Bactericidal activity of flucloxacillin against Staphylococcus aureus in primary keratinocyte cultures of lesional and unaffected skin of patients suffering from atopic dermatitis

More than 90% of patients with atopic dermatitis (AD) are colonized by Staphylococcus aureus. Due to prevalent multi-resistant strains, it is highly difficult to eliminate S. aureus contamination of primary cell cultures of human AD skin by means of the antibiotics commonly used in cell culture. Therefore, an anti-staphylococcal treatment was investigated by which sterile proliferating keratinocyte cultures are attained from lesional and unaffected skin of AD patients by applying flucloxacillin, which in general is systemically used in vivo. The treatment with 1% flucloxacillin for 20 min included both colonized skin samples and contaminated primary cell cultures. In the case of persistent contamination, this step was repeated as often as required until complete decontamination without any cytotoxic indications was achieved. Antibacterial treatment with flucloxacillin permitted the cultivation of sterile, vital, and proliferating primary cultures of human skin keratinocytes from lesional and unaffected skin of AD patients with S. aureus colonization. The method increased the success rate of isolation of AD keratinocytes from 30 to 90%, representing on average 3 vs. 9 successfully isolated, sterile and proliferating cultures out of 10 contaminated skin biopsies, which is comparable to normal human keratinocyte isolation rates.     

A new live‐cell biobank workflow efficiently recovers heterogeneous melanoma cells from native biopsies

Fibroblast contamination can make establishing primary melanoma cell cultures from native biopsies a major challenge, due to fibroblasts overgrowing the melanoma cells. Standard protocols therefore enrich for highly proliferative melanoma cells that grow well in vitro but may not represent the full range of in vivo tumor heterogeneity. Here we apply conditional methods that more effectively retrieve melanoma cells by differential trypsinization or by inducing fibroblast senescence through contact inhibition, serum starvation or deprivation of adhesion. Simple mixing experiments of melanoma and fibroblast cells demonstrated the efficacy of the new protocols in retrieving slow‐growing melanoma cells. Applying our protocols to 20 cultures that had failed to grow by conventional methods, we could retrieve 12 (60%) validated melanoma cell cultures. Further application of the protocols in the live‐cell biobank of 124 early passage cultures significantly improved recovery rates from 13% using standard protocols to 70% overall for the new workflow.     

Spread and persistence of infection with a trachoma biovar strain of Chlamydia trachomatis in multiplying and nonmultiplying McCoy cells

The trachoma biovar of Chlamydia trachomatis enters host cells in culture with difficulty, and cell-to-cell spread resulting in amplification of the initial growth usually does not occur. An experimental model was devised to demonstrate that, by using conditions that more closely approximate those encountered in man, the trachoma biovar of C. trachomatis can readily achieve cell-to-cell passage. Fresh McCoy cells were sequentially added to monolayers that had been inoculated with a trachoma biovar strain of C. trachomatis 3, 6, and 9 days earlier. Subsequent incubation resulted in intercellular propagation, with an increase in the number of inclusions from 500 to 80,000 per coverslip. A second experiment demonstrated the reproducibility of this phenomenon and showed that cell-to-cell spread can occur at a low rate in overcrowded, not overlaid, cell layers; it also showed that, in multiplying cell layers, the infection tends to become persistent.     

Long-term cultivation of canine keratinocytes

The growth characteristics and morphology of canine keratinocytes grown in vitro were studied. Keratinocytes from canine oral mucosa, ear skin, and ventral abdominal skin were grown in culture either as explants or as dispase/trypsin-derived suspensions in the absence of a feeder cell layer. Cholera toxin and epidermal growth factor were essential to the successful long-term growth and propagation of the cells during multiple passages. Keratinocytes from all tissue sources, either as primary cultures or subcultivated for up to 10 passages, had growth characteristic and morphology similar to that reported in other species. The use of cultured canine keratinocytes should provide a suitable model for comparative in vitro studies of the pathogenesis of dermatologic diseases.     

Primary cell culture for biochemical studies of human keratinocytes

Starting with large numbers of small split-thickness explains of human skin, it is possible to grow ample numbers ol keratinocytes in vitro as primary cell cultures to permit biochemical studies without the need of subculturing techniques.     

