Positional cloning of a gene for Hermansky-Pudlak syndrome, a disorder of cytoplasmic organelles.

Hermansky-Pudlak syndrome (HPS) is an often-fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single-gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.

Mutations of keratinocyte transglutaminase in lamellar ichthyosis.

Lamellar ichthyosis is a severe congenital skin disorder characterized by generalized large scales and variable redness. Affected individuals in three families exhibited drastically reduced keratinocyte transglutaminase (TGK) activity. In two of these families, expression of TGK transcripts was diminished or abnormal and no TGK protein was detected. Homozygous or compound heterozygous mutations of the TGK gene were identified in all families. These data suggest that defects in TGK cause lamellar ichthyosis and that intact cross-linkage of cornified cell envelopes is required for epidermal tissue homeostasis.

Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is a hereditary skin disorder characterized by blistering and a marked thickening of the stratum corneum. In one family, affected individuals exhibited a mutation in the highly conserved carboxyl terminal of the rod domain of keratin 1. In two other families, affected individuals had mutations in the highly conserved amino terminal of the rod domain of keratin 10. Structural analysis of these mutations predicts that heterodimer formation would be unaffected, although filament assembly and elongation would be severely compromised. These data imply that an intact keratin intermediate filament network is required for the maintenance of both cellular and tissue integrity.

Confirmation of the origin of NISCH syndrome.

Neonatal ichthyosis-sclerosing cholangitis (NISCH) syndrome, a rare autosomal recessive ichthyosis syndrome characterized by scalp hypotrichosis, scarring alopecia, ichthyosis, and sclerosing cholangitis, was described for the first time in 2002. It is caused by a mutation in the gene coding for the tight junction protein claudin-1. Only four patients carrying the same mutation of the CLDN1 gene have been described until now. We report a patient presenting with the clinical characteristics of NISCH syndrome and carrying a novel mutation in the CLDN1 gene.

The melanin pigmentary disorder in a family with Hermansky-Pudlak syndrome.

The albinotic skin and hair of 2 patients with Hermansky-Pudlak syndrome were investigated by light and electron microscopy. Incubation of hairbulbs and epidermis in 1-dopa revealed a weak tyrosinase activity. The epidermal melanocyte population was of normal density. The most striking feature was the presence of numerous giant melanosomes resembling those mainly reported in various hyperpigmented skin lesions. The association of this melanosomal disorder with the platelet dysfunction and ceroid storage typical of the autosomal recessive Hermansky-Pudlak syndrome might provide new insights into the mechanism leading to formation of giant melanosomes.

Neurofibromatosis of von Recklinghausen: a quantitative study of the epidermal keratinocyte and melanocyte populations.

The numerical keratinocyte to melanocyte relation was studied in café au lait spots and adjacent normally pigmented skin of 9 patients with classical neurofibromatosis. Compared to normal skin of healthy individuals, the keratinocyte:melanocyte ratio distributions obtained in neurofibromatosis indicated a shift to lower values in the biopsies of café au lait spots and normally pigmented skin. These results are evidence in favor of an impaired tissue organization of the epidermis in neurofibromatosis with regard to the keratinocyte-melanocyte interrelation.

Thermolysin treatment: a new method for dermo-epidermal separation.

The epidermis of superficial human skin samples could easily be separated from the dermis following incubation at +4 degrees C for 1 h in a solution containing 250-500 micrograms/ml thermolysin, a proteolytic enzyme hitherto mostly used for protein analysis. Light and electron microscopy revealed that the dermo-epidermal separation occurred at the basement membrane between the sites of bullous pemphigoid antigen and laminin and that the hemidesmosomes were selectively disrupted. The cohesion and morphology of the separated epidermis as well as the immunologic parameters investigated were not altered by this procedure. The clear cut dermo-epidermal separation produced by thermolysin treatment differed from the separation obtained with trypsin, which predominantly occurred between basal and suprabasal cells by disruption of desmosomes.

Evidence that ferritin is UV inducible in human skin: part of a putative defense mechanism.

