ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situimmune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

Effect of histamine H1 and H2 receptor antagonists, microinjected into cerebellar vermis, on emotional memory consolidation in mice

This study investigated the effects of histamine H1 or H2 receptor antagonists on emotional memory consolidation in mice submitted to the elevated plus maze (EPM). The cerebellar vermis of male mice (Swiss albino) was implanted using a cannula guide. Three days after recovery, behavioral tests were performed in the EPM on 2 consecutive days (T1 and T2). Immediately after exposure to the EPM (T1), animals received a microinjection of saline (SAL) or the H1 antagonist chlorpheniramine (CPA; 0.016, 0.052, or 0.16 nmol/0.1 µL) in Experiment 1, and SAL or the H2 antagonist ranitidine (RA; 0.57, 2.85, or 5.7 nmol/0.1 µL) in Experiment 2. Twenty-four hours later, mice were reexposed to the EPM (T2) under the same experimental conditions but they did not receive any injection. Data were analyzed using one-way ANOVA and the Duncan test. In Experiment 1, mice microinjected with SAL and with CPA entered the open arms less often (%OAE) and spent less time in the open arms (%OAT) in T2, and there was no difference among groups. The results of Experiment 2 demonstrated that the values of %OAE and %OAT in T2 were lower compared to T1 for the groups that were microinjected with SAL and 2.85 nmol/0.1 µL RA. However, when animals were microinjected with 5.7 nmol/0.1 µL RA, they did not show a reduction in %OAE and %OAT. These results demonstrate that CPA did not affect behavior at the doses used in this study, while 5.7 nmol/0.1 µL RA induced impairment of memory consolidation in the EPM.

Expressão da proteína nm23 em carcinoma de células escamosas de língua metastático e não-metastático

O carcinoma de células escamosas oral (CCEO) exibe prognóstico desfavorável em decorrência da capacidade de invasão aos tecidos vizinhos e elevada incidência de metástases. OBJETIVO: O presente trabalho objetiva analisar a expressão imunohistoquímica da proteína nm23 em CCEs de língua metastáticos e não-metastáticos. METODOLOGIA: A técnica da imunohistoquímica para a proteína nm23-h1 foi realizada em 35 casos de CCE de língua com metástase em 15 casos. Atribuiu-se escore 0, para ausência de marcação; 1, marcação focal e 2 para marcação difusa. RESULTADOS: Observou-se marcação focal para a proteína nm23 em 9 casos, difusa em 15, e ausência de marcação em 11 espécimes. O teste exato de Fischer foi aplicado, não havendo diferença estatisticamente significativa para positividade desta proteína nos casos metastáticos e não-metastáticos (p=0.365), apesar de que em 66.7% dos casos com metástase não houve marcação. CONCLUSÕES: A presença da proteína nm23 não esteve relacionada de forma positiva aos casos de CCE de língua sem metástase. Dessa forma, vários outros fatores inerentes à célula neoplásica e ao hospedeiro podem estar relacionados aos mecanismos supressores do processo metastático nesta entidade.

Reações de hipersensibilidade imediatas após uso intravenoso de soros antivenenos: valor prognóstico dos testes de sensibilidade intradérmicos

