RESUMEN
Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.
Asunto(s)
Células Epidérmicas , Epidermólisis Ampollosa de la Unión/terapia , Regeneración , Células Madre/citología , Células Madre/metabolismo , Transgenes/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Linaje de la Célula , Autorrenovación de las Células , Rastreo Celular , Niño , Células Clonales/citología , Células Clonales/metabolismo , Dermis/citología , Dermis/patología , Epidermis/patología , Epidermólisis Ampollosa de la Unión/genética , Epidermólisis Ampollosa de la Unión/metabolismo , Epidermólisis Ampollosa de la Unión/patología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/trasplante , Masculino , Provirus/genética , KalininaRESUMEN
BACKGROUND: Sri Lankan Russell's viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell's vipers. This study aimed to investigate evidence of myotoxicity in Russell's viper envenoming, response to antivenom and the toxins responsible for myotoxicity. METHODOLOGY AND FINDINGS: Clinical features of myotoxicity were assessed in authenticated Russell's viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman's correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 µg/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 µM, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity. CONCLUSION: The study shows that myotoxicity in Sri Lankan Russell's viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties.