Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos

RESUMO:A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação à proteína CAV-2 e que as proteínas CAV-1 e -2 são parte da composição das cavéolas, sendo estruturas importantes e relacionadas com a transferência de moléculas para o feto, realizando a nutrição do mesmo mediante endocitose e pinocitose.

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001

Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 ± 5.85% (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 vs 91.2 ± 0.9%, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 µM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

Immunoexpression of cyclooxygenase-1 and -2 in ulcerative colitis

Ulcerative colitis (UC) is a disease of the colon and rectum characterized by a nonspecific chronic inflammation mediated by the concerted response of cellular and humoral events. Prostaglandins are synthesized by cyclooxygenase (COX)-1 and -2 and exhibit both pro- and anti-inflammatory activity. To evaluate COX-1 and COX-2 immunoexpression in 42 cases of UC and to correlate it with clinicopathological parameters, COX-1 and COX-2 expression was investigated by the immunohistochemistry method. Only patients with all pertinent clinical and evolutive data as well as with adequate biopsy material were included in the study. Fifteen samples of colorectal adenocarcinoma and 14 of large bowel with no histological changes were used for positive and negative controls, respectively. UC patients showed COX-1 immunoreactivity in epithelial cells in 29% of the cases and in inflammatory cells in 43%. COX-2 positivity in epithelial and inflammatory cells was found in 69% of the samples. The comparison between UC and the control groups revealed that the UC group had significantly more positive cases for COX-1 and COX-2 in inflammatory cells. Immunohistochemistry allowed the identification of COX-1 and COX-2 expression in epithelial and inflammatory cells in UC biopsies. No significant difference between COX-1 and COX-2 immunoreactivity in epithelial and inflammatory cells was observed regarding the clinicopathological parameters. COX-2 presented low expression in normal colon and high expression in colorectal adenocarcinoma. COX-2 might play a role in the pathophysiologic processes of inflammatory bowel disease and the development of neoplasia. Treatment with selective COX-2 inhibitors might be an additional option for therapy.

Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

Angiotensin-converting enzymes 1 (ACE1) and 2 (ACE2) are key enzymes of the renin-angiotensin system, which act antagonistically to regulate the levels of angiotensin II (Ang II) and Ang-(1-7). Considerable data show that ACE1 acts on normal skeletal muscle functions and architecture. However, little is known about ACE1 levels in muscles with different fiber compositions. Furthermore, ACE2 levels in skeletal muscle are not known. Therefore, the purpose of this study was to characterize protein expression and ACE1 and ACE2 activities in the soleus and plantaris muscles. Eight-week-old female Wistar rats (N = 8) were killed by decapitation and the muscle tissues harvested for biochemical and molecular analyses. ACE1 and ACE2 activities were investigated by a fluorometric method using Abz-FRK(Dnp)P-OH and Mca-YVADAPK(Dnp)-OH fluorogenic substrates, respectively. ACE1 and ACE2 protein expression was analyzed by Western blot. ACE2 was expressed in the skeletal muscle of rats. There was no difference between the soleus (type I) and plantaris (type II) muscles in terms of ACE2 activity (17.35 ± 1.7 vs 15.09 ± 0.8 uF·min-1·mg-1, respectively) and protein expression. ACE1 activity was higher in the plantaris muscle than in the soleus (71.5 ± 3.9 vs 57.9 ± 1.1 uF·min-1·mg-1, respectively). Moreover, a comparative dose-response curve of protein expression was established in the soleus and plantaris muscles, which indicated higher ACE1 levels in the plantaris muscle. The present findings showed similar ACE2 levels in the soleus and plantaris muscles that might result in a similar Ang II response; however, lower ACE1 levels could attenuate Ang II production and reduce bradykinin degradation in the soleus muscle compared to the plantaris. These effects should enhance the aerobic capacity necessary for oxidative muscle activity.