Culture grafted leg ulcers

The culture of keratinocytes from normal skin was attempted for many years but was largely hampered by a tendency for keratinocytes to differentiate in vitro rather than proliferate, and problems with fibroblast overgrowth. However, the report from Rheinwald and Green (1975), which utilizes a mouse 3 T 3 substrate and mitogens, led major advances in culture techniques permitting serial passaging of keratinocyte cultures without fibroblast contamination. Other systems using defined media (Boyce & Ham, 1983) and additives such as epidermal growth factor (Rheinwald & Green, 1977) and bovine pituitary extract (Peehl & Ham, 1980) provide expanded growth without a feeder system. This ability to expand, greatly, a given population of keratinocytes is of obvious clinical application in the repair of epidermal defects and already has been used in the treatment of young burns patients in the States (O'Connor et al., 1981). We report the use of cultured skin in the grafting of an elderly patient with stasis ulceration.     

Modified epidermal lipid composition in air-exposed culture of non-bullous congenital ichthyotic erythroderma (NBCIE) keratinocytes

Emerged epidermal cultures on dead de-epidermized dermis (DED) constitute an excellent model for in vitro reproduction of dermatoses linked to a keratinocyte defect. We used such cultures for studies of non-bullous congenital ichthyotic erythroderma (NBCIE). Keratinocytes of normal and pathological origin were expanded in submerged cell cultures and frozen keratinocytes from the resulting cell bank were subsequently used for seeding on DED. Lipid extracts from 14 day emerged cell cultures were assayed qualitatively and quantitatively using thin layer chromatography and compared with the neutral and non-polar lipid profiles obtained from normal epidermis extracts and with those from the plantar stratum corneum of healthy donors and untreated NBCIE patients. The ichthyotic cultures were found to contain significantly elevated levels of n-alkanes, as were the lipid extracts from the patients' plantar horny layer. Our results demonstrate that a major marker of the NBCIE epidermis can be reproduced under the emerged culture conditions. They also indicate that the characteristic n-alkane increase in NBCIE is indeed endogenous and not merely related to possible contamination from topical treatments.This paper was partially presented at the ESDR annual meeting in London, 4–7 May 1992 (selected for the F. Serri Poster Prize)     

The culture of dermal papilla cells from human hair follicles

Dermal papillae were isolated from human hair follicles and primary cell cultures were established from the papilla explants. The cultured papilla cells spread slowly, initially as a monolayer, and eventually formed multi-layered parallel arrays of fibroblast-like cells. At the edges of expanding colonies the cells were large and flattened and showed a tendency to form clumps. The behaviour of human dermal papilla cells in culture is very similar to that reported in cultures of papilla cells from rat vibrissa follicles.     

The nickel content of certain commercially available metallic patch test materials and its relevance in nickel-sensitive subjects

The nickel contamination of the metallic patch test substances from 3 commercial sources was estimated by atomic absorption spectrophotometry. Contamination was generally greatest in the cobalt patch test substances. To determine whether the levels of contamination encountered were sufficient to produce a false positive patch test, 20 nickel-sensitive patients were patch tested to various concentrations of free elemental nickel in aqueous solution. The level of nickel contamination was less than that required to produce a false positive patch test in our patients, but was at a level which could produce false positive reactions in subjects highly sensitive to nickel.     

Dispersed Cell Culture of Human Sweat Duct Cells under Serum-free Conditions

Human eccrine gland duct cells were successfully cultured using a serum-free medium, K-GM medium. Eccrine sweat ducts were isolated from dispase treated skin specimens from palms or soles. After treatment of the isolated ducts with trypsin and EDTA, dispersed cells were cultured in K-GM medium. In primary cultures, small colonies were seen 3 to 4 days after inoculation. Then the cells rapidly proliferated and formed large colonies with a paving stone-like cell arrangement. During the culture, small dome shaped areas were sometimes formed in the centers of colonies. Cultures multiplied for a maximum of 7 passages. The plating efficiencies of the 1st to 6th passage cells were about 20% to 30%. Immunocytochemically, cultured cells were positively stained with anti-carcinoembryonic antigens, K8.37 and K8.13, but not with anti-S100 protein, anti-HLA-DR, 34βB4, or PKK3. An electron micrograph of the cultured cells showed a multilayer of flattened cells linked by desmosomes. These results indicate that the cultured cells possessed the staining properties compatible with those of the ductal portion of eccrine sweat glands. No contamination by other mesenchymal cells, such as fibroblasts, was seen during the culture.     

