Extracellular protease profile of Acanthamoeba after prolonged axenic culture and after interaction with MDCK cells.

Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.

OxLDL plasma levels in patients with Alzheimer's disease.

OBJECTIVE: The objective of this study was to characterize the conventional lipid profile, oxLDL levels and ApoE polymorphism in patients with Alzheimer's disease (AD) and in elderly individuals without cognitive impairment. METHODS: Eighty elderly individuals were selected and the levels of oxLDL were determined using the ELISA kit, and ApoE gene polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Significantly reduced levels of oxLDL were observed in patients with AD compared to the control group. A higher frequency of the ApoE ε4 allele was observed in patients with AD compared to controls. No difference was observed for total cholesterol, HDL-C, and LDL-C levels between the two groups, while triglyceride levels were higher in controls compared with patients with AD. CONCLUSION: The data analyzed together did not reveal significant differences in lipid profiles, including oxLDL levels. However, the importance of lipid changes in the genesis of the disease cannot be ruled out. Nevertheless, the ApoE ε4 allele was significantly more frequent in patients with Alzheimer's dementia in agreement with previous findings in the literature, but this genetic component did not change the levels of oxLDL.

OxLDL plasma levels in patients with Alzheimer's disease / Níveis plasmáticos de LDL-ox em pacientes com doença de Alzheimer

ABSTRACT Objective: The objective of this study was to characterize the conventional lipid profile, oxLDL levels and ApoE polymorphism in patients with Alzheimer's disease (AD) and in elderly individuals without cognitive impairment. Methods: Eighty elderly individuals were selected and the levels of oxLDL were determined using the ELISA kit, and ApoE gene polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism. Results: Significantly reduced levels of oxLDL were observed in patients with AD compared to the control group. A higher frequency of the ApoE ε4 allele was observed in patients with AD compared to controls. No difference was observed for total cholesterol, HDL-C, and LDL-C levels between the two groups, while triglyceride levels were higher in controls compared with patients with AD. Conclusion: The data analyzed together did not reveal significant differences in lipid profiles, including oxLDL levels. However, the importance of lipid changes in the genesis of the disease cannot be ruled out. Nevertheless, the ApoE ε4 allele was significantly more frequent in patients with Alzheimer's dementia in agreement with previous findings in the literature, but this genetic component did not change the levels of oxLDL.

RESUMO Objetivo: O objetivo deste estudo foi caracterizar o perfil lipídico convencional, os níveis de LDL-ox e o polimorfismo da ApoE em pacientes com doença de Alzheimer (DA) e em indivíduos idosos sem comprometimento cognitivo. Métodos: Foram selecionados oitenta indivíduos idosos. Os níveis de LDL-ox foram determinados usando o kit ELISA e a investigação do polimorfismo do gene da ApoE por PCR-RFLP. Resultados: Níveis significativamente reduzidos de LDL-ox foram observados em pacientes com DA comparado ao grupo controle. Uma maior frequência do alelo ε4 da ApoE foi observada nos pacientes com DA em relação aos controles. Nenhuma diferença foi observada para os níveis de colesterol total, HDL-C e LDL-C entre os dois grupos, enquanto níveis de triglicérides foram mais altos em controles comparados aos pacientes com DA. Conclusão: Os dados analisados em conjunto não revelaram diferenças significativas no perfil lipídico, incluindo os níveis de LDL-ox. No entanto, não se pode excluir a importância de alterações lipídicas na gênese da doença. Não obstante, o alelo ε4 da ApoE foi signicativamente mais frequente nos pacientes com demência de Alzheimer em concordância com achados prévios da literatura, mas esse componente genético não interferiu nos níveis de LDL-ox.

In vitro study of putative genomic biomarkers of nephrotoxicity through differential gene expression using gentamicin.

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs.