Longitudinal muscle shows abnormal relaxation responses to nitric oxide and contains altered levels of NOS1 and elastin in uncomplicated diverticular disease.

OBJECTIVE: Recent evidence challenges the 'low-fibre/high-colonic intraluminal pressure' hypothesis of diverticular disease (DD) and raises the possibility that other mechanisms are involved. Although bowel wall smooth muscle is known to be hypercontractile in DD, the nature of its relaxation is unknown. The present study investigated colonic smooth muscle responses to nitric oxide, as well as the smooth muscle contents of neural nitric oxide and elastin associated with the disease. METHOD: Immunohistochemical/image analysis of antibodies to nitric oxide synthase (NOS1), co-localized with protein gene product (PGP) and to elastin, was performed on three histological sections of sigmoid colons from 20 patients (10 DD, 10 controls) following resections for rectal tumours. Organ bath experiments examined smooth muscle responsiveness to nitroprusside, a nitric oxide donor. RESULTS: Uncomplicated diverticular longitudinal muscle showed lower nitric oxide immunoreactivity compared with controls: median percentage surface area of NOS1 over PGP was 26.0% (range 0.5-58.3), controls 45.0% (35.0-70.1; P = 0.018). Median percentage surface area of elastin was elevated, 21.3% (10.6-45.6), controls 8.2% (1.7-13.5; P = 0.0002), together with a low sensitivity to nitroprusside [mean - log EC(50) 5.3 (SD 0.5), controls 6.6 (SD 0.5), difference 1.3, 95% CI 0.8-1.7; P < 0.0001] and there were lower maximum relaxation responses to nitroprusside compared with controls: median percentage (relaxation induced by nitroprussside/contraction induced by bethanecol) was 52.0%, range (20.0-92.0), controls 100.0% (71.0-125.0), P < 0.0001. No statistically significant differences were found in circular muscle, at the sample size studied. CONCLUSION: This study established, for the first time, specific abnormalities in longitudinal muscle relaxation and contents of neural nitric oxide and elastin in uncomplicated DD. These findings may have important implications for both colon structure and function in the disease.

Polypyrrole-based conducting polymers and interactions with biological tissues.

Polypyrrole (PPy) is a conjugated polymer that displays particular electronic properties including conductivity. In biomedical applications, it is usually electrochemically generated with the incorporation of any anionic species including also negatively charged biological macromolecules such as proteins and polysaccharides to give composite materials. In biomedical research, it has mainly been assessed for its role as a reporting interface in biosensors. However, there is an increasing literature on the application of PPy as a potentially electrically addressable tissue/cell support substrate. Here, we review studies that have considered such PPy based conducting polymers in direct contact with biological tissues and conclude that due to its versatile functional properties, it could contribute to a new generation of biomaterials.

A study to evaluate primary dressings for the application of cultured keratinocytes.

Despite the recent improvements in cell culture and dermal regeneration methods, tissue engineering of skin has yet to receive widespread acceptance in the management of burn injuries. The reasons for this are complex and include not only the inherent costs of (particularly) setting up and running such a system but also the continuing difficulties in achieving successful engraftment of the neoepidermis. The latter has previously been addressed in a number of ways, including improving the recipient bed and using pre-confluent delivery systems to allow earlier application of cells to that wound bed. One area that has received little attention is that of the optimal wound dressing to use with this technology; the cells are very poorly attached at early time points, and, in this context, the traditional dressing of paraffin gauze has never been formally assessed in comparison with newer materials. Using a porcine acute wound chamber model, we performed a prospective randomised trial to assess four different wound dressings with reference to the amount of epidermal cover gained and the histological quality of the regenerated skin after 3 weeks. Out of the four materials tested, polyurethane foam (Allevyn) was superior histologically (although equal in take rate with paraffin gauze), whilst polythene sheet (Opsite) and silicone sheet were substantially inferior. We conclude that the traditional dressing used with this technology should be compared with polyurethane foam in a clinical trial. In the future, novel dressings should be formally tested against traditional methods before being adopted.

In vitro characterization of an artificial dermal scaffold.

The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.

Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis.

