Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy.

OBJECTIVE: To define the molecular basis of a multisystem phenotype with progressive musculoskeletal disease of the hands and feet, including camptodactyly, subluxation, and tendon rupture, reminiscent of Jaccoud's arthropathy. METHODS: We identified 2 families segregating an autosomal-dominant phenotype encompassing musculoskeletal disease and variable additional features, including psoriasis, dental abnormalities, cardiac valve involvement, glaucoma, and basal ganglia calcification. We measured the expression of interferon (IFN)-stimulated genes in the peripheral blood and skin, and undertook targeted Sanger sequencing of the IFIH1 gene encoding the cytosolic double-stranded RNA (dsRNA) sensor melanoma differentiation-associated protein 5 (MDA-5). We also assessed the functional consequences of IFIH1 gene variants using an in vitro IFNß reporter assay in HEK 293T cells. RESULTS: We recorded an up-regulation of type I IFN-induced gene transcripts in all 5 patients tested and identified a heterozygous gain-of-function mutation in IFIH1 in each family, resulting in different substitutions of the threonine residue at position 331 of MDA-5. Both of these variants were associated with increased IFNß expression in the absence of exogenous dsRNA ligand, consistent with constitutive activation of MDA-5. CONCLUSION: These cases highlight the significant musculoskeletal involvement that can be associated with mutations in MDA-5, and emphasize the value of testing for up-regulation of IFN signaling as a marker of the underlying molecular lesion. Our data indicate that both Singleton-Merten syndrome and neuroinflammation described in the context of MDA-5 gain-of-function constitute part of the same type I interferonopathy disease spectrum, and provide possible novel insight into the pathology of Jaccoud's arthropathy.

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells.

BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of cas-pase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.