As ferritin has been identified as an important factor in antioxidant defense in cultured human skin cells we evaluated the presence of ferritin in human skin in vivo and the modifications following irradiation with UVA I, UVA I + II, and solar simulating light by immunohistochemical analysis. We report that the putative protective protein ferritin is regularly present in the basal layer of unirradiated epidermis in vivo and that the induction of ferritin was dependent on wavelength and cell type. Following UVA I radiation, ferritin increased both in epidermal and in dermal tissue. The same response occurred, although to a lesser extent, with UVA I + II but did not occur following solar simulating radiation. Quantitative analysis for ferritin in cultured keratinocytes and fibroblasts from seven individuals following each UV spectra were also assessed by enzyme-linked immunosorbent assay. The induction of ferritin by UV was highly dependent on the waveband and cell type. UVA I and UVA I + II radiations induced ferritin expression in dermal fibroblasts up to 260% and 200% over basal levels, respectively. Solar simulating radiation produced only a small induction of approximately 130% over basal ferritin levels in dermal fibroblasts. Ferritin increased in cultured fibroblasts as early as 3 h post-UVA with a peak at 6 h that remained until 48 h; there was no observable qualitative or quantitative increase seen in the undifferentiated cultured epidermal keratinocytes. Our findings indicate that the putative defense system of ferritin exists in human skin in vivo and its induction is dependent on UV spectra and cell type. The increased concentrations of this antioxidant in human skin following acute UV radiation could afford increased protection against subsequent oxidative stress.

Cellular sensitivity to oxidative stress in the photosensitivity dermatitis/actinic reticuloid syndrome.

Skin fibroblasts from certain patients with the photosensitivity dermatitis/actinic reticuloid syndrome show enhanced sensitivity to ultraviolet radiation compared to normal fibroblasts. To probe further the link between oxidative damage and this disease, we have obtained a more extensive set of cell lines from patients with a severe form of the disease and examined their sensitivity towards oxidative stress by measuring cell survival following UVA radiation (330-450 nm) or hydrogen peroxide treatment (0.1-2.4 mM). The activation of the stress gene, heme oxygenase, has also been assessed by measuring the accumulation of mRNA after hydrogen peroxide treatment. Our studies have confirmed that a slight ultraviolet sensitivity is a characteristic of photosensitivity dermatitis/actinic reticuloid syndrome cell strains and we further demonstrate that these cell lines are particularly sensitive to hydrogen peroxide with up to a three- to fourfold increased sensitivity as compared to normal controls. We also show that certain ataxia telangiectasia strains that are especially sensitive to hydrogen peroxide are also slightly sensitive to ultraviolet radiation. Hydrogen peroxide induces accumulation of mRNA for the oxidant-inducible stress protein, heme oxygenase, with similar kinetics (maximum mRNA accumulation 2-4 h following treatment) and with a similar range of magnitudes in both normal (6.6-20.6 times mRNA increase over basal levels) and photosensitivity dermatitis/actinic reticuloid (2.9-12.8 times) skin cells. Because cells from photosensitivity dermatitis/actinic reticuloid patients show increased sensitivity towards oxidative stress but show no significant change in oxidant activation of the heme oxygenase gene, we propose that the defect involves a late stage of processing of oxidative damage rather than a compromised free radical scavenging system.

Abnormal keratin 1 and 10 cytoskeleton in cultured keratinocytes from epidermolytic hyperkeratosis caused by keratin 10 mutations.

Epidermolytic hyperkeratosis is caused by mutations of the differentiation-specific keratins K1 and K10. These mutations produce a weakened cytoskeleton that is prone to collapse resulting in cell fragility and lysis. In this study we have analyzed cultured keratinocytes from EHK patients bearing 10R-to-H and 15L-to-S mutations within the 1A segment of the K10 rod domain. Keratinocytes were grown submerged in serum-free medium and induced to differentiate by growing to confluence and increasing the Ca++ concentration in the medium. Cultures were either harvested for mRNA sequence analysis or subjected to immunofluorescence microscopy. Differentiating keratinocytes from these patients were found to express these K10 mutations in their mRNA. Moreover, these cells could be distinguished from normal keratinocytes by their aberrant morphology. EHK keratinocytes frequently exhibited a collapsed perinuclear network of K1/K10 filaments and sometimes peripheral granules of K1 and K10 aggregates, reminiscent of the cells of the suprabasal layers in these patients. This report documents the expression of mutant keratin 10 in cultured EHK keratinocytes.

Contour-clamped homogeneous electric field gel electrophoresis as a powerful epidemiologic tool in yeast infections.

To examine the longitudinal and cross-sectional patterns of yeast colonization in critically ill patients using genotypic characteristics defined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis, 322 clinical isolates of Candida species were prospectively collected from 29 critically ill patients under routine surveillance over a 6-month period. All isolates, recovered from multiple anatomic sites and from the same sites on different days, were characterized by several identification methods (germ tube test), phenotyping (API system), and genotyping (electrophoretic karyotyping). Electrophoretic karyotype (EK) was determined using pulsed field electrophoresis with the CHEF technique. We used a karyotyping system for Candida albicans (EK code) that facilitated intraspecies delineation. C. albicans colonized 83% of the 29 patients. Candida sp. strains isolated from an individual patient had an identical EK pattern, even when isolated from different body sites, and remained the same over a prolonged period, up to 140 days. EK delineated not only the different Candida species, but also different strains of C. albicans. Strains of C. albicans isolated from different patients were distinguished using the EK pattern, but not API system. Minor variations in EK pattern could be demonstrated in a minority of strains recovered from four patients and were interpreted as chromosomal rearrangements between parent strains. Severe candidal infections, including eight episodes of fungemia, occurred in 11 of 29 patients (38%). All patients had been previously colonized with strains with identical EK patterns. Infection occurred a mean of 25 days after initial surveillance cultures grew yeast. No horizontal transmission could be demonstrated during the study period. In conclusion, EK is a reproducible, stable marker allowing inter-, as well as, intraspecies Candida strain delineation. EK strain delineation is a useful tool in candidal epidemiologic and pathogenic studies. Yeast colonization with the same strain preceded infection in critically ill patients.

Isolation and characterisation of an extracellular alkaline protease of Aspergillus fumigatus.

Aspergillus fumigatus secreted an inducible alkaline protease (AlPase) when cultivated in the presence of collagen (200 micrograms/ml) as sole nitrogen and carbon source. Proteolytic activity was maximum at pH 9.0 with azocollagen as substrate. The enzyme, which was the major protein found in the supernate of a liquid culture, was purified by ammonium sulphate precipitation and gel filtration. The Mr was determined to be 33 Kda by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The isoelectric point was estimated to be pH 8.2. Divalent cations strongly inhibited enzyme activity, whereas non-ionic detergents and reducing agents had no effect. A. fumigatus AlPase was totally inhibited by phenylmethanesulphonyl fluoride, antipain, chymostatin and alpha-2-macroglobulin. A. fumigatus AlPase is closely related to the A. oryzae AlPase, a serine protease of the subtilisin family, as attested by the antigen pattern seen by immunoblotting. The high collagenic activity and the ability of A. fumigatus AlPase to digest elastin could play a role in the invasion of the tissues by the fungus.

The identification of pathogenic yeast strains by electrophoretic analysis of their chromosomes.

Epidemiological studies require characterisation of pathogenic yeasts at and below the species level. The chromosomes of 130 strains of four pathogenic species of the genus Candida, isolated from clinical material, were separated by pulsed field electrophoresis with the clamped homogeneous electric field (CHEF) technique. Each species was characterised by a distinct electrophoretic karyotype (EK). Furthermore, smaller variations of the EK amongst strains belonging to the same species appeared to offer a useful means of strain differentiation. A karyotyping system is proposed for C. albicans. The EKs were assigned to a code of four numbers which designated the number of bands that could be resolved in each of four sets of chromosomes. Morphotypes of the colonies of C. albicans on malt agar plates, which did not correlate with the EK, could provide a complementary means of strain characterisation in epidemiological studies.

Disseminated spiked hyperkeratosis. An unusual discrete nonfollicular keratinization disorder.

An unusual nonfollicular keratinization disorder was observed in a father and his son, and in an unrelated woman. The disorder began during the second decade of life and gradually became more widespread and more pronounced thereafter. The dermatosis is characterized by tiny, rough, keratotic spikes giving the skin a raspy fell on palpation. Microscopically, the lesions showed a thick compact corneum without structural changes in the underlying epidermis except for moderate epidermal cell hyperplasia and some reduction in keratohyalin content.

Analysis of the cornified cell envelope in lamellar ichthyosis.

BACKGROUND: Loricrin and involucrin are major precursor proteins to the cornified cell envelope expressed late in epidermal differentiation. Involucrin expression starts in the upper spinous layers in normal human epidermis and precedes loricrin expression, which is restricted to the granular layer. Subsequently, both proteins become cross-linked by the activity of transglutaminases TGK/E as major components of the cornified cell envelope by N epsilon-(gamma-glutamyl)lysine isopeptide bonds. In this study, three cases of lamellar ichthyosis were analyzed by immunohistologic study with antibodies to loricrin, involucrin, filaggrin, and transglutaminase TGK. OBSERVATIONS: A high expression of loricrin and involucrin with a peculiar and abnormal cytoplasmic staining concurred with a diminished cytoplasmic staining of transglutaminase TGK as assessed by antibodies B.C.1 and K.D.3. This pattern was absent in a collodion baby at birth but present 2 weeks later before a phenotype of lamellar ichthyosis appeared clinically. CONCLUSIONS: The results suggest that in our cases of lamellar ichthyosis, (1) disturbed membrane anchorage of transglutaminase TGK could alter loricrin and involucrin cross-linkage and the formation of the cornified cell envelope and that (2) immunohistologic study might serve as an early diagnostic and prognostic tool in the treatment of collodion babies.

Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress.

Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.

Relationship between melanogenesis, glutathione levels and melphalan toxicity in human melanoma cells.

