Search

We present a case of a 78-year-old man suffering from a chronic psoriasiform eruption, with rapid deterioration over the previous 8 weeks. Langerhans' cell histiocytosis with skin and bone involvement was diagnosed, and there was evidence of liver and lung dysfunction. The patient was treated with prednisolone and etoposide, and initially experienced a partial improvement. Three weeks later, haemophagocytic lymphohistiocytosis and subsequently a large pulmonary abscess with sepsis attributed to opportunistic gram-negative enterobacteriaceae Serratia marcescens developed, and the patient died. The present case of Langerhans' cell histiocytosis is of particular interest because of the previously unreported development of haemophagocytic lymphohistiocytosis in the elderly population.

The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.

Background/purpose: Ultraviolet (UV) radiation; induces a variety of responses in the skin, including tanning and inflammation, and may also act as a carcinogen. As epidermal melanocytes are seen as the major targets of UV light, the present study was conducted to evaluate the direct effects of UVA and UVB irradiation on melanocytes in vitro., Methods: Normal human epidermal melanocytes (NHM) were exposed on 3 consecutive days to UVA (0.072-7.2 J/cm2) and UVB (7.2-48 mJ/cm2), respectively, and changes of morphology, cell number, melanin synthesis and antigen expression (APAAP technique) were determined 5 days after the first exposure., Results: UVA radiation caused only minimal effects on NHM by slightly inducing expression of the activation marker HMB-45 and decreasing expression of the proliferation marker Ki-67. No changes of morphology, cell number or melanin synthesis were detectable with any of the applied doses. On the other hand, UVB radiation significantly induced dendrite formation and decreased the number of NHM in a dose-dependent manner (74% of the controls at 7.2 mJ/cm2, 64% at 14.4 mJ/cm2 and 28% at 36 mJ/cm2). Significant induction of the activation marker HMB-45 was found in parallel to decreased expression of the differentiation marker K.1.2.58. UVB doses >or=9.6 mJ/cm2 also resulted in significant downregulation of the proliferation marker Ki-67, confirming the data of the cell counts, and melanin content was increased in NHM (20% over the controls, P<0.01) after applying 7.2 mJ/cm2 UVB., Conclusion: Our results may suggest that the effect of UVB radiation in skin is due to direct activation of melanocytes, whereas skin tanning caused by UVA is mediated rather in an indirect way.

We report a case of Netherton syndrome manifested as congenital ichthyosiform erythroderma, trichorrhexis invaginata and atopy, who in early adulthood developed multiple, aggressive epithelial neoplasms in sun-exposed areas of the skin, in areas with papillomatous skin hyperplasia and at the left parotid region. The occurrence of cutaneous neoplasia has been reported in syndromes with congenital ichthyosis and suggests that the underlying genetic defects may cause the development of cancer in prone patients., (Copyright 2003 S. Karger AG, Basel)

Protein kinase C (PKC), a calcium and phospholipid-dependent kinase, has been implicated in carcinogenesis of melanocytic cells. However, its role in melanoma cell growth remains controversial. We therefore investigated the growth dependence of PKC isozyme expression in human normal melanocytes and melanoma cells. Logarithmic and stationary growth phases in culture were clearly distinguished by nuclear cell staining with the proliferation marker Ki-67. PKC-beta I and -beta II were expressed exclusively in normal melanocytes but not in melanoma cells, whereas PKC-gamma was not found in any of the cultures studied. Low PKC-delta, -epsilon and -zeta mRNA levels were detected by RT-PCR in proliferating melanoma cells and higher in confluent non-proliferating cells, whereas levels of PKC-alpha mRNA remained rather stable. Subcellular fractionation and immunoblotting revealed accordingly low expression of PKC-alpha, -delta, -epsilon, and -zeta in the logarithmic growth phase of melanoma cells, with subsequent increase of expression and of membrane association in the stationary phase. Only weak differences were detected between the growth phases in normal melanocytes for the respective PKC isozymes, except for membrane-associated PKC-beta I and -beta II which were clearly elevated in confluent melanocyte cultures. These data suggest that certain PKC isozymes are involved in the intracellular signalling that regulates melanoma cell proliferation, and may function as suppressors of tumour cell growth.

