Hepatocyte-like cells differentiated from methylmalonic aciduria cblB type induced pluripotent stem cells: A platform for the evaluation of pharmacochaperoning.

Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B12 increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.

Modelling the dynamics of dengue real epidemics.

In this work, we use a mathematical model for dengue transmission with the aim of analysing and comparing two dengue epidemics that occurred in Salvador, Brazil, in 1995-1996 and 2002. Using real data, we obtain the force of infection, Λ, and the basic reproductive number, R(0), for both epidemics. We also obtain the time evolution of the effective reproduction number, R(t), which results in a very suitable measure to compare the patterns of both epidemics. Based on the analysis of the behaviour of R(0) and R(t) in relation to the adult mosquito control parameter of the model, we show that the control applied only to the adult stage of the mosquito population is not sufficient to stop dengue transmission, emphasizing the importance of applying the control to the aquatic phase of the mosquito.

Domestic cats constitute a natural reservoir of human enteropathogenic Escherichia coli types.

Feces of 70 diarrhoeic and 230 non-diarrhoeic domestic cats from Sao Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non-diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae-theta (three strains), eae-kappa (n = 3), eae-alpha1 (n = 2), eae-iota (n = 2), one eae-alpha2, eae-beta1 and eae-eta each, and two were not typeable. The majority of the EPEC isolates adhered to HEp-2 cells in a localized adherence-like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.

Fatores ambientais que condicionam a infecção pelo vírus da Dengue / Environmental factors that condictionate of dengue virus infection

O dengue, atualmente, se constitui em um dos maiores problemas de saúde pública do mundo, sendo o Brasil o país que mais notificou casos nos últimos anos. A possibilidade de haver diferenças na potencialidade de cada área em produzir casos de dengue, pela sua capacidade de desenvolver populações de mosquitos, entretanto, ainda não está esclarecida. Este estudo, que tem como o bjetivo identificar fatores referentes ao individuo, ao imóvel e a área de residência para ocorrência de infecção pelo vírus do dengue em área urbana, acompanhou 1069 indíviduos, residentes em 30 áreas sentinelas, através de inquéritos sorológicos e coleta de dados das caracteristicas do ambiente, de 1998 a 2000. Verificou-se uma soroincidência média de 85% no periodo, sem diferença significativa para as diferentes áreas...

Conditioned immunosuppression of lipopolysaccharide-induced antibody response of orally immunized mice.

BALB/C mice previously conditioned in a paired regimen in which cycophosphamide was given in association with a novel taste in the drinking water (saccharin, SAC) subsequently received LPS (1mg/mouse) by oral gavage. Subgroups of mice were reexposed to SAC or water respectively, and anti-LPS antibody production measured 6 days later using an ELISA/ELISPOT assay. Our data show that the immune response to this T-independent antigen, administered orally, is amenable to conditioned immunosuppression in this model system.

Mediastinitis necrotizante descendente / Descendent necrotizing mediastinitis

Se presenta una serie de 4 enfermos con mediastinitis necrotizante descendente (MND) de origen dental operados entre 1988 y 1993. Del análisis retrospectivo de su etiología, métodos diagnósticos, tratamiento y evolución surge la importancia del diagnóstico temprano y del tratamiento oportuno y completo, destacando la importancia del abordaje por toracotomía para el tratamiento de la supuración mediastinal, pleural y frecuentemente pericárdica. La morbilidad fue alta por múltiples complicaciones: diabrosis de los vasos del cuello, obstrucción de la vía áerea, broncoaspiración, colecciones pleurales y neumotórax postoperatorios, perforación de úlcera gástrica. La muerte de los primeros 3 de estos 4 enfermos destaca la gravedad de la afección.

Conditioned immunosuppression in orally immunized mice.

Mice were given oral immunization after pretreatment with a regimen (cyclophosphamide and a novel taste in the drinking water, chocolate milk (CHM), which leads to suppression of the antibody response to intravenously administered antigens given concurrently with CHM. Following this treatment mice were reexposed to CHM and IgM and IgA antibody forming cells (AFC) were measured in spleen and Peyer's patch cells. Conditioned immunosuppression of AFC production was most marked (> fivefold) for IgA-AFC in Peyer's patch, with effects of lesser magnitude for IgM-AFC in Peyer's patch (twofold) and both IgM- and IgA-AFC in spleen. Analysis of cytokine production from stimulated Peyer's patch and splenic T cells in vitro showed significant decreased production of both IL-2 and IL-4, with the latter being the predominant cytokine produced in Peyer's patch cells of control animals.

Production of the Alzheimer's beta-amyloid peptide by C6 glioma cells.

The beta-amyloid peptide (A beta) is a 4 kDa proteolytic fragment derived from the beta-amyloid precursor protein (beta APP) which is deposited as amyloid fibrils in the brains of patients with Alzheimer's disease. beta APP processing was investigated in C6 glioma cells using several affinity-purified anti-peptide antibodies raised against different domains of the protein. Both direct immunoblot analysis of C6 glioma conditioned medium and metabolic labeling of cells followed by immunoprecipitation of extracellular medium with specific antibodies revealed that these glial cells normally produce and release a soluble 4 kDa peptide which co-migrates with synthetic A beta (1-40) and is specifically recognized by antibodies raised against N- or C-terminal domains of the beta-amyloid peptide. Our results further suggest that glial cells may prove a major source of beta-amyloid production in the nervous tissue.

Differential effects of ethanol in mouse lines genetically selected for high and low antibody production.

There is considerable evidence of a relationship between the central nervous system and the immune system, and that several drugs with primary action at the central nervous system can affect different immune responses. In an attempt to obtain a better understanding of the interaction between ethanol's effects and immune functions, we used two mouse lines selected according to their high (HGP) or low (LGP) antibody production. These animals were injected with ethanol (20% w/v, 3.6 g/kg, ip); the latency for the loss of the righting reflex (LORR) and the time for the recovery of the righting reflex (RORR) were measured. Body temperature of both lines was also recorded before the injections and at RORR. Blood samples were taken at RORR to determine blood ethanol concentration. The results showed that the HGP mice presented shorter latency for sleep, longer sleeping time, and lower blood ethanol levels than the LGP mice. These data suggest that the HGP line is more sensitive to the effects of ethanol, and provide an additional approach for studying interactions between the immune and the central nervous systems.

The cellular pathology associated with Alzheimer beta-amyloid deposits in non-demented aged individuals.

In this study, we have compared the cellular pathology associated with beta-amyloid (beta A) deposits which characterize Alzheimer's disease (AD) in demented patients with pathologically confirmed AD, with that in non-demented aged individuals. Brain sections from two severely demented AD cases, six non-demented individuals with beta A deposits, and six age-matched controls devoid of beta A deposits were double-immunostained with antibodies against beta A, and antibody markers for neurofibrillary tangles (NFT), astrocytes and microglial cells. We found that the severely demented patients displayed numerous plaques of variable morphology, most of which were associated with NFT, hypertrophied astrocytes and reactive microglial cells. In contrast, non-demented patients showed fewer plaques, few or not NFT and less astroglial and microglial reaction. The number of plaques with associated abnormal cellular elements were much lower in non-demented than in demented cases. Furthermore, classical plaques were more likely to be associated with abnormal cellular elements than diffuse plaques, which were most often devoid of any associated cellular change. These findings suggest that: (i) beta A plaques in non-demented individuals may represent an early stage of AD; (ii) beta A deposition is the first recognizable pathological abnormality of AD; and (iii) NFT, and astro- and microglial proliferation are later features, possibly secondary to the known dystrophic effects of the beta A peptide and other fragments of its precursor protein.

Biopsia pulmonar toracoscópica: alternativas / Thoracoscopic lung biopsy: alternatives

El desarrollo de aparatos de endosutura permite realizar biopsias pulmonares toracoscópicas con instrumento abierto o asistidas por video. Las técnicas descriptas aquí logran obtener piezas de un tamaño más apto para el diagnóstico de las enfermedades intersticiales que a las pequeñas biopsias transbronquiales, en condiciones seguras aún en pacientes con suficiencia respiratoria limitada

Effects of indomethacin, aspirin, and acetaminophen on ethanol diuresis in rats.

