In Vitro Complement-Binding on Cytoplasmic Structures in Normal Human Skin: I. Immunofluorescence Studies

Incubation of cryostat sections of normal human skin with normal human serum (NHS) at 37°C followed by fluorescein isothiocyanate labeled rabbit antihuman C3 (FITC-R/Hu-C3) yields cytoplasmic staining of various cell types including keratinocytes of the upper epidermal layers, melanocytes, fibroblasts, smooth muscle cells, and cells lining vascular structures.Deposition of C3 on the respective cytoplasmic structures is most likely due to activation of the classical complement (C) cascade on these structures since no fluorescent staining is observed when serum of patients with hereditary C4-deficiency is used instead of NHS or when incubation with NHS is performed in the presence of EDTA or EGTA in concentrations known to inhibit classical C pathway activation. Further evidence suggesting the involvement of the classical C pathway comes from the finding that incubation of cryostat skin sections with NHS followed by FITC labeled rabbit antihuman Clq (FITC-R/Hu-Clq) results in a fluorescent staining pattern remarkably similar to that seen after exposure of cryostat skin sections to NHS and FITC-R/ Hu-C3.Although formal proof is lacking, our investigations strongly indicate that binding to and activation of C components on cytoplasmic structures occur independently of the presence of circulating antibodies. This assumption is based on the finding that in 17 out of 20 NHS we were not able to detect any skin reactive antibodies by indirect immunofluorescence (IF) techniques. More conclusive evidence for a direct, antibody-independent interaction between C components and cytoplasmic structures is provided by the observation that incubation of the substrate with purified Clq followed by FITC-R/ Hu-Clq results in cytoplasmic staining of some of the skin cell populations described above.The phenomenon of C-binding adn activation on cytoplasmic structures of normal human skin cells may be a critical event in the initiation of complement mediated pathopysiological reactions of the skin

A novel screening system improves genetic correction by internal exon replacement

Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5′, 3′ or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered ‘RNA trans-splicing molecules’ (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51–intron 51–exon 52–intron 52–exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement

Inherited epidermolysis bullosa

Inherited epidermolysis bullosa (EB) encompasses a number of disorders characterized by recurrent blister formation as the result of structural fragility within the skin and selected other tissues. All types and subtypes of EB are rare; the overall incidence and prevalence of the disease within the United States is approximately 19 per one million live births and 8 per one million population, respectively. Clinical manifestations range widely, from localized blistering of the hands and feet to generalized blistering of the skin and oral cavity, and injury to many internal organs. Each EB subtype is known to arise from mutations within the genes encoding for several different proteins, each of which is intimately involved in the maintenance of keratinocyte structural stability or adhesion of the keratinocyte to the underlying dermis. EB is best diagnosed and subclassified by the collective findings obtained via detailed personal and family history, in concert with the results of immunofluorescence antigenic mapping, transmission electron microscopy, and in some cases, by DNA analysis. Optimal patient management requires a multidisciplinary approach, and revolves around the protection of susceptible tissues against trauma, use of sophisticated wound care dressings, aggressive nutritional support, and early medical or surgical interventions to correct whenever possible the extracutaneous complications. Prognosis varies considerably and is based on both EB subtype and the overall health of the patient

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days

Over-the-Counter Monocyclic Non-Steroidal Anti-Inflammatory Drugs in Environment—Sources, Risks, Biodegradation

Recently, the increased use of monocyclic non-steroidal anti-inflammatory drugs has resulted in their presence in the environment. This may have potential negative effects on living organisms. The biotransformation mechanisms of monocyclic nonsteroidal anti-inflammatory drugs in the human body and in other mammals occur by hydroxylation and conjugation with glycine or glucuronic acid. Biotransformation/biodegradation of monocyclic non-steroidal anti-inflammatory drugs in the environment may be caused by fungal or bacterial microorganisms. Salicylic acid derivatives are degraded by catechol or gentisate as intermediates which are cleaved by dioxygenases. The key intermediate of the paracetamol degradation pathways is hydroquinone. Sometimes, after hydrolysis of this drug, 4- aminophenol is formed, which is a dead-end metabolite. Ibuprofen is metabolized by hydroxylation or activation with CoA, resulting in the formation of isobutylocatechol. The aim of this work is to attempt to summarize the knowledge about environmental risk connected with the presence of over-the-counter antiinflammatory drugs, their sources and the biotransformation and/or biodegradation pathways of these drugs