Identification of mutations in the uroporphyrinogen III cosynthase gene in German patients with congenital erythropoietic porphyria.

The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes along the heme biosynthetic pathway. Congenital erythropoietic porphyria is a very rare disease that is inherited as an autosomal recessive trait and results from a profound deficiency of uroporphyrinogen III cosynthase, the fourth enzyme in heme biosynthesis. The degree of severity of clinical symptoms mainly depends on the amount of residual uroporphyrinogen III cosynthase activity. In this study, we sought to characterize the molecular basis of congenital erythropoietic porphyria in Germany by studying four patients with congenital erythropoietic porphyria and their families. Using PCR-based techniques, we identified four different mutations: C73R, a well-known hotspot mutation, the promoter mutation -86A that was also described previously, and two novel missense mutations, designated G236V and L237P, the latter one encountered in the homozygous state in one of the patients. Our data from the German population further emphasize the molecular heterogeneity of congenital erythropoietic porphyria as well as the advantages of molecular genetic techniques as a diagnostic tool and for the detection of clinically asymptomatic heterozygous mutation carriers within families.

Specific induction of metallothionein in hairless mouse skin by zinc and dexamethasone.

The stimulation of metallothionein (MT) synthesis in mouse skin after i.p. treatments with various doses of dexamethasone or zinc was demonstrated. Specific MT mRNA induction was determined by Northern analysis. Zinc was a more efficient inducer than dexamethasone. The maximal MT accumulation occurs after i.p. injections of 50-100 mg dexamethasone/kg body weight. The possible role of MT in the skin is briefly discussed.

Type III collagen aminopropeptide levels in serum of patients with progressive systemic scleroderma.

Sera from 101 patients with progressive systemic scleroderma were analyzed for circulating aminopropeptides of type III collagen using a radioimmunoassay which measures the intact and degraded forms (Fab assay). About 41% of the patients were found to have values above the normal range. A good correlation was observed between elevated levels of aminopropeptides and the degree of involvement of the skin and internal organs in the patients. Most patients (89%) with an active progression of the disease but not those in a stationary phase showed increased serum levels of aminopropeptides. Treatment with corticosteroids apparently normalized the levels of aminopropeptides. Only minor changes were observed with an antibody-based radioimmunoassay which measures primarily the intact form of the aminopropeptide.

Photosensitization of uroporphyrin augments the ultraviolet A-induced synthesis of matrix metalloproteinases in human dermal fibroblasts.

Porphyria cutanea tarda is characterized by severe connective tissue damage in sun-exposed skin. The regulated synthesis and degradation of the extracellular matrix by various matrix metalloproteinases (MMPs) determine its amount and composition within the skin. In this study, we therefore asked whether long-wave ultraviolet irradiation (340-450 nm) in conjunction with uroporphyrin I could modulate the synthesis of MMPs with substrate specificities for dermal (collagens I, III, V; proteoglycans) and basement membrane components (collagens IV, VII; fibronectin; laminin) and whether synthesis of the counteracting tissue inhibitor of metalloproteinases is also affected. After irradiation of uroporphyrin-pretreated fibroblasts, specific mRNAs of MMP-1 and MMP-3 increased concomitantly up to 2.7-fold compared with ultraviolet-irradiated cells and up to 10-fold compared with mock-irradiated or uroporphyrin I-treated controls. In contrast, mRNA levels of tissue inhibitor of metalloproteinases remained unaltered. Similar results were obtained by immunoprecipitation. Gelatin and casein zymography revealed increased proteolytic activity of MMP-2 and MMP-3 in blister fluids of patients with porphyria cutanea tarda, indicating that similar events may occur in vivo. Using deuterium oxide as enhancer and sodium azide as quencher of singlet oxygen, we could increase or reduce MMP synthesis, suggesting that singlet oxygen is the major intermediate in the upregulation of MMPs after irradiation of uroporphyrin-pretreated fibroblasts. Taken together, our results show that ultraviolet irradiation alone, and to a greater extent in conjunction with uroporphyrin I, results in an unbalanced synthesis of MMPs that may contribute to the destruction of the dermis and basement membrane, leading to blistering and accelerated photoaging in porphyria cutanea tarda patients.

