Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

Conserved (CT) n·(GA) n Repeats in the Non-coding Regions at the Gpdh Locus are Binding Sites for the GAGA Factor in Drosophila melanogaster and its Sibling Species

The (CT)n.(GA)n rich sequences in the upstream and 5' intron enhancer regions of the sn-glycerol-3-phosphate dehydrogenase (Gpdh) gene in Drosophila melanogaster, its sibling and distantly related species are conserved in their position and in the number of repeats. Using in vitro DNA-footprint analyses we show that the GAGA factor binds to these multiple closely spaced and overlapping conserved (CT)n.(GA)n repeats in D. melanogaster and D. erecta.     

An Ex Vivo Study of Congenital Pigmented Nevi in Epidermal Reconstructs

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

Higher number of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> CagA EPIYA C phosphorylation sites increases the risk of gastric cancer,but not duodenal ulcer

Background  

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis.     

Informed consent and ethical re-use of African genomic data

Rapid advances in human genomic research are increasing the availability of genomic data for secondary analysis. Particularly in the case of vulnerable African populations, ethics and informed consent processes need to be transparent-both to ensure participant protection, as well as to share skills and to evolve best practice for informed consent from a shared knowledge base. An open dialogue between all stakeholders can facilitate this.     

Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.     

Herd-level risk factors associated with the presence of Phage type 21/28 E. coli O157 on Scottish cattle farms

Background  

E. coli O157 is a bacterial pathogen that is shed by cattle and can cause severe disease in humans. Phage type (PT) 21/28 is a subtype of E. coli O157 that is found across Scotland and is associated with particularly severe human morbidity.     

The influence of serum,glucose and oxygen on intervertebral disc cell growth <Emphasis Type="Italic">in vitro</Emphasis>: implications for degenerative disc disease

Introduction  

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.