Foram admitidos no Hospital das Clínicas da FMRPUSP, durante os anos de 1983 a 1988, 494 pacientes vítimas de acidentes ofídicos e escorpiônicos que receberam soro antiveneno (SAV) e nos quais foi avaliada a freqüência e tipo das manifestações imediatas, bem como o valor prognóstico do teste de sensibilidade. Do total de pacientes vítimas de ofidismo, 82 (25,6%) apresentaram reações imediatas, das quais as mais comuns foram as cutâneas, isoladas (40%) ou associadas com sintomas respiratórios (19%), seguidas de manifestações gastrintestinais (17%). Choque anafilático foi detectado em 10 pacientes (12%). Em relação aos acidentes escorpiônicos, reações imediatas foram observadas em 13 pacientes (7,5%), também com predomínio de lesões cutâneas. Sintomatologia cardiocirculatória foi detectada em apenas 1 paciente. Devido ao baixo valor preditivo positivo (31,8%) e à baixa sensibilidade (54,6%), propomos que o teste de sensibilidade intradérmico seja abolido da rotina de atendimento de urgência aos pacientes vítimas de acidentes por animais peçonhentos. Anti-histamínicos (bloqueadores dos receptores H1 e H2) e corticosteróides devem ser administrados por via parenteral anteriormente à soroterapia, no sentido de prevenir ou minimizar as reações de hipersensibilidade imediatas que podem ocorrer. O soro antiveneno deve ser sempre administrado por via intravenosa, de preferência sem diluição, gota a gota, durante 15 a 30 minutos, sob contínua supervisão da equipe médica.

Proposal of abolition of the skin sensitivity test before equine rabies immune globulin application

An epizootic outbreak of rabies occurred in 1995 in Ribeirão Preto, SP, with 58 cases of animal rabies (54 dogs, 3 cats and 1 bat) confirmed by the Pasteur Institute of São Paulo, and one human death. The need to provide care to a large number of people for the application of equine rabies immune globulin (ERIG) prevented the execution of the skin sensitivity test (SST) and often also the execution of desensitization, procedures routinely used up to that time at the Emergency Unit of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (EU-UHFMRP-USP), a reference hospital for the application of heterologous sera. In view of our positive experience of several years with the abolition of SST and of the use of premedication before the application of antivenom sera, we used a similar schedule for ERIG application. Of the 1489 victims of animal bites, 1054 (71%) received ERIG; no patient was submitted to SST and all received intravenously anti-histamines (anti-H1 + anti-H2) and corticosteroids before the procedure. The patients were kept under observation for 60 to 180 minutes and no adverse reaction was observed. On the basis of these results, since December 1995 ERIG application has been decentralized in Ribeirão Preto and has become the responsibility of the Emergency Unit of the University Hospital and the Central Basic Health Unit, where the same routine is used. Since then, 4216 patients have received ERIG (1818 at the Basic Health Unit and 2398 at the EU-UHFMRP), with no problems. The ideal would be the routine use of human rabies immune globulin (HRIG) in public health programs, but this is problematic, because of their high cost. However, while this does not occur, the use of SST is no longer justified at the time of application of ERIG, in view of the clinical evidence of low predictive value and low sensitivity of SST involving the application of heterologous sera. It is very important to point out that a negative SST result may lead the health team to a feeling of false safety that no adverse reaction will occur, but this is not true for the anaphylactoid reactions. The decision to use premedication, which is based on knowledge about anaphylaxis and on the pharmacology of the medication used, is left to the judgment of health professionals, who should always be prepared for eventual untoward events.

Outbreak of influenza type A (H1N1) in Iporanga, São Paulo State, Brazil

From June to July 1999 an outbreak of acute respiratory illness occurred in the town of Iporanga. Out of a total of 4,837 inhabitants, 324 cases were notified to the Regional Surveillance Service. Influenza virus was isolated from 57.1% of the collected samples and 100% seroconversion to influenza A (H1N1) was obtained in 20 paired sera tested. The isolates were related to the A/Bayern/07/95 strain (H1N1). The percentages of cases notified during the outbreak were 28.4%, 29.0%, 20.7%, 6.2% and 15.7% in the age groups of 0-4, 5-9, 10-14, 15-19 and older than 20 years, respectively. The highest proportion of positives was observed among children younger than 14 years and no cases were notified in people older than 65 years, none of whom had been recently vaccinated against influenza. These findings suggest a significant vaccine protection against A/Bayern/7/95, the H1 component included in the 1997-98 influenza vaccine for elderly people. This viral strain is antigenically and genetically related to A/Beijing/262/95, the H1 component of the 1999 vaccine. Vaccines containing A/Beijing/262/95 (H1N1) stimulated post-immunization hemagglutination inhibition antibodies equivalent in frequency and titre to both A/Beijing/262/95-like and A/Bayern/7/95-like viruses. Thus, this investigation demonstrates the effectiveness of vaccination against influenza virus in the elderly.

Snakebites by Bothrops spp in children in Campinas, São Paulo, Brazil

From January, 1984 to March, 1999, 73 children under 15 y old (ages 1-14 y, median 9 y) were admitted after being bitten by snakes of the genus Bothrops. Twenty-six percent of the children were classified as mild envenoming, 50.7% as moderate envenoming and 20.6% as severe envenoming. Two patients (2.7%) showed no signs of envenoming. Most of the patients presented local manifestations, mainly edema (94.5%), pain (94.5%) ecchymosis (73.9%) and blisters (11%). Local and/or systemic bleeding was observed in 28.8% of the patients. Before antivenom (AV) administration, blood coagulation disorders were observed in 60.7% (incoagulable blood in 39.3%) of the 56 children that received AV only in our hospital. AV early reactions, most of which were considered mild, were observed in 44.6% of these cases (in 15/30 patients not pretreated and in 10/26 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). The main clinical complications observed were local infection (15.1%), compartment syndrome (4.1%), gangrene (1.4%) and acute renal failure (1.4%). No deaths were recorded. There were no significant differences with regard to severity of envenoming versus the frequency of blood coagulation disorders among the three categories of envenoming (p = 0.75) or in the frequency of patients with AV early reactions between the groups that were and were not pretreated (p = 0.55). The frequency of local infection was significantly greater in severe cases (p < 0.001). Patients admitted more than 6 h after the bite had a higher risk of developing severe envenoming (p = 0.04).

Snakebites by Crotalus durissus ssp in children in Campinas, São Paulo, Brazil

From January, 1984 to March, 1999, 31 children under 15 y old (ages 1-14 y, median 8 y) were admitted after being bitten by rattlesnakes (Crotalus durissus ssp). One patient was classified as "dry-bite", 3 as mild envenoming, 9 as moderate envenoming and 18 as severe envenoming. Most patients had neuromuscular manifestations, such as palpebral ptosis (27/31), myalgia (23/31) and weakness (20/31). Laboratory tests suggesting rhabdomyolysis included an increase in total blood creatine kinase (CK, 28/29) and lactate dehydrogenase (LDH, 25/25) levels and myoglobinuria (14/15). The main local signs and symptoms were slight edema (20/31) and erythema (19/31). Before antivenom (AV) administration, blood coagulation disorders were observed in 20/25 children that received AV only at our hospital (incoagulable blood in 17/25). AV early reactions were observed in 20 of these 25 cases (9/9 patients not pretreated and 11/16 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). There were no significant differences in the frequency of patients with AV early reactions between the groups that were and were not pretreated (Fisher's exact test, p = 0.12). Patients admitted less than and more than 6 h after the bite showed the same risk of developing severe envenoming (Fisher's exact test, p = 1). No children of the first group (< 6 h) showed severe complications whereas 3/6 children admitted more than 6 h post-bite developed acute renal failure. Patients bitten in the legs had a higher risk of developing severe envenoming (Fisher's exact test, p = 0.04). There was a significant association between both total CK and LDH blood enzyme levels and severity (p < 0.001 for CK and p < 0.001 for LDH; Mann-Whitney U test). No deaths were recorded.

Diversity of Trypanosoma cruzi stocks and clones derived from Chagas disease patients: I-Behavioral characterization in vitro

In this study, we isolated Trypanosoma cruzi from chronic Chagas heart disease and from megaesophagus patients. The parasite stock hSLU239 (heart disease) yielded clones h1 and h2, whereas stock mSLU142 (megaesophagus) yielded clones m1, m2, m3 and m4. The parasite growth kinetics, doubling time and differentiation in axenic liquid medium showed broad behavioral diversity. It was shown that a particular pattern of behavior for a parental stock could not necessarily be assigned for subsequent clones. This study indicates that i) each Chagas disease patient is infected with several T. cruzi populations; ii) clonal lines derived from patient samples may have different biological characteristics from the original isolate; and that iii) additional behavioral and/or molecular markers are required for further characterization of Trypanosoma cruzi stocks and clones derived from Chagas disease patients in order to identify correlations with pathology.