Anticorpos fixadores de complemento para o vírus respiratório sincicial e adenovírus e inibidores da hemaglutinação para os vírus parainfluenza 1, 2 e 3 numa população infantil brasileira

Apresentaram-se os resultados obtidos na pesquisa de anticorpos fixadores de complemento para o vírus respiratório sincicial e adenovírus, assim como de anticorpos inibidores da hemaglutinação para os vírus parainfluenza dos tipos 1, 2 e 3, num grupo de 972 crianças de idade compreendida entre 3 meses e 14 anos. A técnica de colheita de sangue foi a de embebição em papel de filtro. Do total de crianças examinadas, considerando o conjunto de todas as idades, 34,6% apresentavam anticorpos para o vírus respiratório sincicial; as porcentagens com anticorpos para adenovírus, parainfluenza 1, parainfluenza 2 e parainfluenza 3, foram respectivamente 47,7%, 46,8%, 54,1% e 66,6%. Foram estudadas as distribuições dos anticorpos em função da idade, do sexo e da localização do domicílio. Em relação aos dois últimos atributos obtiveram-se os seguintes resultados: dos indivíduos do sexo masculino, 32,3% apresentavam anticorpos contra o vírus respiratório sincicial, 49,2% contra adenovírus, 60,1%, 65,1% e 78,3%, respectivamente, contra os vírus parainfluenza 1, 2 e 3; nas crianças do sexo feminino as porcentagens de positividade encontradas foram, respectivamente, 37,4%, 45,9%, 31,1%, 41,2% e 52,9%; em relação à localização do domicílio, 44,8% do total de crianças da zona rural mostraram possuir anticorpos contra o vírus respiratório sincicial, 70,1% contra adenovírus, 43,8% contra vírus parainfluenza 1 e 46,8% e 65,4% contra os vírus parainfluenza dos tipos 2 e 3; as porcentagens de positividade na zona urbana foram, respectivamente, 30,5%, 38,7%, 47,9%, 57,1% e 67,1%.

Confirming the presence of HTLV-1 infection and the absence of HTLV-2 in blood donors from Arequipa, Peru

Epidemiological studies conducted in Peru disclosed HTLV-1 to be prevalent in different ethnic groups, and found HTLV-2 in some Amazonian Indians and in men who have sex with men. No data concerning HTLV-1/2 infection in blood donors from Arequipa, a highlands region in southern Peru, is available. We searched for the presence of HTLV-1 and HTLV-2 antibodies in 2,732 serum samples obtained from blood donors from this geographic area. HTLV-1/2-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) and were confirmed by Western blot (WB). Reactive sera had their blood bags discarded from donation, and the demographic characteristics of the donors were analyzed. Thirty-five sera (1.2%) were HTLV seroreactive by ELISA, and 25 were confirmed HTLV-1-positive by WB. One serum disclosed HTLV-positivity, and the remaining nine serum samples showed indeterminate results by WB; three of which had an HTLV-1 indeterminate Gag profile. The median age of HTLV-positive individuals was 34.6 years; 27 were male and eight were female. All individuals were from southern Peru: 27 from Arequipa, five from Puno, and three from Cuzco. HTLV co-positivity with hepatitis B (five sera) and syphilis (one serum) were detected. Previous transfusion and tattooing were observed in two and one individuals, respectively. No serum was positive for HTLV/HIV co-infection. This study confirmed, for the first time, HTLV-1 infection and the absence of HTLV-2 infection in blood donors from Arequipa, Peru and suggests vertical transmission as the major route of HTLV-1 transmission and acquisition in this geographic region.

ORIGIN AND PREVALENCE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AND TYPE 2 (HTLV-2) AMONG INDIGENOUS POPULATIONS IN THE AMERICAS

Human T-lymphotropic virus type 1 (HTLV-1) is found in indigenous peoples of the Pacific Islands and the Americas, whereas type 2 (HTLV-2) is widely distributed among the indigenous peoples of the Americas, where it appears to be more prevalent than HTLV-1, and in some tribes of Central Africa. HTLV-2 is considered ancestral in the Americas and is transmitted to the general population and injection drug users from the indigenous population. In the Americas, HTLV-1 has more than one origin, being brought by immigrants in the Paleolithic period through the Bering Strait, through slave trade during the colonial period, and through Japanese immigration from the early 20th century, whereas HTLV-2 was only brought by immigrants through the Bering Strait. The endemicity of HTLV-2 among the indigenous people of Brazil makes the Brazilian Amazon the largest endemic area in the world for its occurrence. A review of HTLV-1 in all Brazilian tribes supports the African origin of HTLV-1 in Brazil. The risk of hyperendemicity in these epidemiologically closed populations and transmission to other populations reinforces the importance of public health interventions for HTLV control, including the recognition of the infection among reportable diseases and events.

