An improved method for culture of epidermal keratinocytes from newborn mouse skin

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca²⁺ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin v6. The expression level of v6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.     

Clinical trial of allogeneic cultured dermal substitutes for intractable skin ulcers of the lower leg

The efficacy of allogeneic cultured dermal substitute (CDS) on wound healing was evaluated in six patients with intractable skin ulcers on the lower extremities. Allogeneic CDS was repeatedly applied to wounds at intervals of 4-7 days to prepare a wound bed acceptable for skin grafting or to induce resurfacing through the granulation tissue formation associated with epithelialization. In one patient with a leg ulcer, the wound size decreased to 32% of the original size within 10 weeks and skin grafting was conducted. In the other five patients with leg, ankle, or foot ulcers, the wound size decreased to 9%-25% of the original size within 6 weeks.     

Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines

Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.     

Alcohol metabolizing enzymes: Studies of isozymes in human biopsies and cultured fibroblasts

Rapid and sensitive micromethods for the study of alcohol dehydrogenase and adehyde dehydrogenase isozymes in skin extracts, cultured fibroblasts and other organs are presented. Possibilities for the application of these techniques to the study of interindividual variations in response to alcohol are discussed. While fibroblasts cultured from a skin biopsy from one Japanese individual revealed a heterodimer (ADH2 2-1) of alcohol dehydrogenase, skin extract from another Japanese showed a homodimer (ADH2 2-2). Up to four isozyme sets for aldehyde dehydrogenase (ALDH) were detected in various human organs and at least three sets were found in skin and fibroblasts extracts. Our preliminary data on liver, stomach, and skin indicate that ALDH is polymorphic and several loci are concerned in the determination of these isozyme sets.     

Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5?days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6?days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3?days, but decreased slightly after 5?days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3–5?days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.     

Comparative study of adult human skin fibroblasts and umbilical fibroblast-like cells

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton’s jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 53–59, January, 2006     

Positive skin tests to aero-allergens and month of birth

The month of birth distribution for 1301 French patients born between 1953 and 1975 with at least one positive skin test was compared to that of the whole population. A chisquare test was performed, and the expected birth month distribution of the groups calculated from the INSEE * data for 1953-1975. All patients underwent skin testing with house dust, Dermatophagoides pternyssinus (Dpt), cat and dog allergens, grass, tree and weed pollens, and moulds. The only study criterion was a positive skin test unrelated to any specific disorder. A significant difference in month of birth distribution was observed 1) for patients with positive skin test to grass pollen, with a high rate of births from January to May, and 2) for patients with mould sensitization, with a low rate of births in April, May and December. Tree and weed pollens, house dust and Dpt showed no significant relation with month of birth. For cat and dog allergens, the observed and expected distributions of birth month were similar. For the whole sensitized population the birth rate tended to be low in December except for the cat and dog sensitized. Our study confirms the well-known seasonal peak of births in the first 5 months of the year for grass pollen sensitized patients. No consistent monthly or seasonal tendency could be statistically demonstrated for other allergens except moulds.     

Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis

BACKGROUND: Mast cells, the key cells of immediate hypersensitivity type reactions, have also been postulated to have a central role in influencing tissue remodelling and fibrosis occurring in the skin. OBJECTIVE: Our aim was to investigate the direct role of human mast cells (HMC) in skin fibrotic processes, by assessing the effects of the addition of the human mast cell line HMC-1 to human skin fibroblasts, and to identify the responsible mediators. METHODS: HMC-1 sonicates were added to human skin fibroblasts and the following parameters were evaluated: proliferation ([3H]-thymidine), collagen synthesis ([3H] proline), activity of matrix metalloproteinases (MMPs) (zymography) and tissue inhibitors of metalloproteinases (TIMPs) (reverse zymography), and collagen gel contraction. RESULTS: HMC-1 sonicate increased significantly both proliferation and collagen production in the human skin fibroblasts and these properties were not affected by heating of the sonicate (56 degrees C, 30 min, or 100 degrees C, 3 min). Two main mast cell mediators, histamine and tryptase, were found to be responsible for the increase in fibroblast proliferation and collagen production. HMC-1 sonicate did not display any pre-formed gelatinase activity, and its addition to the fibroblasts did not change their pro-MMP-2 and MMP-2 activity. On the other hand, HMC-1 were found to possess TIMP-1 and TIMP-2. Addition of HMC-1 had no effect on fibroblasts TIMP-1 but induced a dose-dependent increase of TIMP-2 activity. In addition, HMC-1 sonicate seeded together with the fibroblasts in tri-dimensional collagen gel significantly enhanced their contraction. CONCLUSION: We have shown that human mast cells, by granule-stored and therefore quickly releasable mediators, increase human skin fibroblast proliferation, collagen synthesis, TIMP-2 and collagen gel contraction. Therefore, mast cells have a direct and potentiating role in skin remodelling and fibrosis.     

Sister chromatid exchange with and without in vitro mutagen induction in cultured skin fibroblasts from patients with adenomatosis of the colon and rectum

Fibroblast cell strains were obtained from skin biopsies taken from patients with adenomatosis of the colon and rectum (ACR), and their relatives. A total of 50 different fibroblast strains were tested for their frequencies of sister chromatid exchange (SCE) in vitro. These strains included nine from patients with the Gardner syndrome, 21 from patients with non-Gardner ACR, and 20 cell strains from healthy relatives who were not at an increased risk for ACR. In 23 strains, the SCE frequencies after in vitro exposure to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) were also determined. Both with and without MNNG induction, SCE values in the Gardner strains were found to be significantly higher than in the control strains (p less than 0.02 and p less than 0.03, respectively). Non-Gardner ACR strains differed only slightly from controls, thus making the difference between the control group and the pooled Gardner + non-Gardner ACR group not significant. In all groups, there was a significant increase in SCE after MNNG exposure, and those strains which had low SCE values spontaneously, also tended to have relatively moderate SCE values after MNNG induction. There was no significant difference between the ratios of SCE values with and without MNNG exposure in the different groups.     