An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells

Abstract:  Human keratinocyte primary cultures are commonly established by tissue dissociation and often rely on feeder cell supports and culture medium that is not defined. Further, contamination by unwanted fibroblasts can be problematic. Here, we developed a skin explant method for growing primary keratinocytes that was rapid, simple, and reliably generated keratinocyte cultures free of fibroblast contamination. The process capitalized on the observation that fibroblasts migrate out of adult skin explants later than epidermal cells, allowing the early harvesting of keratinocytes by trypsinization. When grown subsequently in defined medium in the absence of feeder cells, the explant-derived cells grew rapidly and could be cultured for multiple passages. Immunofluorescence microscopy revealed that a high percentage of cells harvested from the explant outgrowths expressed K15, while very few expressed the differentiation marker K10. Cells that were stained while migrating out from explants strongly expressed markers associated with progenitor cells, including p63, K15 and CD133, and displayed intense K6 expression, indicative of activated keratinocytes in wound-healing epidermis. By replenishing the explants with fresh medium after harvesting, further epidermal outgrowths could be obtained, offering the possibility of greatly increased keratinocyte yields for clinical applications.     

Blastomycosis: a case report and review of the literature

Background Blastomycosis is a chronic granulomatous and suppurative mycosis caused by the fungus Blastomyces dermatitidis. This is a dimorphic fungus, which exists as a non‐pathogenic mold in mycelial form in nature and converts to pathogenic yeast at body temperature. Infection is acquired by either inhalation or inoculation. We report a case of blastomycosis with severe involvement of the scalp, face, and neck, with no evidence of systemic involvement. Methods Biopsy specimen was stained with hematoxylin and eosin, periodic acid‐Schiff (PAS), PAS with diastase digestion, and Grocott. Culture was performed on a Sabouraud’s dextrose agar plate using an aseptic technique as per standard operating procedure for processing mycology specimens at our institution. A lactophenol cotton blue preparation from the cultured material was performed. Results Histopathologic examination showed pseudoepitheliomatous hyperplasia and a granulomatous inflammation with round to oval organisms, with refractile cell walls in the cytoplasm of giant cells. PAS, PAS with diastase digestion, and Grocott stains enhanced the organisms. Cultured material showed growth after 10 days, and the lactophenol cotton blue preparation from the cultured material showed the organism to be Blastomyces dermatitidis. Sensitivity studies favored treatment with itraconazole. Radiological examination of the patient showed no evidence of systemic involvement. Conclusions Our case may represent the rare primary cutaneous inoculation blastomycosis as lesions started on an area of previous trauma. Treatment with itraconazole was successful.     

Alopecic and aseptic nodule of the scalp in a girl

Alopecic and aseptic nodule of the scalp is a rare entity characterized by the presence of nodules or cysts with sterile punctured material and negative cultures accompanied by nonscarring alopecia in the scalp of young men. We describe a case in which an 11‐year‐old girl presented with a nodular, fluctuant, round lesion on the vertex with localized alopecia. High‐resolution ultrasound showed a hypoechoic lesion with increased flow on Doppler imaging and culture of the citrine‐yellowish material obtained by puncture was negative. The patient showed complete clinical response to treatment with topical indomethacin.     

Localized lymphomatoid papulosis: Unilesional lymphomatoid papulosis,regional lymphomatoid papulosis,and persistent agmination of lymphomatoid papulosis

