Effects of dexamethasone on the Li-pilocarpine model of epilepsy : protection against hippocampal inflammation and astrogliosis

Background: Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy. Methods: Initially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels. Results: Basal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model. Conclusions: Changes in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present

Molecular Identification of Burkholderia Cepacia Complex and Species Distribution Among Cystic Fibrosis Patients Seen at the Reference Center in Southern Brazil

Background: Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) patients are associated with decline in lung function and reduced survival. The potential transmissibility of Bcc among CF patients has been reported, indicating that strict segregation of CF patients with Bcc is crucial.Aims: To standardize the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay in order to identify Bcc species and to establish the prevalence of Bcc species and their susceptibility profile among CF patients seen at the Hospital de Clínicas de Porto Alegre (HCPA).Methods: The classification of the clinical isolates recovered from respiratory tract specimens of CF patients as Bcc was achieved using the API-20NE® phenotypic commercial system. The identification of the Bcc species was performed using PCR-RFLP. The antimicrobial disk diffusion susceptibility testing was performed according to the CLSI (2006).Results: API-20NE® was able to identify Bcc isolates (244 specimens), such as B. cepacia, indicating that it was not able to distinguish among the Bcc species. The PCR-RFLP molecular method discriminated the eight reference Bcc species, thus validating the method for clinical isolates. Bcc prevalence determined by PCR-RFLP was 10.6% (26/244). The molecular analysis identified B. cenocepacia in 53.8% (14/26) of infected patients, B. multivorans in 15.4% (4/26), and B. vietnamiensis and B. ambifaria in 7.7% (2/26). The antibiotic resistance profile was variable among Bcc species.Conclusions: The PCR-RFLP method was validated for the identification of Bcc species. B. cenocepacia proved to be the most prevalent species among the CF patients seen at the HCPA

Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance

Variantes genômicas do complexo Burkholderia cepacia em pacientes com fibrose cística no Hospital de Clínicas de Porto Alegre

A Fibrose cística (FC) é uma condição genética que predispõe os portadores a infecções crônicas repetidas do trato respiratório. O complexo Burkholderia cepacia (CBC), formado por espécies (variantes genômicas) intimamente relacionadas, são microorganismos comumente associados a essas infecções. A variante genômica do CBC envolvida na colonização de um determinado paciente influencia diretamente na progressão da sua doença e sobrevida. A identificação fenotípica do CBC é difícil por se tratar de um bacilo Gram-negativo não fermentador e a identificação de suas espécies é ainda mais complexa devido à similaridade fenotípica que existe entre elas. Os principais objetivos do estudo foram a padronização de uma técnica molecular de PCR-RFLP ("polymerase chain reaction restriction fragment length polymorphism") para identificação do CBC e de suas espécies e o estabelecimento da prevalência dessas espécies entre os pacientes com FC atendidos no Hospital de Clínicas de Porto Alegre no período de fevereiro a dezembro de 2006. Na análise por PCR foram utilizados iniciadores específicos para o gene recA (BCR1 e BCR2). Para o RFLP foram utilizadas as enzimas de restrição HaeIII e Mnll (New England Biolabs Inc., Hitchin, England). No período do estudo foram submetidas ao laboratório amostras de 244 pacientes com FC, sendo que em amostras de 26 pacientes foram identificadas bactérias pertencentes ao CBC. Nesse período, 10.6% (26/244) dos pacientes com fibrose cística que entraram no critério de inclusão estavam colonizados pelo CBC. A análise molecular do gene recA mostrou que B. cenocepacia foi isolada de 53.8% (14) dos pacientes, B. multivorans; 15.4% (4); B. vietnaminsis 7.7% (2) e B. ambifaria 7.7% (2). Dois pacientes 7.7% (2) estiveram colonizados por B. cenocepacia IIIA and IIIB em diferentes momentos. Em dois pacientes não foi possível realizar a identificação em nível de espécie. As técnicas moleculares, como PCR-RFLP são mais eficientes para a identificação do CBC e as únicas capazes de identificar suas variantes genômicas, o que é imprescindível para o acompanhamento clínico dos pacientes portadores de fibrose cística

Variantes genômicas do complexo Burkholderia cepacia em pacientes com fibrose cística no Hospital de Clínicas de Porto Alegre

A Fibrose cística (FC) é uma condição genética que predispõe os portadores a infecções crônicas repetidas do trato respiratório. O complexo Burkholderia cepacia (CBC), formado por espécies (variantes genômicas) intimamente relacionadas, são microorganismos comumente associados a essas infecções. A variante genômica do CBC envolvida na colonização de um determinado paciente influencia diretamente na progressão da sua doença e sobrevida. A identificação fenotípica do CBC é difícil por se tratar de um bacilo Gram-negativo não fermentador e a identificação de suas espécies é ainda mais complexa devido à similaridade fenotípica que existe entre elas. Os principais objetivos do estudo foram a padronização de uma técnica molecular de PCR-RFLP ("polymerase chain reaction restriction fragment length polymorphism") para identificação do CBC e de suas espécies e o estabelecimento da prevalência dessas espécies entre os pacientes com FC atendidos no Hospital de Clínicas de Porto Alegre no período de fevereiro a dezembro de 2006. Na análise por PCR foram utilizados iniciadores específicos para o gene recA (BCR1 e BCR2). Para o RFLP foram utilizadas as enzimas de restrição HaeIII e Mnll (New England Biolabs Inc., Hitchin, England). No período do estudo foram submetidas ao laboratório amostras de 244 pacientes com FC, sendo que em amostras de 26 pacientes foram identificadas bactérias pertencentes ao CBC. Nesse período, 10.6% (26/244) dos pacientes com fibrose cística que entraram no critério de inclusão estavam colonizados pelo CBC. A análise molecular do gene recA mostrou que B. cenocepacia foi isolada de 53.8% (14) dos pacientes, B. multivorans; 15.4% (4); B. vietnaminsis 7.7% (2) e B. ambifaria 7.7% (2). Dois pacientes 7.7% (2) estiveram colonizados por B. cenocepacia IIIA and IIIB em diferentes momentos. Em dois pacientes não foi possível realizar a identificação em nível de espécie. As técnicas moleculares, como PCR-RFLP são mais eficientes para a identificação do CBC e as únicas capazes de identificar suas variantes genômicas, o que é imprescindível para o acompanhamento clínico dos pacientes portadores de fibrose cística

Effects of dexamethasone on the Li-pilocarpine model of epilepsy : protection against hippocampal inflammation and astrogliosis

Background: Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy. Methods: Initially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels. Results: Basal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model. Conclusions: Changes in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present

Molecular Identification of Burkholderia Cepacia Complex and Species Distribution Among Cystic Fibrosis Patients Seen at the Reference Center in Southern Brazil

Background: Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) patients are associated with decline in lung function and reduced survival. The potential transmissibility of Bcc among CF patients has been reported, indicating that strict segregation of CF patients with Bcc is crucial.Aims: To standardize the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay in order to identify Bcc species and to establish the prevalence of Bcc species and their susceptibility profile among CF patients seen at the Hospital de Clínicas de Porto Alegre (HCPA).Methods: The classification of the clinical isolates recovered from respiratory tract specimens of CF patients as Bcc was achieved using the API-20NE® phenotypic commercial system. The identification of the Bcc species was performed using PCR-RFLP. The antimicrobial disk diffusion susceptibility testing was performed according to the CLSI (2006).Results: API-20NE® was able to identify Bcc isolates (244 specimens), such as B. cepacia, indicating that it was not able to distinguish among the Bcc species. The PCR-RFLP molecular method discriminated the eight reference Bcc species, thus validating the method for clinical isolates. Bcc prevalence determined by PCR-RFLP was 10.6% (26/244). The molecular analysis identified B. cenocepacia in 53.8% (14/26) of infected patients, B. multivorans in 15.4% (4/26), and B. vietnamiensis and B. ambifaria in 7.7% (2/26). The antibiotic resistance profile was variable among Bcc species.Conclusions: The PCR-RFLP method was validated for the identification of Bcc species. B. cenocepacia proved to be the most prevalent species among the CF patients seen at the HCPA