hundred seventy-five selected candidates were tested by RT-PCR in six tissues, and expression could be verified in 43 cases (24.6%).

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy.

Autophagy or "self eating" is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to gamma-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific target-siRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells.