Sinais clínicos e patologia da intoxicação crônica experimental de caprinos por Palicourea marcgravii

Palicourea marcgravii é considerada a principal planta tóxica de interesse pecuário no Brasil, porém, até o momento, é conhecido apenas o quadro agudo da intoxicação. Esse estudo avaliou as alterações clínico-patológicas de 10 caprinos cronicamente intoxicados por P. marcgravii. Os animais receberam, diariamente, 0,2g/kg de peso corporal da planta fresca por 6 a 38 dias. Os caprinos apresentaram como principais sinais clínicos anorexia, apatia, taquicardia, arritmia, pulso venoso jugular positivo e decúbito. Nas necropsias, os achados macroscópicos foram hidropericárdio, palidez dos rins e do miocárdio, atrofia gelatinosa da gordura cardíaca, evidenciação do padrão lobular hepático e edema pulmonar. Os principais achados microscópicos foram tumefação e vacuolização de cardiomiócitos, necrose de fibras cardíacas e infiltrado inflamatório mononuclear no miocárdio. Nos rins foi encontrada marcante degeneração hidrópico-vacuolar difusa. Os achados demonstraram nos caprinos cronicamente intoxicados, quadro clínico-patológico com características distintas do observado na forma aguda da intoxicação por P. marcgravii. Essas observações comprovam o risco para caprinos da ingestão da planta, mesmo que em pequenas doses, resultando no surgimento de quadro clínico e graves lesões ainda pouco conhecidas, e que poderiam ser confundidas com outras enfermidades.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Prevalence of diarrheogenic Escherichia coli and rotavirus among children from Botucatu, São Paulo State, Brazil

In a one-year prospective study carried out to define the role of rotavirus and Escherichia coli in local childhood diarrhea, we determined the prevalence of both agents in 54 diarrheic children attending a health center in Botucatu. Diarrheogenic E. coli (DEC) strains were characterized by O:H serotyping, a search for virulence genetic markers, and assays of adherence to HEp-2 cells. Except for enteroaggregative E. coli (EAEC), no other DEC category was detected in the children's stools. Both EAEC and rotavirus were isolated from 22 of the 54 (41.0%) diarrheic children as single agents or in combination with other enteropathogens. However, when considering the presence of a single agent, EAEC was dominant and isolated from 20.4% of the patients, whereas rotavirus was detected in 14.8%. These results indicate that rotavirus and EAEC play a significant role as agents of childhood diarrhea in the local population.

Biological activity of Serratia marcescens cytotoxin

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 µg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.

Dipyridamole increases the cytotoxicity of cisplatin in human larynx cancer cells in vitro

This paper describes the effect of dipyridamole (DIP) on the cytotoxicity of cisplatin in HEp-2 human larynx cancer cells in vitro and the nature of the interaction between cisplatin and dipyridamole. Cytotoxic assays were performed to obtain the IC50 for cisplatin. The cells were treated with 0, 20, 40, 80, 120 or 200 µM cisplatin, with or without a single concentration of DIP and incubated for 60 min at 37ºC and 5% CO2 for 3 days and then counted with a hemocytometer. The accumulation of cisplatin in the cells was measured by atomic absorption and fluorescence was used to determine the membrane binding constant of DIP. In the presence of 10, 20 and 30 µM DIP, the IC50 of cisplatin was reduced by 25, 60 and 82% in HEp-2 cells. Combination index analysis revealed that cisplatin and DIP interact synergistically. In larynx cancer cells, the accumulation of cisplatin increased by 13, 27 and 65% as the DIP concentration was increased from 10 to 20 and 30 µM, respectively. The binding constant of DIP to the cell membrane was estimated to be (0.36 ± 0.12 mg/ml)-1 (N = 2) by fluorescence and cisplatin did not suppress DIP fluorescence. These results suggest that DIP significantly enhances cisplatin cytotoxicity in HEp-2 cells by increasing cisplatin accumulation, probably by altering the cell membrane as suggested by its binding constant. The results obtained reinforce the importance of combination therapy to reduce the doses of chemotherapeutic drugs and therefore the side effects of chemotherapy.

Immunoglobulin E and systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by intense polyclonal production of autoantibodies and circulating immune complexes. Some reports have associated SLE with a Th2 immune response and allergy. In the present study 21 female patients with SLE were investigated for total IgE and IgE antibodies to dust house aeroallergens by an automated enzyme-linked fluorescent assay, and were also evaluated for antinuclear IgE autoantibodies by a modified indirect immunofluorescence test using HEp-2 cells as antigen substrate. Additionally, immunocapture ELISA was used to investigate serum anti-IgE IgG autoantibodies. Serum IgE above 150 IU/ml, ranging from 152 to 609 IU/ml (median = 394 IU IgE/ml), was observed in 7 of 21 SLE patients (33%), 5 of them presenting proteinuria, urinary cellular casts and augmented production of anti-dsDNA antibodies. While only 2 of 21 SLE patients (9.5%) were positive for IgE antibodies to aeroallergens, all 10 patients with respiratory allergy (100%) from the atopic control group (3 males and 7 females), had these immunoglobulins. SLE patients and healthy controls presented similar anti-IgE IgG autoantibody titers (X = 0.37 ± 0.20 and 0.34 ± 0.18, respectively), differing from atopic controls (0.94 ± 0.26). Antinuclear IgE autoantibodies were detected in 17 of 21 (81%) sera from SLE patients, predominating the fine speckled pattern of fluorescence, that was also observed in IgG-ANA. Concluding, SLE patients can present increased IgE levels and antinuclear IgE autoantibodies without specific clinical signs of allergy or production of antiallergen IgE antibodies, excluding a possible association between SLE and allergy.

Potential pathogenic role of aggregative- adhering Corynebacterium diphtheriae of different clonal groups in endocarditis

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

Doenca renal policistica autossomica dominante em pacientes em hemodialise no sul do Brasil

Introdução: A doença renal policística autossômica dominante é a enfermidade renal hereditária mais comum em seres humanos. Objetivo: Analisar a prevalência, características clínicas e laboratoriais de pacientes com rins policísticos e relacionar as manifestações da doença por gênero. Métodos: Trata-se de um estudo observacional e retrospectivo. Foram revisados todos os prontuários médicos de pacientes com rins policísticos admitidos para hemodiálise entre 1995 e 2012, em quatro centros que atendem a área de abrangência da 15ª regional de saúde do Paraná, Brasil. Resultados: Fizeram parte do estudo 48 pacientes com rins policísticos, causa primária da doença renal crônica (DRC) estágio 5. A prevalência da doença foi de um em 10.912 habitantes. A média de idade de ingresso na hemodiálise (50,7 anos) e o tempo de seguimento em hemodiálise até o transplante (36,5 meses) foi menor nos homens. A hipertensão arterial foi o diagnóstico mais frequente em 73% dos pacientes, com predominância em mulheres (51,4%). O cisto hepático foi a manifestação extrarrenal mais frequente nos homens (60,0%). Foram a óbito 10,4% dos pacientes que faziam uso de hemodiálise, sendo 60% de homens. A classe de droga anti-hipertensiva mais utilizada foi a que atua no sistema renina-angiotensina, com maior frequência de uso nas mulheres (53,3%). A ureia pós-diálise foi significativamente maior em homens. Conclusão: A prevalência da doença é baixa entre pacientes em hemodiálise no sul do Brasil. As diferenças observadas entre os gêneros, com exceção da ureia pós, não foram significantes. Os dados encontrados são diferentes dos reportados na América do Norte e Europa.