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim-/fla-) strain, a fimbriate but aflagellate (fla-) strain and a fimbriate/flagellate but non-motile (mot-) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues with an intact mucus layer. A smooth swimming chemotaxis-defective (che-) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants. However, the possession of active flagella did appear to be an important factor in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.     

Comparison of five new antihistamines (H1-receptor antagonists) in patients with allergic rhinitis using nasal provocation studies and skin tests

BACKGROUND: It was the aim of the authors to compare all of the latest second-generation antihistamines and to see if there were significant differences in their efficacy. It is important for ENT specialists to know if these differences exist, as it is for general practitioners trying to choose between these drugs. METHODS: In 12 confirmed grass pollen allergic patients the authors performed nasal smears to asses eosinophilia, histamine/grass pollen skin tests, and grass pollen nasal provocation tests. All tests were performed before and after administration of one of five different antihistamines (cetirizine, loratadine, ebastine, fexofenadine, mizolastine) or placebo. The order of administration of antihistamines and placebo was randomised, and patients were not aware of which drug they were given. A decrease in nasal eosinophilia (nasal smear), or nasal or skin reactivity (provocation tests) was looked for. RESULTS: A significant decrease in nasal eosinophilia was observed for all antihistamines but not for placebo. For the grass pollen nasal provocation tests, the decrease was significant for nasal blockage and sneezing; for rhinorrhea there was an insignificant decrease that was true for all antihistamines. A significant reduction in histamine/grass pollen skin test reactivity was also observed for all antihistamines, during an 8 h observation period. A significant difference in efficacy between the different antihistamines could not be found with any of the tests performed. CONCLUSIONS: For the newer nonsedating H1-antagonists there appears to be no clinically relevant differences in activities--at least not in our study. Preference of the patient may be the most important factor in making a choice between these drugs.     

Wheat allergy: diagnostic accuracy of skin prick and patch tests and specific IgE

BACKGROUND: Food allergy makes an important contribution to the pathogenesis of atopic eczema in infants. However, clinical data on cereal allergy are scanty. The objective was to study the relevance of patch testing, skin prick tests, and the concentration of wheat-specific IgE antibodies (CAP RAST) in correlation with oral wheat challenge in infants with suspected wheat allergy. In particular, we aimed to determine whether the patch test could increase the diagnostic accuracy in detecting wheat allergy. METHODS: The study material comprised 39 infants under the age of 2 years. Of these patients, 36 were suffering from atopic eczema and three had only gastrointestinal symptoms. The patients were subjected to a double-blind, placebo-controlled or open wheat challenge. Wheat-specific IgE was measured by CAP RAST, and skin prick and patch tests were performed. RESULTS: Of the total 39 wheat challenges, 22 (56%) were positive. Of the positive reactions, five involved immediate-type skin reactions over a period of 2 h from the commencement of the challenge. In 17 patients, delayed-onset reactions of eczematous or gastrointestinal type appeared. Of the infants with challenge-proven wheat allergy, 20% showed elevated IgE concentrations to wheat, 23% had a positive skin prick test, and 86% had a positive patch test for wheat. The specificities of CAP RAST, skin prick tests, and patch tests were 0.93, 1.00, and 0.35, respectively. CONCLUSIONS: Our study demonstrated that patch testing with cereals will significantly increase the probability of early detection of cereal allergy in infants with atopic eczema and is helpful in the planning of successful elimination diets before challenge. The specificity of the patch test was lower than that of other tests. Therefore, confirmation of the diagnosis with the elimination-challenge test is essential in patients with positive patch test results.     

The diagnosis of Brazil nut allergy using history, skin prick tests, serum-specific immunoglobulin E and food challenges

BACKGROUND: Allergy to Brazil nut is a relatively common nut allergy and can be fatal. However, the evidence is lacking regarding the best approach to its diagnosis. OBJECTIVE: We sought to determine the relative merits of history, skin prick testing, measurement of serum-specific IgE and challenge in the diagnosis of Brazil nut allergy. METHODS: Fifty-six children and adults with a history of an allergic reaction to Brazil nut or evidence of sensitization were investigated by questionnaire (n=56), skin prick tests (SPTs) (n=53), measurement of serum-specific IgE to Brazil nut (n=54) and double-blind, placebo-controlled labial, and if necessary oral, challenges (n=19). RESULTS: Brazil nut allergy occurred in highly atopic individuals of any age with a strong family history of atopy. In 24 of 56 (43%), the history of an immediate reaction was sufficient to make a diagnosis with confidence and an oral challenge was considered unsafe. Of the 19 subjects undertaking the 'gold standard' test of a double-blind, placebo-controlled, food challenge, all six subjects with a SPT of at least 6 mm had a positive challenge and all three subjects with a SPT of 0 mm had a negative challenge. In the remaining 10 (53%) subjects, where SPT was between 1 and 5 mm and serum-specific IgE was less than 3.5 kU/L, an oral challenge was performed resulting in three positive and seven negative challenges. CONCLUSION: A combination of history, SPT and serum-specific IgE was adequate in achieving a diagnosis in the majority (77%) patients with suspected Brazil nut allergy. However, a doubtful history with SPT between 1 and 5 mm, or a serum-specific IgE less than 3.5 kU/L may require an oral challenge to help determine the risk of a Brazil nut allergic reaction.