HUMAN T-LYMPHOTROPIC VIRUS 1 (HTLV-1) AND HUMAN T-LYMPHOTROPIC VIRUS 2 (HTLV-2): GEOGRAPHICAL RESEARCH TRENDS AND COLLABORATION NETWORKS (1989-2012)

Publications are often used as a measure of research work success. Human T-lymphotropic virus (HTLV) type 1 and 2 are human retroviruses, which were discovered in the early 1980s, and it is estimated that 15-20 million people are infected worldwide. This article describes a bibliometric review and a coauthorship network analysis of literature on HTLV indexed in PubMed in a 24-year period. A total of 7,564 documents were retrieved, showing a decrease in the number of documents from 1996 to 2007. HTLV manuscripts were published in 1,074 journals. Japan and USA were the countries with the highest contribution in this field (61%) followed by France (8%). Production ranking changed when the number of publications was normalized by population (Dominican Republic and Japan), by gross domestic product (Guinea-Bissau and Gambia), and by gross national income per capita (Brazil and Japan). The present study has shed light on some of the defining features of scientific collaboration performed by HTLV research community, such as the existence of core researchers responsible for articulating the development of research in the area, facilitating wider collaborative relationships and the integration of new authors in the research groups.

Carga proviral do HTLV-1 e HTLV-2: um método simples através da PCR quantitativa em tempo real

Os vírus linfotrópicos de células T humanas, quando integrados ao genoma da célula hospedeira, provírus, têm como marcador de replicação seu DNA proviral. A carga proviral parece ser um importante fator no desenvolvimento de patologias associadas a estes retrovírus. Neste estudo foi desenvolvida uma metodologia para quantificação absoluta da carga proviral dos HTLV-1 e HTLV-2 através da PCR em tempo real. Cinqüenta e três amostras de doadores de sangue com teste de ELISA reagente foram submetidas à metodologia, que utilizou o sistema TaqMan® para três seqüências alvo: HTLV-1, HTLV-2 e albumina. A quantificação proviral absoluta foi determinada através da proporção relativa entre o genoma do HTLV e o genoma da célula hospedeira, levando em consideração o número de leucócitos. O método apresentado é sensível (215 cópias/mL), prático e simples para quantificação proviral, além de eficiente e adequado para confirmação e discriminação da infecção pelos tipos virais.

Absence of HTLV-1/2 infection and dermatological diseases in Manaus, State of Amazonas, Brazil

Introduction The prevalence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) infection is heterogeneous across different populations. We tested the hypothesis that HTLV-1/2 infection occurs more often in dermatological patients. Methods A total of 1,091 patients from a tropical dermatology clinic were tested for HTLV-1/2. In parallel, 6865 first-time blood donors from the same geographic area were screened for HTLV-1/2; HTLV-1/2 positive blood donors underwent dermatological examinations. Results The prevalence of HTLV-1/2 in first-time blood donors was 0.14%. No co-occurrence of HTLV-1/2 infection and dermatological conditions was observed. Conclusions Our results challenge the hypothesis that HTLV-1/2 infection occurs more often in dermatological patients.

Biology of amazonian mosquitoes. III. Esterase isozymes in Anopheles darlingi.

Six esterase isozymes were studied during the development of Anopheles darlingi by using polyacrylamide gel electrophoresis and two different substrates, a-naphthylcelate and a-naphthylpropionate. Esterases 5 and 6'were detected in all developmental stages esterases 1 and 2 were more intensively stained if larvae, while esterases 3 and 4 were better visualized in pupae and adults. Strong differences in intensity of some of the isozymes were observed during the pupal stage.Four out of the six isozymes showed variation in the electrophoretic mobility. Esterase-2 was choosed for genetic studies, because was the best stained isozyme in the gels. Two codominant alleles {Est2*S and Est2*F) code for this polymorphic system, with the Est*S frequency equal to 0.521. Phenotypic distribution is in agreement with hardy-Weinberg expectations.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

Métodos de concentração de minerais 2:1 secundários na fração argila visando sua identificação por difratometria de raios x

Além dos baixos teores normalmente encontrados na fração argila dos solos sob clima tropical e subtropical, o tamanho reduzido e a baixa cristalinidade dos minerais 2:1 secundários dificultam sua identificação por difratometria de raios X (DRX). Este estudo objetivou avaliar métodos químicos e físico de concentração de minerais 2:1 secundários na fração argila para facilitar a identificação por DRX, incluindo a natureza dos minerais quanto ao local de formação de cargas permanentes (lâmina tetraedral ou octaedral). Coletaram-se amostras de dois Cambissolos originados de argilito da Formação Guabirotuba na Bacia Sedimentar de Curitiba (PR): horizontes A, Bi, C1 (1,2 a 1,5 m), C2 (2,2 a 2,5 m), C3 (3,2 a 3,5 m) e C4 (4,2 a 4,5 m). Após remoção da matéria orgânica e dispersão da terra fina seca ao ar, a fração argila foi submetida a tratamentos sequenciais com ditionito-citratobicarbonato (DCB) (amostra desferrificada - remoção de óxidos de Fe pedogenéticos) e com soluções de NaOH a quente, em diferentes concentrações (0,5; 1,0; 1,5; 2,5; 3,5; 4,0; 4,5 e 5,0 mol L-1), para extração de gibbsita e caulinita, em diferentes graus. A fração argila desferrificada também foi submetida à separação física (centrifugação) em argila grossa (0,2 a 2 m) e fina (< 0,2 m). Foram realizados tratamentos auxiliares para identificar as espécies minerais 2:1 na fração argila: saturação com Mg e solvatação com etilenoglicol; saturação com K e secagem ao ar e aquecimento a 550 ºC; e saturação com Li (teste de Greene-Kelly). Os resultados mostraram que o método clássico de extração da caulinita, com solução de NaOH 5,0 mol L-1 a quente, não deve ser aplicado para concentração de minerais 2:1 secundários, pois também removeu grande parte desses minerais. O tratamento com DCB e com solução de NaOH 3,5 mol L-1 possibilitou, com maior eficiência, a concentração e identificação de minerais 2:1 secundários por DRX nas amostras dos horizontes A, Bi e C1. Nas amostras tomadas em maiores profundidades (horizontes C2, C3 e C4), devido aos maiores teores desses minerais e ao menor tamanho dos cristais (argila fina), a solução menos concentrada de NaOH (1,5 mol L-1) foi mais eficiente para esse propósito. No horizonte A, os minerais 2:1 concentraram-se na fração argila grossa, compatível com o maior grau de intemperismo desse horizonte. Identificou-se esmectita com hidroxi-Al entrecamadas nos horizontes mais superficiais (A e Bi) e esmectita nas amostras do horizonte C. A saturação com Li permitiu a identificação das esmectitas dioctaedrais montmorilonita e beidelita/nontronita. As adaptações ao métodopadrão (NaOH 5 mol L-1) favoreceram a concentração de minerais 2:1 secundários na fração argila dos solos; a concentração da solução de NaOH deve ser maior para horizontes com menor teor do mineral.

A ultra-sonografia do pâncreas é eficaz em diagnosticar o diabete melito tipo 1 e tipo 2?

Este trabalho foi realizado para verificar se a ultra-sonografia do pâncreas oferece dados auxiliares na classificação de diabéticos adultos dos tipos 1 e 2. O tamanho e a ecogenicidade do pâncreas foram determinados pela ultra-sonografia em 81 diabéticos, sendo 20 do tipo 1 e 61 do tipo 2 (53 obesos e oito não-obesos). Os pacientes tipo 2 obesos diferiram dos demais por apresentarem área total e diâmetro ântero-posterior do corpo do pâncreas significativamente maiores. Quanto à ecogenicidade pancreática, esta estava aumentada com maior freqüência nos diabéticos tipo 2 obesos que nos diabéticos tipo 1. Consideramos, assim, que a ultra-sonografia do pâncreas constitui metodologia auxiliar na classificação de diabéticos entre os tipo 1 e 2, sendo menos eficaz quando os últimos não são obesos.