Variegate porphyria: identification of a nonsense mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders of porphyrin metabolism that result from inherited or acquired aberrations in the control of the heme biosynthetic pathway. Variegate porphyria is characterized by a partial reduction in the activity of protoporphyrinogen oxidase. In this study, we identified the first nonsense mutation in a family with variegate porphyria. The mutation consisted of a previously unreported G-to-T transversion in exon 5 of the protoporphyrinogen oxidase gene, resulting in the substitution of glutamic acid by a nonsense codon, designated E133X. Our investigation establishes that a nonsense mutation in the protoporphyrinogen oxidase gene is the underlying mutation in this family with variegate porphyria.

UVA-induced autocrine stimulation of fibroblast-derived-collagenase by IL-6: a possible mechanism in dermal photodamage?

Like other cytokines, IL-6 has been reported to stimulate collagenase. In this study we were interested in whether IL-6 is involved in the ultraviolet (UV) mediated up-regulation of fibroblast-derived collagenase. Confluent fibroblast monolayers were irradiated under standardized conditions. Following UVA irradiation the bioactivity of IL-6 increased up to fiftyfold in the supernatants of irradiated compared to mock-irradiated fibroblasts. As determined by Northern blot analysis this was also reflected on the pre-translational level by a tenfold increase of IL-6-specific mRNA following UVA irradiation. Induction of IL-6-specific mRNA was maximal at 6 h post-irradiation, thus clearly preceding the maximal induction of collagenase mRNA at 24 h post-irradiation. To elucidate the regulatory role of IL-6 in the UVA induction of fibroblast-derived collagenase, monospecific polyclonal neutralizing antibodies directed against recombinant human IL-6 and antisense oligonucleotides specifically inhibiting the translation of IL-6 mRNA were used at various concentrations. The amount of UVA-induced collagenase mRNA was reduced in a dose-dependent manner when antibodies or specific antisense oligonucleotides were present during and after irradiation. Taken together our data provide first evidence that UVA enhances IL-6 synthesis and secretion in fibroblasts. IL-6 induces via an autocrine mechanism collagenase and may thus contribute to the actinic damage of the dermis.

Migration of a human keratinocyte cell line (HACAT) to interstitial collagen type I is mediated by the alpha 2 beta 1-integrin receptor.

The migratory response of the human keratinocyte cell line HaCaT to collagen type I and the molecular mechanism underlying collagen-mediated migration have been analyzed. The migratory response of HaCaT cells to collagen type I consisted of a dose-dependent migration to insoluble step gradients of substratum-bound collagen (haptotaxis) and to gradients of soluble collagen (chemotaxis). Checkerboard analysis demonstrated a minor chemokinetic component. Denatured collagen type I was less chemoattractive than the native triple-helical form. Pre-treatment of cells with 25-250 micrograms/ml of synthetic peptides containing the fibronectin cell-recognition sequence RGD (Arg-Gly-Asp) resulted in a concentration-dependent inhibition of fibronectin-mediated chemotaxis, whereas chemotaxis to collagen was not affected. We then investigated the role of VLA/collagen-receptors for collagen type I-induced chemotaxis. Monoclonal antibody (MoAb) 5E8, which selectively blocks function of the alpha 2 subunit of the VLA-2/collagen receptor, dose-dependently inhibited the chemotactic response of HaCaT cells to collagen. This effect was specific for collagen-mediated chemotaxis because the chemotactic response to fibronectin remained unaffected. In contrast, a function blocking MoAb directed to the alpha 3 subunit of the coexpressed VLA-3 receptor, which is also capable of binding collagen, had no effect. However, function blocking MoAb directed to the beta 1-chain of integrins completely inhibited chemotaxis to collagen type I. Based on our results, we propose that the chemotactic migration of the human keratinocyte cell line (HaCaT) to collagen type I is specifically mediated by the RGD independent VLA-2/collagen receptor (alpha 2 beta 1) of the integrin family.

Wavelength dependency of photodynamic effects after sensitization with 5-aminolevulinic acid in vitro and in vivo.