A number of studies have indicated that prostaglandin synthetase inhibitors (PGSI) can antagonize several effects of ethanol. This study examines the influence of intraperitoneal pretreatment with the PGSI indomethacin (5.0 or 10.0 mg/kg), aspirin (15.0 or 30.0 mg/kg) and acetaminophen (15.0 or 30.0 mg/kg) on the diuretic effect produced by orally administered ethanol (4.0 g/kg) in female rats. Pretreatments with indomethacin and aspirin led to an antagonism of ethanol's diuretic effect. Nevertheless, acetaminophen failed to antagonize this effect. These results are in agreement with data from the literature on PGSI versus ethanol and suggest possible involvement of prostaglandins in the diuretic effect of ethanol.

Sulfhydryl groups modulate the allosteric interaction between glycine binding sites at the inhibitory glycine receptor.

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents.

Using 'inside-out' membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or beta-alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor.

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical and pharmacological studies on Talauma ovata St. Hil. (Magnoliaceae).

Antidiabetic activity of crude extract of leaves of Talauma ovata St. Hil. (Magnoliaceae) was analysed as part of a general pharmacological screening of this plant. Chemical analysis demonstrated the presence of phytosteroids, saponins, alkaloids and tannins in the crude extract. Pharmacological studies failed to demonstrate hypoglycemic effect of this plant in normoglycemic, hyperglycemic or alloxan-diabetic rats. The low LD50 obtained for this plant strongly suggests that its consumption by the population may be hazardous.

Functional reconstitution of the glycine receptor.

The functional reconstitution of the chloride channel coupled glycine receptor is described. Glycine receptors were purified from the cholate extract of rat spinal cord membranes by affinity chromatography and incorporated into phospholipid vesicles by the addition of phosphatidylcholine and removal of detergent by gel filtration. The reconstituted vesicles showed the same polypeptide composition as the purified receptor (proteins of Mr 48,000 and 58,000). The pharmacological characteristics of the glycine receptor were also preserved in the proteoliposomes, as demonstrated by the displacement of [3H]strychnine binding by several glycinergic ligands and by photoaffinity labeling experiments. In order to observe functional responses (i.e., specific agonist-induced anion translocation), we have developed an assay based on the fluorescence quenching of an anion-sensitive entrapped probe, SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium]. Reconstituted vesicles were loaded with the fluorescent probe during a freeze-thaw-sonication cycle in the presence of added liposomes containing cholesterol. In such a reconstituted system, glycine receptor agonists are able to increase the rate of anion influx into the vesicles. The action of agonists is blocked by the simultaneous presence of strychnine or other glycine antagonists. Our results show that the purified 48,000- and 58,000-dalton polypeptides reconstituted into phospholipid vesicles can bind ligands and promote specific ion translocation in a way similar to the glycine receptor in its native environment.

Salvage of angioaccess after late thrombosis of radiocephalic fistulas for hemodialysis.

A retrospective study of 44 surgical procedures performed in 39 patients with late occlusions of radiocephalic fistulas for hemodialysis is reported. In 6 cases (13.6%) no apparent anatomical cause was found, and a simple thrombectomy was performed as a corrective procedure. Some sort of arterial and/or venous stenosis was present in the other 38 cases. In 11 cases with evidence of mild localized vascular stenosis we performed a thrombectomy and then an endoluminal angioplasty with a balloon catheter and Bakes dilators. In 26 cases, with more severe degrees of stenosis, we made a proximal new fistula in the distal portion of the forearm, bypassing the vascular stenosis, either end-to-end radiocephalic or with interposition of a short segment of PTFE graft. The best results were achieved with the creation of a proximal new fistula, which resulted in almost 50% cumulative patency rates at 2 years, with no immediate failures. We think that an attempt at angioaccess salvage should be made in every case of late thrombosis of radiocephalic fistulas.