Variabilidade diária da precipitação em regiões de floresta e pastagem na Amazônia

As estações automáticas do Projeto ABRACOS instaladas na Amazônia Ocidental forneceram os dados horários de precipitação em áreas de pastagem e floresta, durante o trimestre mais chuvoso (j/f/m) no período de 1992 a 1995. A variabilidade diária de precipitação maior que 1 mm h1 mostrou uma frequência relativa de 6,7% dos casos às 14 horas local (hl) na pastagem e 5,7% às 16 hl na floresta. O período da tarde (13-18 hl) registra acima de 30% dos eventos de precipitação em ambos os sítios. Há uma boa regularidade na distribuição horária dos eventos, principalmente na floresta. Para as precipitações maiores que 10 mm h1, existe uma concentração dos casos no final da tarde. Na pastagem 12,3%, e na floresta 11,5% dos eventos ocorrem às 18 hl. O intervalo entre 7-18 hl detém 64,9% dos casos na pastagem, e 57,7% na floresta. Do total de eventos de precipitação, 14% são maiores que 10 mm h1, tanto na floresta como na pastagem. Comparando-se o total de chuva ocorrido nas áreas de floresta e pastagem, o volume precipitado na floresta é cerca de 28% em média superior ao da pastagem.

Ablação por cateter do flutter atrial. Caracterização eletrofisiológica da interrupção da condução pelos istmos posterior e septal

OBJETIVO: Avaliar os tipos de bloqueio obtidos nos istmos posterior (entre o anel tricuspídeo e veia cava inferior) e septal (entre o anel tricuspídeo e óstio do seio coronário), após ablação do flutter atrial (FLA). MÉTODOS: Foram submetidos à ablação por radiofreqüência (RF) 14 pacientes com FLA tipo I (9 homens) em 16 procedimentos. A ativação atrial ao redor do anel tricuspídeo foi avaliada em ritmo sinusal utilizando-se cateter "Halo" com 10 pares de eletrodos (H1-2 a H19-20), durante estimulação do seio coronário proximal (SCP) e região póstero-lateral do átrio direito (H1-2), antes e após ablações lineares. De acordo com a frente de programação do impulso definiu-se: ausência de bloqueio (condução bidirecional), bloqueio incompleto (condução bidirecional com retardo num dos sentidos) e bloqueio completo (ausência de condução pelo istmo). O intervalo desta ativação (deltaSCP/H1-2) foi analisado. RESULTADOS: Bloqueio completo foi obtido em 7 procedimentos (44%) e incompleto em 4 (25%). O deltaSCP/H1-2 foi de 74 ± 26ms no primeiro grupo e de 30,5 ± 7,5ms no segundo (p<0,05). Em 5 casos não demonstrou-se bloqueio. Num seguimento médio de 12 meses, ocorreu recorrência do FLA nos 6 pacientes com bloqueio incompleto ou ausente e em nenhum com bloqueio completo (p<0,001). CONCLUSÃO: A verificação de bloqueio completo pelo mapeamento multipolar atrial é uma estratégia eficaz para definir o sucesso e recorrência clínica na ablação do FLA tipo I.

Caracterização funcional da hipertrofia miocárdica induzida pelo isoproterenol e de sua regressão

OBJETIVO: Analisar as disfunções da hipertrofia miocárdica induzida pelo isoproterenol e de sua regressão. Corações isolados hipertrofiados por isoproterenol (ISO) (8 dias) e após 22 dias de sua suspensão (regressão) foram distendidos. MÉTODOS: Até pressão de repouso (Pr) de 60mmHg, analisaram-se: pressão desenvolvida máxima (PDmáx.); estresse sistólico (sigmamáx); inclinação da reta estresses/deformações; constante de relaxamento; rigidez da câmara e rigidez miocárdica. RESULTADOS: Nos corações hipertrofiados (H) as variações de volume (deltaV) necessárias para Pr=60mmHg foram heterogêneas. Em alguns (H1; n=10) deltaV equivaleu à dos controle (C) enquanto em outros (H2; n=10) foi inferior, e também diferiram quanto ao peso seco, complacência ventricular, rigidez miocárdica, constante de relaxamento,e sigmamáx. PDmáx dos grupos H1 e H2 foram superiores às de C (n=8) e Regressão (R) (n=8). Contudo, sigmamáx de H2 foi menor que C, H1 e R. O mecanismo de Frank-Starling foi deprimido nos corações hipertrofiados. A constante de relaxamento de H2 indicou retardo no decaimento da pressão associado a menor complacência ventricular e rigidez miocárdica acentuada. CONCLUSÃO: Hipertrofia miocárdica induzida pelo ISO não é homogênea. Alguns corações têm alterações pouco expressivas; outros têm comprometimento das funções sistólica e diastólica. A hipertrofia miocárdica reduz a capacidade de gerar força e aprimora a capacidade em variar pressão por aumento da relação massa/volume. Há, também, comprometimento da complacência ventricular e da rigidez muscular.

Resposta cronotrópica ao teste cardiopulmonar após o uso de cimetidina

OBJETIVO: Testar a hipótese, por meio de ensaio clínico randomizado, de que a administração de cimetidina altera a resposta cronotrópica ao exercício. MÉTODOS: Foram selecionados 24 indivíduos saudáveis, com idade entre 20 e 68 anos, não-atletas, os quais concordaram em ser submetidos a testes cardiopulmonares, após uso de placebo e de cimetidina, 400 mg, duas vezes ao dia, durante uma semana. Os testes foram realizados em esteira rolante, com protocolo de rampa com análises diretas dos gases expirados. Foi avaliada freqüência cardíaca máxima atingida, além da freqüência cardíaca de repouso e do limiar anaeróbico. RESULTADOS: Os indivíduos estudados foram igualmente distribuídos por sexo, com idade média (± desvio padrão) de 43 ±11 anos. Os exames com placebo e com cimetidina tiveram igual duração (578±90 seg vs 603±131 seg) e igual VO2 pico (35±8 ml/kg.min vs 35±8 ml/kg.min). A administração de cimetidina não apresentou efeito significativo na freqüência cardíaca de repouso (75±10 vs 74±8 bpm), no pico do esforço (176±12 vs 176±11 bpm) e, da mesma forma, também não houve diferença entre as variações das freqüências cardíacas (pico - repouso), nos dois estudos (101±14 vs 101±13 bpm). CONCLUSÃO: A administração de cimetidina por sete dias não altera a resposta cronotrópica ao exercício.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Evaluation of the natural killer cytotoxicity and the levels of cytokines in rats with type I diabetes mellitus

Type I diabetes mellitus (insulin-dependent DM = IDDM) is a chronic disease characterized by specific destruction of pancreatic beta cells, resulting in an absolute lack of insulin. Immune mechanisms, genetic susceptibility, and environmental factors are all implicated in the pathogenesis of Type 1 diabetes. This study was aimed at determining the efficiency of cytokines, natural killer (NK) cells in the pathophysiology of IDDM. Therefore, we evaluated the plasma levels of cytokines by specific enzyme-linked immunosorbent assay (ELISA) and the cytotoxicity activity of NK cells by anti-candididal index in rats with type I diabetes. We found that the cytotoxicity activity of NK cells in IDDM groups significantly decreased compared to the control groups. The levels of interferon-g (IFN-g) in IDDM groups were slightly higher than in healthy controls. These results indicate that the changes of T H1 type cytokines such as IFN-g and NK cell activity can play a role in the etiology of IDDM. The data may provide new strategies for the treatment of IDDM.