A unique series of epidermal cell lines representing different stages of malignant transformation were spontaneously derived from a single adult immunosuppressed individual. Four keratinocyte lines (PM1-4) established from forehead skin are here compared with 4 squamous cell carcinoma (SCC) lines (MET1-4) derived respectively from a primary cutaneous tumour, two local recurrences and a distant metastasis of invasive SCC. Despite altered growth properties, the PM lines retained many features of normal keratinocytes including keratin phenotype, differentiation capacity and non-tumorigenicity in athymic mice. In contrast, from early passage, the MET lines displayed markedly reduced growth requirements, abnormal differentiation, aberrant K18 expression and tumorigenicity in athymic mice. The abnormal keratin profile of individual MET lines closely reflected the keratin phenotype of the tumour of origin. Although unusual HPV types were identified in the original tissue, there was no evidence of persistent virus within any cell line and it appears that HPV is not critical for maintenance of the immortal phenotype. The PM lines were distinctly different from invasive SCC lines and are likely to be useful for studies of mutations important early in neoplastic progression. The SCC series represent primary, recurrent and metastatic carcinoma. Availability of such a series from the same individual will facilitate genetic analysis of the malignant process.

Nerve and blood vessel growth in response to grafted dermis and cultured keratinocytes.

The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.

A hyaluronic acid membrane delivery system for cultured keratinocytes: clinical "take" rates in the porcine kerato-dermal model.

The clinical take rates of cultured keratinocyte autografts are poor on a full-thickness wound unless a dermal bed is provided. Even under these circumstances two important problems are the time delay in growing autografts and the fragility of the grafts. A laser-perforated hyaluronic acid membrane delivery system allows grafting at early confluence without requiring dispase digestion to release grafts from their culture dishes. We designed this study to investigate the influence of this membrane on clinical take rates in an established porcine kerato-dermal grafting model. The study demonstrated a significant reduction in take as a result of halving the keratinocyte seeding density onto the membrane. The take rates, however, of grafts grown on the membrane at half or full conventional seeding density and transplanted to a dermal wound bed were comparable, if not better, than those of keratinocyte sheet grafts.

The region coding for the helix termination motif and the adjacent intron 6 of the human type I hair keratin gene hHa2 contains three natural, closely spaced polymorphic sites.

Mutations in distinct sites of epidermal keratins, in particular in the helix initiation and termination regions, cause human genodermatoses due to faulty intermediate filament formation. Extension of this observation to human hereditary hair and nail diseases includes population analyses of human hair keratin genes for natural sequence variations in the corresponding sites. Here we report on a large-scale genotyping of the short helix termination region (HTR) of the human type I cortical hair keratins hHa1, a3-I, and a3-II, and the cuticular hair keratin hHa2. We describe two polymorphic loci, P1 and P2, exclusively in the cuticular hHa2 gene, both creating dimorphic protein variants. P1 is due to a C to T mutation in a CpG element leading to a threonine to methionine substitution; P2 concerns a serine codon AGT that also occurs as an asparagine coding variant AAC. A third polymorphism, P3, is linked with a C to T point mutation located at the very beginning of intron 6. The three polymorphic sites are clustered in a 39-nucleotide sequence of the hHa2 gene. Both allelic frequency calculations in individuals of different races and pedigree studies indicate that the two-allelic hHa2 variants resulting from P1 and P2 occur ubiquitously in a ratio of about 1:1 (P1) and 2:1 (P2) respectively in our survey, and are clearly inherited as Mendelian traits. A genotype carrying both mutations simultaneously on one allele could not be detected in our sampling, and there was no association of a distinct allelic hHa2 variant with the known ethnic form variations of hairs. Sequence comparisons of the HTR of hHa2 with those of other type I hair keratins including the hHa2-ortholog from chimpanzee provide evidence that the P1- and P2-linked mutations must have occurred very early in human evolution and that the two P2-associated codon variants may be the result of two independent point mutations in an ancestral AGC serine codon. These data describe natural polymorphisms in the HTR of a member of the keratin multigene family.

Epidermal differentiation and dermal changes in healing following treatment of surgical wounds with sheets of cultured allogeneic keratinocytes.

AIMS: To establish the structural changes that occur in deep surgical wounds engrafted with allogeneic sheets, their time course and inter-relation. METHODS: Deep surgical wounds following shave excision of tattoos (down to deep dermis/subcutaneous fat) were treated with sheets of sex mismatched allogeneic keratinocytes in 19 patients and then biopsied weekly until wound healing was complete. More superficial surgical wounds--that is, 20 standard skin graft donor sites, were biopsied at seven to 10 days (all healed) following application of keratinocyte allografts. All biopsy specimens were examined with a large panel of monoclonal antibodies to keratins, envelope proteins, basement membrane components, and to extracellular matrix components. RESULTS: The hyperproliferative keratin pair K6/16 was expressed in all wounds, for up to six weeks in keratinocyte grafted deep wounds, and up to six months in split thickness skin grafted wounds. CONCLUSIONS: Keratins 6 and 16 have not been detected in normal skin, although the relevant mRNA has. This raises the possibility of regulation at a post-transcriptional level allowing a rapid response to injury with cytoskeletal changes that may aid cell migration. This keratin pair offers the most sensitive marker for altered epidermis following wounding.

Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients.

Point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex, in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma. We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex, the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture-induced keratins and in conditions where filament fragility is not otherwise obvious. SV40-T antigen and HPV16 (E6--E7) immortalised keratinocyte cell lines were examined, established from control and epidermolysis bullosa simplex-affected individuals with either severe (Dowling-Meara) or mild (Weber-Cockayne) forms of the disease. In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated. However after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines, with filaments breaking into aggregates similar to those seen in skin from EBS patients. These aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes. Differences were also seen in the cells during respreading after replating: cells containing mutant keratins were slower to respread than controls and fine aggregates were seen at the cell margins in the Dowling-Meara derived cell line. Such delays in restoring the normal intermediate filament network after physiological processes involving cytoskeleton remodelling may render the cells vulnerable to cytolysis in vivo if physically challenged during this time window. The steady reduction in the mitotic index of the epidermis during the first few years of life could then explain the clinical improvement which is frequently observed in growing children.

Novel mutations in keratin 16 gene underly focal non-epidermolytic palmoplantar keratoderma (NEPPK) in two families.

Keratins K6 and K16 are expressed in suprabasal interfollicular epidermis in wound healing and other pathological conditions associated with hyperproliferation, such as psoriasis and are induced when keratinocytes are cultured in vitro. However, these keratins are also constitutively expressed in normal suprabasal mucosal and palmoplantar keratinocytes. Mutations in keratins have been reported in the basal keratin pair K5 and K14 in epidermolysis bullosa simplex and in suprabasal epidermal keratins K1, K2 and K10 in epidermolytic ichthyoses. Two families with autosomal dominant disorder of focal non epidermolytic palmoplantar keratoderma, have oral mucosal and follicular lesions in addition to the palmoplantar hyperkeratosis. Previous studies have shown linkage in these families to the type I keratin gene cluster at 17q12-q21 and this report shows that the cDNA of affected members of both families have novel heterozygous mutations in the expressed keratin 16 gene. These mutations (R10C and N8S) lie in the helix initiation motif of the 1A domain. These mutations do not appear to cause epidermolysis on light or electron microscopy, which may reflect differences in function, assembly or interaction of the 'hyperproliferative' or 'mucoregenerative' keratins from other major types of keratins. The mutations reported here are the first to describe the molecular pathology of focal non epidermolytic palmoplantar keratoderma.

Culturing skin in vitro for wound therapy.

Current tissue-culture techniques enable keratinocytes from a small piece of skin to be grown into sheets of epithelium, or cultured keratinocyte grafts, that are suitable for treating wounds. Serial subculture enables rapid expansion of a cell population, such that grafts of a total area equivalent to that of the surface of an adult can be obtained from an initial skin biopsy of approximately 2 cm2 in under one month. In this article, the methods currently used for culturing keratinocytes, the search for a fully functional replacement for the dermal elements of skin, and the prospects for clinical development of these technologies in the near future are discussed.

Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma.

Palmoplantar keratoderma of Voerner type (or epidermolytic palmoplantar keratoderma) is an autosomal dominant inherited disorder of keratinization with histologic features of epidermolytic hyperkeratosis. We studied members of two large unrelated kindreds with epidermolytic palmoplantar keratoderma, and biopsy specimens of lesional palmar skin from both families confirmed the histologic changes of epidermolytic hyperkeratosis. Whorls of abnormally aggregated keratin filaments were seen ultrastructurally to be associated with signs of cellular disintegration in spinous and granular cells. Direct sequencing of genomic DNA samples obtained from several members of each family established the substitution of a highly conserved arginine by tryptophan (R162W) in the 1A region of the alpha-helical rod domain of keratin 9. This arginine residue in a highly conserved region of keratins 1 and 10 is affected by disruptive missense point mutations in many patients with bullous ichthyosiform erythroderma. An equivalent position in the sole and palm restricted keratin 9 appears to be the mutation hot spot in epidermolytic palmoplantar keratoderma. To date, R162W is the most prevalent genetic defect reported in this genodermatosis.