A promising new therapeutic modality for skin cancer, administration of the heme precursor 5-aminolevulinic acid followed by light irradiation, is known as photodynamic therapy. Photofrin, the only clinically approved sensitizer, has an absorption maximum at 630 nm, the wavelength used in most experimental and clinical trials with 5-aminolevulinic acid. We investigated photodynamic efficacy of irradiation with coherent light at wavelengths ranging from 622 to 649 nm in vitro and in vivo as well as the content and distribution of intracellular porphyrin after administration of 5-aminolevulinic acid. HaCaT immortalized human keratinocytes were sensitized with 30 micrograms/ml 5-aminolevulinic acid for 24 h in vitro. By cell viability determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, the best cell-killing effects were observed after irradiation at 635 nm. Using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, we confirmed these results in vivo: tumor growth was markedly delayed in animals treated with 100 mg/kg 5-aminolevulinic acid intravenously and irradiated with coherent light at 635 nm as compared to animals irradiated at 630 nm. This photodynamic effect is probably mediated by large amounts of the photosensitizing porphyrin, protoporphyrin IX, localized in cell membranes as visualized by confocal laser scan microscopy and as determined by high pressure liquid chromatography in vitro. The results suggest that irradiation at 635 nm with a coherent light source is more effective than irradiation at 630 nm for photodynamic therapy with 5-aminolevulinic acid.

Effect of hexachlorobenzene on the activities of hepatic alcohol metabolizing enzymes.

To study the effect of experimental hepatic porphyria on the activities of hepatic alcohol metabolizing enzymes, female rats received a chow diet containing 0.05% hexachlorobenzene (HCB). After long-term HCB treatment for 60 days hepatic porphyria developed as evidenced by increased hepatic delta-aminolevulinic acid synthase activity and enhanced urinary excretion of delta-aminolevulinic acid, porphobilinogen and total porphyrins. Concomitantly, the activities of the hepatic microsomal ethanol oxidizing system (MEOS) were strikingly augmented by 213% (P less than 0.05) and 177% (P less than 0.01) when expressed per g of liver wet weight or per 100 g of body weight, respectively, whereas hepatic alcohol dehydrogenase activities remained virtually unchanged. Moreover, hepatic catalase showed only a trend for a slightly lower enzymic activity under these experimental conditions. The present data therefore show that experimental hepatic porphyria is associated with alterations of hepatic MEOS activities, which in turn may be a factor for the manifestation of human hepatic porphyrias in the course of alcohol consumption.

Influence of chronic alcohol consumption on hepatic heme and porphyrin metabolism.

To study the effect of prolonged alcohol consumption on hepatic heme and porphyrin metabolism, female Wistar rats were fed for 60 days a nutritionally adequate liquid diet containing 36% of total calories as ethanol, whereas the control diet was isocaloric and contained no alcohol. Compared to pair-fed controls, the administration of the alcohol diet resulted in an increased hepatic activity of delta-aminolevulinic acid synthase by 223% (112.3 +/- 19.6 nmoles/hr/100 g b.wt. vs. 362.8 +/- 42.5; P less than 0.01), an enhanced urinary excretion of delta-aminolevulinic acid by 101% (64.8 +/- 11.8 nmoles/day vs. 130.8 +/- 22.4; P less than 0.05), and an augmented urinary output of total porphyrins by 142% (1.2 +/- 0.2 nmoles/day vs. 2.9 +/- 0.5; P less than 0.05). Concomitantly, the hepatic content of cytochrome P-450 was significantly enhanced and that of hepatic catalase activity marginally increased, whereas the hepatic iron content remained unaltered. In summary, the feeding of rats with a liquid alcohol diet for 60 days results in changes of hepatic heme and porphyrin metabolism which are associated and may be causally related with an induction of hepatic hemoproteins and subsequent derepression of hepatic delta-aminolevulinic acid synthase, whereas hepatic iron appears to play no pathogenic role.

Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria.

The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Photodynamic therapy in dermatology.

Photodynamic therapy (PDT) uses exogenously administered or endogenously formed photosensitizers activated by light to induce cell death via formation of singlet oxygen and other free radicals. Photodynamic therapy is increasingly used for the treatment of skin cancers and other indications. The efficacy of PDT depends on the structure of the photosensitizer, the administration modality, the light source, and the treatment procedure. We reviewed the most recent clinical and experimental developments in PDT research related to dermatology. The substrate under most intense investigation in PDT research is delta-aminolevulinic acid (ALA). Photodynamic therapy with topically applied ALA has been shown to be highly efficient in the treatment of cutaneous neoplasms by using intralesionally formed porphyrins as photosensitizers. For solar keratoses, best response rates have been described. delta-Aminolevulinic-PDT is also efficient in the treatment of superficial basal cell and squamous cell carcinomas. In addition, the fluorescence of ALA-induced porphyrins under a Wood light is highly selective in neoplastic cutaneous tissue and offers a useful technique in detecting and delineating skin tumors with ill-defined borders.

Hemorrhagic bullous amyloidosis. A histologic, immunocytochemical, and ultrastructural study of two patients.

In patients with bullous hemorrhagic amyloidosis of the skin, the skin lesions were the first manifestations of a plasma cell dyscrasia. Both cases were characterized by similar clinical, histologic, and ultrastructural findings showing an intradermal blister within deposits of amyloid substances. Immunohistologic investigations with a panel of antibodies directed against amyloid fibril proteins showed reactivity of the amyloid deposits with an anti-A lambda serum in both patients.

Monoclonal gammopathy in scleredema. Observations in three cases.

A monoclonal gammopathy was observed in three patients with long-term and widespread scleredema (Buschke's disease). There was no evidence of multiple myeloma in any patient. Deposition of monoclonal immunoglobulins in the skin was not detected by direct immunofluorescence microscopy. In contrast to scleromyxedema (lichen myxedematosus), from which scleredema can be distinguished clinically and histologically, the monoclonal immunoglobulins in two cases were of IgG2-kappa and IgG3-kappa type. Only one of the three patients had IgG1-lambda paraproteinemia, which is frequently seen in scleromyxedema. Our findings suggest that diffuse scleredema may be characterized by paraproteinemia but that the possible role of monoclonal immunoglobulins in the pathogenesis of this disease has yet to be resolved.

Cutaneous bacillary angiomatosis in a patient with chronic lymphocytic leukemia.

BACKGROUND: Bacillary angiomatosis is a recently described vascular disorder that is associated with infection by Bartonella henselae (formerly known as Rochalimaea henselae) and Bartonella quintana (formerly known as Rochalimaea quintana); this disorder usually occurs in patients with human immunodeficiency virus infection. We report a case of cutaneous bacillary angiomatosis that occurred in a patient with chronic lymphocytic leukemia. OBSERVATIONS: A 55-year-old man with chronic lymphocytic B-cell leukemia, Rai stage IV, presented with multiple angiomatous papules that clinically resembled pyogenic granulomas. Histopathologic examination revealed circumscribed lobules of small vessels with plump endothelial cells, numerous neutrophils, and abundant nuclear dust; these features were diagnostic for bacillary angiomatosis. The diagnosis was confirmed by the Grocott-Gomori methenamine-silver nitrate stain that revealed argyrophilic bacteria and by ultrastructural demonstration of bacillary structures with trilaminar walls. Treatment with clarithromycin led to complete resolution of the lesions within 4 weeks. CONCLUSIONS: This case emphasizes that (1) bacillary angiomatosis must be considered in the differential diagnosis of vascular lesions in immunocompromised patients without human immunodeficiency virus infection, (2) Grocott-Gomori methenamine-silver nitrate stain is a simple and satisfactory alternative to the Warthin-Starry stain for the demonstration of bacilli in this condition, and (3) clarithromycin is an effective oral antibiotic for the treatment of this disease.

Multiple exercise-related mononeuropathy with abdominal colic.

Hereditary neuropathy with liability to pressure palsies (NLPP) is a rare disease characterized by recurrent sensory-motor deficits precipitated by exposure to minor pressure. This report describes a variant of this neuropathy in 5 siblings suffering from painful palsies after strenuous work with concurrent episodes of abdominal colic resembling that of acute intermittent porphyria. Electrophysiological studies of the index case showed the typical abnormalities of motor and sensory nerve conduction, including clinically non-affected nerves. Light and electron-microscopic examination showed the characteristic lesions of the NLPP with sausage-like swelling of the myelin sheaths. In addition, non-compacted, "loose" myelin lamellae were frequently observed in association with distended Schmidt-Lantermann incisures. Non-compacted myelin was a prominent finding in this type of demyelinating neuropathy. We suggest that an unknown metabolic factor may induce both demyelination of peripheral nerve fibers and functional disturbance in autonomic nerves leading to attacks of abdominal pain.

Ex vivo application of delta-aminolevulinic acid induces high and specific porphyrin levels in human skin tumors: possible basis for selective photodynamic therapy.

In photodynamic therapy with topically applied delta-aminolevulinic acid porphyrins are acting as photosensitizers. The profile of porphyrin metabolites in normal or in neoplastic skin after administration of delta-aminolevulinic acid has not been determined in detail yet. Thus, to study porphyrin biosynthesis in human skin an organ culture model was developed. Explant pieces of normal skin, keratoacanthoma, and basal cell carcinoma were incubated with 1 mM delta-aminolevulinic acid for 36 h. Levels of delta-aminolevulinic acid, porphyrins and porphyrin metabolites were measured in tissues and supernatants. After incubation with delta-aminolevulinic acid, higher porphyrin levels were demonstrated in tumors as compared to normal skin. In supernatants, most of formed porphyrins, preferentially highly carboxylated porphyrin metabolites, were measured. The pattern of synthesized porphyrins differed between normal and neoplastic skin explants. In tissues of basal cell carcinomas protoporphyrin was preferentially shown and tissues of keratoacanthomas were characterized by a predominance of coproporphyrin as compared to normal skin. The results show that explant cultures offer an easy approach to examine the porphyrin biosynthesis of various tissues. The tumor-specific delta-aminolevulinic acid metabolism indicates additional porphyrin metabolites such as coproporphyrin apart from protoporphyrin as effective photosensitizers and may offer a novel approach to tumor-selective photodynamic damage.

Gradient centrifugation analysis of steroid-hormone binding in human malignant melanoma.

Vertical tube rotor sucrose gradient centrifugation in the presence of the protease inhibitor sodium molybdate provide to give a deeper insight into the steroid hormone receptor status of human malignant melanoma (MM) as did previous studies using dextran-coated charcoal procedure only. As compared to endocrine dependent breast cancer, the oestrogen binding capacity of MM is low. Although in some biopsies fairly high concentrations of progestin binders were detected, the sedimentation properties were not receptor typic. Androgen binding was found to be negligible. In contrast, the presence of glucocorticoid receptors is a common feature of human MM. A human MM cell line was demonstrated to contain glucocorticoid receptors only.

[Spun glass hair (author's transl)]. / Das Glaswoll-Haar ("Spun Glass" Hair).

A 5-year-old girl with the characteristic clinical picture of "spun glass hair" is described; additionally she suffers from atopic dermatitis. This case is of special interest since the child not only suffers also from endogenic eczema, but that another member of the family (child's aunt) also suffers from the same hair anomaly. The diagnosis is based upon the clinical symptom that the hair cannot be managed, and is proved by observation of triangular and kidney-shaped cross-sections taken from the child's hair. Electron scan microscopic examination of the hair revealed grooves on the surface of the hair which exhibit similarity to the hair-condition pili canaliculi. The following physical properties of these irregularly shaped hairs were measured: Fibre thickness, tensile strength, extent of fibre expansion shortly before breakage, and form elasticity. None of these properties were found to differ from those of normal hair.

Influence of chloroquine on the porphyrin metabolism.

Rats were treated with the well-known porphyrogen hexachlorobenzene (HCB) to induce experimental porphyria. At the same time another group of rats was treated with chloroquine in addition to HCB. The HCB-induced increase of the urinary excretion of porphyrin precursors could thereby be reduced to normal levels and the porphyrin excretion rates were decreased significantly in comparison to those of the other group. The delta-aminolevulinate synthase in the liver of the animals was slightly increased by exclusive treatment with chloroquine, which in the HCB-treated rats chloroquine led to a dramatic decrease in the key enzyme of the porphyrin (heme)-biosynthesis. The influence of chloroquine on the HCB-induced increase of the cytochrome P-450 content and the dependent enzymatic activities were different. The 7-ethoxycumarin deethylase and the arylhydrocarbon hydroxylase activities were not influenced, whereas the increased aminopyrine-N-demethylase activity was reduced to nearly normal levels. Our findings indicate that chloroquine acts by reduction of the delta-aminolevulinate synthase activity, probably by influencing the regulation of the key enzyme of the heme biosynthesis, which is enhanced in human porphyria cutanea tarda, as well as in the HCB-induced porphyria of the rats.