Differentiation of infectious bursal disease virus (IBDV) genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis

Molecular characterization of IBDV usually relies on the analysis of segment A of the bi-segmented, double-stranded RNA genome. Although segments B of classical and very virulent IBDVs differ significantly, re-assortment of genome segments does occur, and molecular epidemiological studies demand the analysis of both segments. An RT-PCR and restriction enzyme analysis for molecular discrimination between genome segment B of classical and very virulent IBDVs is described. Tested on eight IBDV strains/isolates, the protocol successfully identified very virulent and classical IBDVs as well as a segment reassortant. This approach is a valuable tool for molecular epidemiological studies on IBDV.     

p38 Mitogen-activated protein kinase signaling regulates streptococcal M1 protein-induced neutrophil activation and lung injury

M1 serotype of Streptococcus pyogenes can cause STSS and acute lung damage. Herein, the purpose was to define the role of p38 MAPK signaling in M1 protein-induced pulmonary injury. Male C57BL/6 mice were treated with specific p38 MAPK inhibitors (SB 239063 and SKF 86002) prior to M1 protein challenge. Edema, neutrophil infiltration, and CXC chemokines were determined in the lung, 4 h after M1 protein administration. Flow cytometry was used to determine Mac-1 expression. Phosphorylation and activity of p38 MAPK were determined by immunoprecipitation and Western blot. IVM was used to analyze leukocyte-endothelium interactions in the pulmonary microcirculation. M1 protein challenge increased phosphorylation and activity of p38 MAPK in the lung, which was inhibited by SB 239063 and SKF 86002. Inhibition of p38 MAPK activity decreased M1 protein-induced infiltration of neutrophils, edema, and CXC chemokine formation in the lung, as well as Mac-1 up-regulation on neutrophils. IVM showed that p38 MAPK inhibition reduced leukocyte rolling and adhesion in the pulmonary microvasculature of M1 protein-treated mice. Our results indicate that p38 MAPK signaling regulates neutrophil infiltration in acute lung injury induced by streptococcal M1 protein. Moreover, p38 MAPK activity controls CXC chemokine formation in the lung, as well as neutrophil expression of Mac-1 and recruitment in the pulmonary microvasculature. In conclusion, these findings suggest that targeting the p38 MAPK signaling pathway may open new opportunities to protect against lung injury in streptococcal infections.     

Suppression of c-Fos Protein and mRNA Expression in Pentylenetetrazole-Induced Kindled Mouse Brain by Isoxylitones

An early immediate gene c-fos has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes occurring during kindling. We have evaluated the effects of novel anticonvulsant isomeric compounds isoxylitones [(E/Z)-2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine] on the c-Fos protein and mRNA expression in the brain samples of kindled mice and compared it with the normal and untreated kindled groups. Kindling was induced in male NMRI mice by repeated administration of sub-convulsive dose (50 mg/kg) of pentylenetetrazole (PTZ) until a seizure score of 4-5 was achieved. The c-Fos expression was quantified by combination of immunohistochemistry and RT-PCR protocols. Both the immunohistochemical and RT-PCR analysis revealed a marked increase in the expression of c-fos mRNA and protein in the brain regions tested in case of PTZ-kindled control group compared to normal control. In contrast, the isoxylitone (30 mg/kg)-treated group demonstrated significant reduction of c-Fos expression compared to PTZ-kindled control animals. However, low expression of c-fos mRNA was only detected in the thalamus of the isoxylitone-treated brain samples. Based on these observations, we suggest that isoxylitones may have the capacity to control the seizure pattern by mechanism such as the suppression of c-Fos protein and mRNA levels in different regions of the brain. Further investigations to explore the mechanism of action of these compounds are under process.     

Mitochondrial disease and epilepsy

Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven.