RESUMO
Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.
Assuntos
Epidermólise Bolhosa Simples/metabolismo , Resposta ao Choque Térmico/fisiologia , Queratina-5/metabolismo , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Chaperonas Moleculares/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Transformada , Inibidores de Cisteína Proteinase , Inibidores Enzimáticos/farmacologia , Epidermólise Bolhosa Simples/patologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Temperatura Alta/efeitos adversos , Humanos , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/patologia , Leupeptinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To determine if there is evidence of inflammation in the duodenal mucosa in patients with psoriatic arthritis (PsA) and to compare the results with those in patients with psoriasis vulgaris (PsV). METHODS: Nineteen consecutive patients with PsA underwent gastroduodenoscopy, and biopsy specimens were taken from the duodenal and gastric mucosa. In addition to routine processing, the duodenal mucosal specimens were stained for CD3+, CD8+ and CD4+ T lymphocytes, tryptase-positive mast cells, and EG2-positive eosinophil granulocytes. The results were compared with those in duodenal mucosal specimens from patients with PsV and patients with irritable bowel syndrome. RESULTS: Compared with PsV patients (without antibodies against gliadin), patients with PsA had a highly significant increase in intraepithelial CD3+ and CD8+ lymphocytes and also in CD4+ lymphocytes in the lamina propria in the villi. The lymphocyte increase was not related to presence of IgA antibodies against gliadin, endomysium, or transglutaminase, or to concomitant gastritis. Patients with PsA and PsV showed a pronounced increase in mast cells and eosinophil granulocytes. CONCLUSION: The increased lymphocyte infiltration in the duodenal mucosa in PsA, but not in PsV, might indicate different pathogenetic mechanisms in these psoriasis variants.
Assuntos
Artrite Psoriásica/patologia , Duodeno/patologia , Mucosa Intestinal/patologia , Linfócitos/patologia , Psoríase/patologia , Adulto , Artrite Psoriásica/sangue , Artrite Psoriásica/etiologia , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Duodenoscopia , Proteínas Granulares de Eosinófilos/análise , Feminino , Mucosa Gástrica/patologia , Granulócitos/química , Granulócitos/patologia , Humanos , Imuno-Histoquímica , Síndrome do Intestino Irritável/sangue , Contagem de Linfócitos , Linfócitos/química , Masculino , Mastócitos/química , Mastócitos/patologia , Pessoa de Meia-Idade , Psoríase/sangue , Psoríase/etiologia , Serina Endopeptidases/análise , TriptasesRESUMO
BACKGROUND: Intradermal botulinum toxin (Btx) produces long-lasting relief of focal hyperhidrosis, but its mechanism of action is poorly understood. OBJECTIVE: To study the effect of Btx A on the size and innervation of sweat glands in patients with palmar hyperhidrosis. METHODS: Palmar skin biopsy was performed in 26 hyperhidrotic patients before scheduled Btx treatment and in 11 controls. Twelve of the patients also underwent biopsy 1 to 6 months after the Btx injections. Sweat gland morphology was investigated by light microscopy; the cross-sectional area of the secretory tubule and its lumen was measured by image analysis. Immunofluorescence (IF) with antibodies to the neural markers protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP 43), and to vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), was used to analyze the periglandular innervation. RESULTS: The gross morphology of the sweat glands was similar in patients and controls, with no significant differences in tubular and luminal areas between the groups. After Btx treatment, the tubular dimensions remained unchanged, but the lumen tended to be smaller ( P = .07). Around the glands, increased GAP 43 staining indicating sprouting was seen within 3 months after Btx treatment ( P = .016); whereas the PGP 9.5 staining was decreased in most specimens ( P = .09) indicating lack of functional nerve growth. No change in VIP or CGRP immunoreactivity was observed. CONCLUSIONS: The sweat glands appear structurally normal in hyperhidrotic patients before Btx therapy, whereas after therapy the luminal area of the gland is frequently diminished. The IF data GAP 43/PGP 9.5 suggest that Btx therapy induces long-standing functional denervation of the sweat glands, which might explain its anti-transpiratory efficacy.