Resistance to alkylating agents has been correlated with cellular levels of reduced glutathione (GSH) and glutathione-S-transferase (GST). GSH is also involved in regulation of melanin synthesis. Therefore, we examined sensitivity to melphalan as a function of differentiation and GSH/GST levels in three human melanoma cell lines. The Me8 cell line, classified as undifferentiated on the basis of cell shape, absence of pigment, insignificant dopa oxidase activity and presence of inhibitors of dopa-melanin formation, showed the lowest GST activity among the cell lines investigated. GLL19 cells exhibited normal differentiation as indicated by the presence of dendrites, typical eumelanosomes, melanin granules and dopa oxidase activity. These cells showed the highest GSH content and the highest GST activity. The JUSO cell line showed incomplete differentiation, and its dopa oxidase and GST activities were intermediate between the Me8 and GLL19 cell lines. The sensitivity of melanoma cell lines to melphalan increased with their degree of differentiation; it was lowest for Me8, intermediate for JUSO and highest for GLL19. Dibutyryl cyclic AMP (dbcAMP) enhanced melphalan toxicity against Me8 cells. Depletion of intracellular GSH with buthionine sulphoximine (BSO) resulted in a three-fold increase in melphalan sensitivity in all three cell lines. Our results indicate that melphalan toxicity is related to cell differentiation and GSH status of melanoma cells. Based on the observed relationship between dopa oxidase, GSH/GST levels and drug toxicity, it is proposed that competition for the GSH pool between quinonoid melanin intermediates and melphalan could diminish drug conjugation and increase cytotoxicity.

In vivo induction of pyrimidine dimers in human skin by UVA radiation: initiation of cell damage and/or intercellular communication?

Pyrimidine dimers participate as important factors in ultraviolet-induced lethality, mutagenicity and tumorgenicity. Substantial efforts have been made in recent years to understand the induction of pyrimidine photodimers and their repair in human skin cells exposed to low physiological fluences of UV-light. Dimers are known to be efficiently induced after UVC and UVB irradiation, but these photoproducts are also highly induced in DNA isolated from human skin irradiated with UVA. By using a sensitive immunohistochemistry dimer detection assay, we confirm that in vivo UVA radiation induces substantial amounts of these DNA changes in the epidermis; in addition, this technique detects them far into the reticular dermis. A considerable number of these photodimers were also seen in non-irradiated control skin up to two centimeters from the irradiation site. All the lesions persist for at least two days post-irradiation. These results sustain the hypothesis that pyrimidine dimer formation and excision could be a modality of epidermal communication.

Induction of the putative protective protein ferritin by infrared radiation: implications in skin repair.

The modification of ferritin in human skin cells in vitro and in vivo following infrared-A irradiation by immunohistochemical analysis and ELISA were evaluated. In addition, we observed that IR-A is not capable of inducing frank damage to DNA (pyrimidine dimers, p53), induction of oxidative stress proteins (heme oxygenase, nitric oxide, superoxide dismutase, heat shock proteins) or proteases (collagenase, stromelysin, gelatinase) involved in carcinogenesis and photoaging of the skin. in vivo, basal levels of ferritin were heterogeneous for all individuals tested but all showed ferritin to stain precisely in the basal layer of unirradiated epidermis. Following IR-A radiation, the ferritin increase was localized to epidermal tissue and showed an increase from 120 to 220%. Parallel to the in vivo analysis, dermal fibroblasts were cultured from six individuals. Quantitative analysis for ferritin in cultured fibroblasts was assessed by ELISA and increases were seen to be dose-dependent and up to 130% of basal levels of ferritin following infrared-A. Our findings indicate that the putative defense system of ferritin that exists in human skin in vivo can be induced by infrared-A radiation and that these wavelengths may prove to be beneficial for human skin. Importantly, following the same doses of IR-A that induced ferritin levels, there was no alteration seen for nuclear DNA type damage, oxidative stress proteins or proteases involved in the degradation of skin. The increased concentrations of this antioxidant in human skin following acute UV radiation could afford increased protection against subsequent oxidative stress.

Modulatory growth effects of 3T3 fibroblasts on cocultivated human melanoma cells.

Normal epidermal melanocytes are stimulated in vitro by fibroblast- and keratinocyte-derived growth factors. In this work, the paracrine responsiveness of human melanoma cells was investigated by cocultivation with growth-inhibited 3T3 fibroblasts. In short-term assays (3-4 days), melanoma cell growth was found to be improved if the cells were plated onto half-confluent 3T3 cell monolayers. In contrast, long-term cocultivation (greater than 7 days) resulted in significant growth inhibition of melanoma cells. These phenomena were not observed when melanoma cells were grown in 3T3 cell supernatants, which suggests a possible mediation by extracellular matrix components and/or by heterologous junctional communications.