Lymphomatoid papulosis (LYP), the most common primary cutaneous CD30-positive lymphoproliferative disorder, is heralded by multiple papular and nodular lesions at anatomically discontiguous cutaneous sites. The histologic patterns are protean. An uncommon form of LYP is one that is anatomically confined. Cases of unilesional LYP, regional LYP, and persistent agmination of LYP were encountered in the routine and consultative practices of Weill Cornell Medicine, Division of Dermatopathology. The clinical presentation, outcomes, light microscopic findings, and phenotypic profile are reviewed. There were 10 cases of LYP presenting as solitary plaques or nodules primarily occurring in older patients and without a relevant medical history in most. Most cases occurred at an acral site with many localized to the foot; the morphology was one of a necrotizing angiocentric type E pattern and borderline type C morphology. Two of the unilesional patients in our series went on to develop mycosis fungoides, one at the initial site of unilesional type A LYP, and the other at a discontiguous site. Excluding one case, the solitary lesions underwent complete regression; after the lesions regressed, some cases had no apparent recurrence. The second anatomically confined variant of LYP in our series was regional LYP exhibiting a type E morphology in two cases and a hybrid type A and granulomatous eccrinotropic morphology in one case. There was no subsequent development of lymphoma, nor was there any spread to additional anatomic sites. The final category was persistent agmination of LYP, whereby the agminated papules of LYP were superimposed on a plaque of cutaneous T-cell lymphoma represented by mycosis fungoides in two and follicular helper T-cell lymphoma in one. In conclusion, anatomically confined LYP defines an uncommon form of LYP, but it is an important one to recognize because the histology can be worrisome despite an indolent clinical course. The clinical presentation, the infrequent association with lymphoma/leukemia, and histology are similar to conventional LYP, although there appears to be a greater tendency for complete regression without recurrence, excluding cases of persistent agmination of LYP whereby the clinical course warrants categorization as a form of cutaneous T cell lymphoma cutaneous T cell lymphoma (CTCL).     

Modelling the hair follicle dermal papilla using spheroid cell cultures

Please cite this paper as: Modelling the hair follicle dermal papilla using spheroid cell cultures. Experimental Dermatology 2010; 19: 546–548. Abstract: Human dermal papilla (DP) cells grown in two‐dimensional (2D) culture have been studied extensively. However, key differences exist between DP cell activities in vivo and in vitro. Using a suspension method of cell culture to maintain DP cells, we created three‐dimensional (3D) dermal spheres morphologically akin to intact (anagen) DPs. Analysis of these spheres using immunocytochemistry demonstrates that they have expression profiles different from papilla cells cultured in 2D but with many similarities to intact DPs. This method of DP cell culture may provide us with a tool to elucidate our understanding of signalling within the DP as it relates to induction, maintenance or even inhibition of hair growth.     

A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells

A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types.     

Long-term establishment, characterization and manipulation of cell lines from mouse basal cell carcinoma tumors

There have been few reports of successful long-term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long-term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)(+/-) mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long-term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique.     

PAPULONECROTIC TUBERCULID SECONDARY TO DISSEMINATED MYCOBACTERIUM AVIUM COMPLEX

Background. Papulonecrotic tuberculid is a rarely reported cutaneous reaction to the mycobacterium bacillus. It is most often encountered in association with tuberculosis. The clinical and histologic picture of the entity is a distinctive one, but the etiology of the disease process is uncertain. Therapy directed against the causative organism is dramatically successful. Methods. A 35–year-old white man with AIDS was referred to the Dermatology clinic for evaluation of a widespread skin eruption. The skin lesions were biopsied for histopathology and culture. From the cutaneous cultures Mycobacterium avium complex (MAC) organisms were grown. Results. We report the first case of papulonecrotic tuberculid manifestation in an AIDS patient with disseminated MAC. Unusual features seen in this case include the predominance of pruritic eschars rather than asymptomatic papules and the confirmation by special stains of mycobacterium organisms within the skin biopsy. Papulonecrotic tuberculid has not been previously associated with either MAC or AIDS. Conclusions. Papulonecrotic tuberculid should be a diagnostic consideration in immunocompromised patients with MAC whose clinical and histologic features are compatible with this rare entity.     

SURVEY OF MATING TYPES OF CLINICAL ISOLATES OF MICROSPORUM CANIS IN JAPAN

The results of mating experiments with isolates of Microsporum canis isolated in Japan were reported. Of the 120 cultures of M. canis studied, 111 originated from human cases and 9 from pet cats. Ninety-eight of the isolates were compatible with Nannizzia otae and proved to be of the “—” mating type. Cleistothecia were not produced by 22 isolates in combination with the two compatible tester strains.