Regeneration pattern of blood vessels and nerves in cultured keratinocyte grafts assessed by confocal laser scanning microscopy.

The aim of this study was to investigate the pattern of both neovascularization and reinnervation, and the relationship between the two processes, in keratodermal grafts, using confocal laser scanning microscopy, at different time points during the healing process. Keratodermal grafts were prepared in pigs by combining autologous dermis with cultured autologous keratinocytes. Immunohistochemistry was carried out on thick cryostat sections (100-150 microns), using antisera to the endothelial marker von Willebrand factor (vWf) and the pan-neuronal marker protein gene product 9.5 (PGP9.5). The results suggest that the neovascularization and reinnervation in the cultured keratodermal graft is almost complete at 6 weeks. Neovascularization precedes innervation, reaching the surface covered by the keratinocytes at 2 weeks, initially with a linear vascular pattern. From 3 weeks, there is a gradual arborization of the vessels to form a typical vascular plexus. The process of reinnervation is similar in pattern to that of neovascularization, although slower in developing a full network of fibres. In conclusion, the use of confocal microscopy allows the precise definition of complex patterns of neovascularization and nerve growth, which are not fully apparent when using conventional microscopy. Because angiogenesis occurs first, it probably plays a leading role in the survival of keratodermal grafts during wound healing. Indeed, new blood vessels form a pathway for the subsequent innervation process, and quickly reach the epidermal layer which, in turn, may play a key role in the tropism of both blood vessels and nerves.

Nerve growth factor levels in cultured human skin cells: effect of gestation and viral transformation.

Extracts of cultured human keratinocytes and fibroblasts were assayed for nerve growth factor-like immunoreactivity (NGF) by a specific enzyme-linked immunoabsorbant assay. NGF levels were higher in primary cultured keratinocytes than in freshly isolated keratinocytes or culture through multiple passages. Viral transformation of keratinocytes with the human papilloma virus (HPV16) significantly increased NGF levels, whilst transformation with the simian virus (SV40), which induces simple epithelial differentiation, reduced the concentration of NGF. Passaged epidermal keratinocytes contained more than twice as much NGF as did passaged fibroblasts. Oral keratinocytes and fibroblasts, and psoriatic fibroblasts, all from high turnover tissues, did not contain significantly different levels of NGF in culture than dermal keratinocytes or fibroblasts. Foetal fibroblasts contained five times as much NGF as did adult fibroblasts. These results suggest that basal keratinocytes are a major but not sole source of NGF in human skin, and that NGF may play a role in human skin development.

A functional "knockout" of human keratin 14.

The importance of keratins and other intermediate filaments in the maintenance of tissue structure is emphasized by the discovery that many hereditary skin-blistering diseases are caused by mutations in keratin genes. Here, we describe a situation in which keratin 14 (K14) is missing altogether in the epidermis: A homozygous 2-nucleotide deletion in exon I of the K14 gene causes premature termination of the mRNA transcripts upstream from the start of the rod domain and results in a K14 null phenotype. In this individual no keratin intermediate filaments are visible in basal epidermal cells, although filaments are present in the upper layers of the epidermis. No compensating keratin expression is detected in vivo, and K14 mRNA is down-regulated. The individual, diagnosed as Köbner (generalized) EBS, suffers from severe widespread keratinocyte fragility and blistering at many body sites, but although the phenotype is severe, it is not lethal. This K14-/- phenotype confirms that only one K14 gene is expressed in human epidermis and provides an important model system for examining the interdependence of different keratin filament systems and their associated structures in the skin.

Mutations in the rod 1A domain of keratins 1 and 10 in bullous congenital ichthyosiform erythroderma (BCIE).

Bullous congenital ichthyosiform erythroderma is a human hereditary skin disorder in which suprabasal keratinocytes rupture. Recent reports have implicated keratins K1 and K10 in this disease. Here we describe four diverse keratin mutations that are all significantly associated with this disease. Two of these are in the helix 1A subdomain of the type II keratin 1, giving a serine-to-proline substitution in codon 185 and an asparagine-to-serine substitution in codon 187. In the analogous region of type I keratin 10, an arginine-to-proline and an arginine-to-serine transition in codon 156 have been identified. All four mutations create restriction fragment length polymorphisms that were used exclude the mutations from 120 normal chromosomes. Insertional polymorphism (in the V2 subdomains of the non-helical tails of K1 and K10) was excluded as the cause of the phenotypic heterogeneity observed within one family.