The present study assessed whether advances in sleep times and circadian phase in older adults might be due to decreased responsiveness of the aging circadian clock to light. Sixteen young (29.3 ± 5.6 yrs) and 14 older adults (67.1 ± 7.4 yrs) were exposed to 4 hours of control dim (10 lux) or bright light (3,500 lux) during the night. Phase shifts of the melatonin rhythm were assessed from the nights before and after the light exposure. Bright light delayed the melatonin midpoint in both young and older adults (p < 0.001). Phase delays for the older subjects were not significantly different from those of the young subjects for either the bright or dim light conditions. The magnitude of phase delays was correlated with both sleep offset and phase angle in the older, but not the younger subjects. The present results indicate that at light intensities commonly used in research as well as clinical practice older adults are able to phase delay to the same extent as younger subjects.
Netherton Syndrome (NS) is a rare autosomal recessive skin disorder caused by mutations in the gene encoding Serine Protease Inhibitor Kazal-type 5 (SPINK5), a protease inhibitor expressed in the stratum corneum of the epidermis. In NS patients, the inhibitory activities of SPINK5 are abolished, triggering hyperactivity of epidermal proteases which leads to skin dryness, hair shaft defects, elevated IgE levels, chronic skin inflammation and scaling that predisposes the patient to life-threatening infections. Currently, there are no curative treatments for NS, and novel targeted therapeutics are necessitated. To this end, we have developed KB104, a replication-defective HSV-1 gene therapy vector encoding human SPINK5 for direct topical application to the skin. Here we show that KB104 efficiently transduces target skin cells in vitro and in vivo and produces functional SPINK5. Human keratinocytes infected with KB104 at varying multiplicities of infection produced and secreted full-length SPINK5 in a dose-dependent manner, as determined by real time PCR, immunocytochemistry, ELISA, and western blotting. Importantly, the KB104-expressed SPINK5 was functional, being capable of inhibiting a native target of SPINK5, the serine protease Kallikrein 5, in an enzymatic inhibition assay. KB104 also efficiently expressed human SPINK5 after either topical or intradermal administration in immunocompetent mice, as assessed by real time PCR and immunohistochemistry. Human SPINK5 co-localized with mouse filaggrin, a protein expressed in the stratum corneum, indicating that KB104 successfully transduced the targeted epidermal layer. Moreover, no inflammatory infiltration or structural changes were observed at the KB104-treated site, confirming that KB104 is well-tolerated. Taken together, these preclinical studies support the potential for KB104 to be a convenient, safe, and efficacious gene therapy vector for direct molecular correction of SPINK5 deficiency in Netherton Syndrome.
Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by severe epidermal blistering and mucous membrane erosions. PV is caused by antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), and in cases involving the skin, both Dsg3 and Dsg1. PV can be assessed clinically using the Pemphigus Disease Area Index (PDAI) and using an ELISA for Dsg IgG titers in patient sera. In vitro, PV IgG activity can be investigated by incubating normal human keratinocytes with PV patient IgG and monitoring changes in desmosomal protein organization and overall strength of cell-cell adhesion using a dispase-based cell-cell dissociation assay. However, the relationship between clinical PV assessments and in vitro activity of patient IgG have not been reported. In the present study, we compared the relationship between the in vitro pathogenicity of PV IgG from 23 patients to their PDAI score and ELISA titers. Overall, Dsg1 ELISA values showed a stronger correlation with PDAI scores (p<0.01) when compared to Dsg3 ELISA (p<0.02). However, the sum of Dsg3 and Dsg1 ELISA values exhibited a highly significant correlation to PDAI (p<0.0005). In vitro, treatment of keratinocytes with patient IgG resulted in a range of alterations in desmosomal protein organization that included Dsg3 clustering, the formation of linear arraylike structures, and endocytosis of Dsg3-PV IgG complexes. Interestingly, the loss of cell-cell adhesion strength as assessed using in vitro dispase cell dissociation assays exhibited a positive correlation with Dsg3 ELISA titers (p<0.005), supporting the validity of the dispase assay as a measure of PV pathogenicity . These findings confirm the association of Dsg ELISA values with PDAI and establish a relationship between Dsg IgG titers in patients and loss of adhesion in cultured keratinocytes.
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