The primary neuroendocrine carcinoma of the skin or Merkel cell carcinoma (MCC) is a skin tumor with aggressive biological behaviour. Experimental models for investigating the biological properties of the tumor are prerequisite for developing new therapeutic approaches. In this study, we report the establishment and characterisation of a cell line derived from the lymph-node metastasis of a patient with highly aggressive MCC. Merkel carcinoma cells (MCC-1) grew as floating aggregates in suspension cultures for more than two years and over 70 subcultures. The proliferation rate in suspension cultures was rather moderate with a population doubling time of 69 h. The immunocytochemical pattern of the cultured MCC-1 was similar to that of the original tumor with expression of cytokeratin 18, neuron-specific enolase, neurofilaments, and synaptophysin. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) revealed presence of chromogranin A mRNA in the MCC-1 cell line. Furthermore, electron microscopy yielded the rare finding of neuroendocrine granules in the cytoplasm of the cultured cells. The cell line MCC-1 was able to form colonies in soft agar. Nude mice developed solid tumors with similar histology to the original tumor after subcutaneous and intravenous injections of cultured MCC-1, and malignant ascites was seen after intraperitoneal injection. Also, two MCC-1 sublines were established by reculturing cells from the xenografts grown in vivo and immunocytochemistry confirmed their neuroendocrine origin. The MCC-1 line may thus serve as a model for studying the biology and the metastatic potential of Merkel cell carcinoma., (Copyright 2001 Wiley-Liss, Inc.)

Background: Although Merkel cell carcinoma (MCC) exhibits specific clinical and histologic features, differentiation from other cutaneous neoplasms, such as lymphoma, metastatic oat cell carcinoma and malignant melanoma (MM), may sometimes be difficult., Objective: The aim of our study was to immunohistochemically differentiate MCC from MM., Methods: Paraffin sections from 6 cases of primary MCC and 6 cases of primary MM were investigated. For immunostaining, the APAAP method was used., Results: Neuron-specific enolase was positive in all cases of MCC, as well as in 2 cases of MM. Marked positivity for cytokeratins 18, 20 and chromogranin A was observed in the MCC group, whereas a complete absence of expression of these three markers was noted in the MM group. Immunostaining with HMB45 and NKI/C3 was positive in all cases of MM and negative in all cases of MCC. S-100 protein was positive in all but 1 case of MM. In contrast, only 1 case of MCC reacted with S-100 protein., Conclusion: Our results underline the role of immunohistochemistry in the diagnosis and differential diagnosis of MCC. In particular, the combination of neuron-specific enolase, cytokeratins 18, 20 and chromogranin A positivity for MCC and HMB45, NKI/C3 and S-100 protein positivity for MM is of great value in the distinction between these two cutaneous neoplasms., (Copyright 2000 S. Karger AG, Basel)

Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC-1 were added to fibronectin (FN)-, vitronectin (VN)- or, as a control, bovine serum albumin (BSA)-coated wells and were stimulated with phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cell extracts and enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4-hr incubation, mRNA expression of interleukin (IL)-8 (and weakly of IL-6) was up-regulated in matrix-adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High-level de novo expression of IL-3 and of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed mainly in matrix-adherent cells. These changes were paralleled by the secretory pattern of HMC-1 cells after a 24-hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL-8, and both VN- and FN-adherent cells produced, almost invariably, a higher level of cytokines than BSA-exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.

2-Chloracetophenone (CN) is widely used as tear gas by police and civilians for self-defence. It may affect the eyes, respiratory system and skin, sometimes causing serious injuries. Both irritative and allergic contact dermatitis have been described. We report three police officers who experienced accidental escape of CN from their professional tear gas canisters. All of them showed localized dermatitis at the site of contact to CN, while widespread lesions appeared after 4 days in one case. Patch tests with the original involved tear gas dissolved in acetone (at 0.1-0.0001%) indicated an allergic reaction in two patients and an irritative reaction in the third. Occupational contact dermatitis due to CN seems to occur among police officers more often than is generally known. Infrequently, extensive health problems may be caused by CN when lesions spread over the integument. Therefore, an improvement of safety measures in occupational CN gas use is needed, especially aiming at avoidance of accidental leakage of canisters.

Previous studies have suggested that transforming growth factor-beta 1 (TGF-beta1) acts as an autocrine growth inhibitor on normal human melanocytes, while melanoma cells may not respond to this stimulus. The role of other TGF-beta isoforms such as TGF-beta2 and TGF-beta3 remained less well characterized. In the present study, the mRNA and protein levels of all three isoforms of TGF-beta were analyzed in a panel of human melanoma cell lines and in cultures of normal human melanocytes in vitro. Northern analysis showed that the degree of TGF-beta1, -beta2, -beta3 mRNA expression varied considerably in melanoma cells, whereas TGF-beta expression was very low in melanocytes. In melanoma cells, secreted amounts of TGF-beta1 and TGF-beta3 were found increased in comparison to normal melanocytes: 615 pg/ml vs. 118 pg/ml and 193 pg/ml vs. 30 pg/ml (mean values). In addition, low levels of TGF-beta2 were detected (mean value: 28 pg/ml). Although TGF-beta secretion increased, the proliferation of melanoma cells was found to be only moderately inhibited by TGF-beta isoforms, in contrast to its strong antiproliferative effect on normal human melanocytes: - 15%, -11%, and -18% vs. -52%, -46%, and -50% average inhibition at 0.5 ng/ml TGF-beta1, -beta2, and -beta3, respectively. The different efficacy of TGF-beta on melanocyte and melanoma cells was highly significant (P<0.0001); in addition, TGF-beta-dependent growth inhibition of melanoma cells from primary tumors vs. cells from metastases showed a trend for further decreased response for the metastatic populations (P< or = 0.075). Measurements of DNA synthesis revealed even more pronounced differences between melanocytes (-86%, -78%, and -80% inhibition, respectively, for TGF-beta1, -beta2, and -beta3) and melanoma cells (no inhibition). Our data show loss of responsiveness of melanoma cells to the growth-inhibitory function of TGF-beta isoforms but not of melanocytes. Although melanoma cells are not growth-inhibited by all three TGF-beta isoforms, they secrete significantly higher levels of TGF-beta, as compared to melanocytes. The reduced response indicates their escape from TGF-beta surveillance with ongoing tumor progression.

Overexpression of transforming growth factor-beta isoforms (TGF-beta1, -beta2, -beta3) has been previously reported in human melanoma cell lines and tumours. The aim of the present study was to evaluate the plasma levels of TGF-beta isoforms in melanoma patients. Significantly elevated levels of TGF-beta1 (4.2 x the controls, P = 0.0094) and of TGF-beta2 (1.5 x the controls, P = 0.012) but not of TGF-beta3 were measured in patients with disseminated but not locoregional melanoma. These results indicate systemic circulation of potentially immunosuppressive peptides of the TGF-beta family in end-stage melanoma patients.

Reverse transcriptase polymerase chain reaction for the detection of tyrosinase-mRNA-positive cells in peripheral blood of melanoma patients, as a possible marker of hematogenous dissemination, has demonstrated varying detection rates. This study examined the sensitivity and reproducibility of the technique using a protocol of multiple polymerase chain reaction to determine circulating melanocytic cells. For each of the 123 melanoma patients included in this study, four nested polymerase chain reactions were performed from two blood specimens requiring both polymerase chain reactions from at least one blood sample to be positive to consider a patient as positive. Thus, a definitive result was obtained in 98% of the cases, whereas only 1.6% lacked conclusive findings. Thus, we found a correlation between the tyrosinase detection rate and the clinical stage. Circulating tyrosinase-mRNA-positive cells were detected in 13% of patients with primary tumor, 17% with regional skin/lymph node metastasis, and 44% with distant metastasis. Positivity also correlated with known melanoma progression markers such as gender, tumor thickness, and histologic type. Positive results were obtained more frequently in (i) men compared with women, (ii) patients with thick primary melanomas (> 4 mm: 38%) compared with those with thinner tumors (1.1-4 mm, 22%; < or = 1 mm, 5%), and (iii) patients with nonclassifiable (38%), nodular (34%), and occult primary melanomas (30%) compared with those with acrolentiginous (17%), superficial spreading (9%), or lentigo maligna melanoma (0%). These findings suggest that detection of tyrosinase-mRNA-positive cells in peripheral blood is not an adequate marker for identifying melanoma patients with distant metastasis. Reverse transcriptase polymerase chain positivity in early melanoma stages, however, as corresponding to other prognostic parameters, may indicate increased risk for the development of hematogenous metastasis and may be of value as a progression marker.

Background and Objective: Systemic mastocytosis is a rather rare disorder involving the skin and several other organs. The aim of this study was to analyse the extent of extracutaneous manifestations in 14 adult patients who presented with prominent cutaneous involvement within the last 5 years., Results: The cutaneous lesions were clinically diagnosed as telangiectasia macularis eruptiva perstans in 2 patients, urticaria pigmentosa of varying extent in 11 and diffuse erythrodermic mastocytosis in 1 patient. All patients had extracutaneous manifestations with involvement of one additional organ system in 6/14 cases, two in 5/14 and three in 3/14. Ten out of 14 patients suffered from generalized pruritus, and 11/14 reported mild wheal formation, while 3/14 with multi-organ involvement mentioned recurrent flushing episodes. The gastro-intestinal tract was involved in 8/14 cases with an increase in gastric and colon mucosal mast cells in 5/8 cases and gastroduodenitis in 2. Bone marrow involvement was seen in 7/13 patients, hepatosplenomegaly in 2, anaemia in 2 and thrombocytopenia in 3. The disease had a duration of 0.5-32 years, clinical symptoms remaining basically unchanged. Malignant transformation was not seen; only 1 patient developed myelodysplastic syndrome within 2 years after the first cutaneous lesions., Conclusions: Our study shows that extracutaneous involvement should be carefully considered in adult patients with cutaneous mastocytosis. Systemic multi-organ mast cell disease in adults is a long-lasting disorder with recurrent episodes of varying clinical symptomatology. However, the disease shows rather slow progression, and malignant transformation is rare. Satisfactory management is achieved by symptomatic oral drug intake.

Background: The management of systemic sclerosis remains unsatisfactory. Thus far, the action of extracorporeal photopheresis (ECP) in severe systemic scleroderma has been evaluated in short-term studies, and only limited experience has been obtained with long-term application., Objective: The aim of the present study was to evaluate prospectively the long-term effect of ECP in a group of 16 patients suffering from severe scleroderma, showing visceral involvement and progressive clinical course., Methods: Fourteen patients with systemic scleroderma involving several organs, 1 with CREST syndrome and another with scleroderma-myositis overlap syndrome were treated with ECP over a period of 6-45 months. In 3 cases, gamma-IFN was additionally administered. Skin and visceral involvement were assessed by evaluating a series of clinical criteria and results from laboratory, imaging and functional tests., Results: Overall, clear improvement was encountered in 6 patients, mixed response in 2, stable disease in 3 and continuing progressive course in 5 patients. Four out of 6 patients with improvement were treated with ECP early after onset of scleroderma (< or = 2 years), whereas all patients with a progressive course under ECP had had scleroderma for longer than 2 years. Immunosuppressive drugs previously administered could be reduced or fully withdrawn under ECP treatment in 5 patients, but additional oral medication was introduced in 4 patients due to disease progression. Addition of gamma-IFN to ECP did not reveal further benefit. No side-effects were recorded under ECP treatment., Conclusions: Based on this observation, we believe that long-term ECP represents an effective treatment modality in severe scleroderma particularly when started early, with stabilization of the disease course and partial remission of the cutaneous findings, whereas visceral involvement, if present, may rarely improve.

We studied, in vitro, the stimulating effects of a commercial preparation of low molecular thymic peptides (TP) on the immunocytotoxicity of peripheral blood lymphocytes and monocytes from patients with breast tumors, melanoma and colorectal tumors. On average, tumor patients showed a lower natural killer (NK), lymphokine (IL-2) activated killer (LAK) cell and basic tumoristatic activity of monocytes, compared with healthy donors. There was no correlation between the NK-cell number and the NK-cell activity of the tumor patients. The TP showed no effects on the NK-cell activity in any group, yet elevated the deficient LAK-cell activity of tumor patients and that of healthy donors. On monocytes, TP enhanced the deranged tumoristatic activity only in tumor patients, while being slightly inhibitory on control monocytes. Dividing the donors on the basis of the TP effects on cytotoxicity of the mononuclear cells into TP-nonresponders and TP-responders, a higher number of TP-responders was found among tumor patients, compared with healthy donors. Moreover, a higher number of TP-nonresponders were observed with lymphocytes from colorectal tumor patients at advanced tumor stage. Therefore, on the basis of the applied immunocytotoxic assays, these results may provide a basis for selecting tumor patients, who may respond to TP in immunotherapy protocols.

We describe a patient with painful enlargement and severe nail deformity of both great toes. The patient furthermore presented with some minor diagnostic criteria for psoriasis such as nail pitting and had a positive family history of the disease. In the recent literature, such cases have been recognized as a new entity termed psoriatic onycho-pachydermo-periostitis (POPP). In this case report, POPP is discussed in relationship to other forms of psoriatic arthropathy and with special emphasis regarding its diagnostic criteria and therapeutic implications.

The oncogenic potential of translation initiation factors (eIF-4E and eIF-2alpha) has been described in previous studies leading to the definition of translational oncogenes. Two previously isolated cDNA clones, expressed differently in human melanoma cells and normal human melanocytes, were identified in this study as coding for the translation initiation factor eIF-4A1. Northern-blot analysis revealed consistent overexpression of eIF-4A1 mRNA in a panel of 14 melanoma cell lines (on an average 5.6 times higher than in cultures of normal human melanocytes). In contrast, the mRNAs of the other group-4 translation initiation factors (eIF-4A2, eIF-4B, eIF-4E and eIF-4gamma) were less and not consistently elevated. Cultures of congenital melanocytic nevi exhibited intermediate expression of eIF-4A1. Thus, eIF-4A1 overexpression seems to be an important feature of melanoma cells and might contribute to their malignant transformation.

Background: Merkel cell carcinoma (MCC) is an uncommon primary neuroendocrine skin tumor most often seen in the elderly. The clinical course varies. Treatment is controversial and few data on drug sensitivity are available., Objective: We evaluated the clinical course and treatment of 10 MCC patients and determined MCC chemosensitivity., Methods: Clinical records as well as laboratory and histopathologic data from 10 patients with MCC treated in our department were examined. Chemosensitivity to various chemotherapeutic agents and interferons of MCC cells from four patients was determined in a soft agar clonogenic assay., Results: MCC behaved as an aggressive tumor with early and frequent local relapses (4 of 10 patients at a 2.2-month average), regional (4 of 10 patients at 2.5 months), and distant metastases (5 of 10 patients 9.6 months after excision of the primary tumor). In all but one patient, regional metastases preceded distant ones. Metastatic spread was associated with an average survival of 21 months from the initial diagnosis. Long-term survival (53+ and 65+ months) was observed in two women. Wide excision of the primary tumor, alone or combined with adjuvant chemotherapy and radiotherapy, was the most effective treatment. In advanced disease, chemotherapy and radiotherapy were not able to induce long-term remission. In vitro assays for MCC drug sensitivity revealed cisplatin, doxorubicin, and vindesine to be the most active., Conclusion: MCC has a poor prognosis in advanced stages; therefore the primary tumor should be aggressively treated. The in vitro clonogenic assay may help to identify the chemosensitivity profile of MCC and to optimize chemotherapy protocols.

The effects of a thymic peptide preparation (TP) on the immunocytotoxicity and cytokine secretion of peripheral blood lymphocytes and monocytes from patients with colorectal tumors, breast tumors and melanoma were studied in vitro. On average, breast tumor and melanoma patients showed significantly lower natural killer (NK) cell activities than colorectal tumor patients and normal controls. In contrast, the generation of lymphokine (IL-2) activated killer (LAK) cells was found to be comparable within the different tumor diseases (24% cytotoxicity), but lower than in the group of normal controls. TP, being without any effects on NK cell activity in all groups, increased the deficient LAK cell activity of breast tumor and melanoma patients, as well as of normal controls? without significant effects on PBL from colorectal tumor patients. This increase was found to be associated with an increase of the IL-2 induced IFN-gamma and, on a lower level, TNF-alpha secretion, especially from breast tumor and melanoma patients. In addition, monocytes from these patients showed a deranged tumoristatic activity, compared to colorectal tumor patients and normal controls. The stimulation of monocytes by IFN-gamma greatly elevated the mean of the antitumor activity in all groups studied. TP being slightly effective on monocytes from melanoma patients, did not further enhance monocyte-mediated cytotoxicity when applied alone or in combination with IFN-gamma. Reduced basal monocytic chemokine levels were only found in the groups of melanoma (IL-8) and colorectal tumor patients (MCP-1), whereas RANTES secretion was increased, compared to normal controls. TP was active only in reducing the IL-8 secretion of monocytes from colon tumor patients. The results indicate that selected functions of peripheral blood mononuclear cells can be partially improved by the thymic peptide preparation.

Hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane is thought to be involved in the control of normal cell proliferation. The aim of this study was to determine the effect of the naturally occurring quinone analogue capsaicin (8-methyl-N-vanillyl-6-noneamide) on the NADH oxidase activity of plasma membranes and cell growth of human primary melanocytes, the A-375 and SK-MEL-28 human melanoma cell cultures. NADH oxidase activity was inhibited preferentially in the A-375 melanoma cells but not in the primary melanocytes, by capsaicin. Inhibition of growth and the NADH oxidase by capsaicin could be induced in resistant SK-MEL-28 melanoma cells by co-administration of capsaicin with t-butyl hydroperoxide, a mild oxidising agent. Death of the inhibited cells was accompanied by nuclear changes suggestive of apoptosis. With B16 mouse melanoma, capsaicin inhibited both the NADH oxidase activity and growth in culture. Growth of B16 melanoma, transplanted in C57BL/6 mice, was significantly inhibited by capsaicin injected directly into the tumour site when co-administered with t-butyl hydroperoxide. The findings correlate the inhibition of cell surface NADH oxidase activity with inhibition of growth and capsaicin-induced apoptosis, and also suggest that the extent of inhibition may relate to the oxidation state of the plasma membrane.

Scleromyxoedema, a disseminated papular and sclerotic variant of lichen myxoedematosus, is a rare disease with a chronic progressive course, and little tendency towards spontaneous remission. The treatment of scleromyxoedema has been largely ineffective. Aggressive chemotherapeutic agents have been used, often leading to therapy-related morbidity and mortality. We report a 41-year-old woman with scleromyxoedema, associated with a monoclonal gammopathy of IgG-kappa type, whose condition almost completely cleared with 12 monthly sessions of extracorporeal photopheresis. The patient had previously not responded to isotretinoin, and chlorambucil with prednisolone.

A cutaneous metastatic angiosarcoma was diagnosed in a 79-year-old woman 19 years after radiotherapy for a carcinoma of the uterine cervix. The diagnosis was confirmed by immunohistochemical staining (factor VIII-related antigen and BMA 120) and electron microscopic examination. Surgical treatment of the large tumour, which was situated in the gluteal region, was not feasible, but electron beam therapy resulted in complete remission. However, a further metastasis occurred in the inguinal region, and management by total excision, radiotherapy, and interferon-alpha treatment was unsuccessful. The patient died 28 months after the initial diagnosis of the neoplasm.

In the present study the action of various cytokines as regulators of human melanocyte growth and differentiation was examined in vitro. Primary melanocyte cultures were obtained in complete medium free of 12-O-tetradecanoylphorbol-13-acetate or serum. First passage melanocytes were treated with various concentrations of recombinant tumour necrosis factor alpha and beta (rTNF-alpha, rTNF-beta), as well as with various recombinant interleukins (rIL-1a, rIL-1b, rIL-2, rIL-3, rIL-4 and rIL-6) for 6 days in complete medium and for 6 and 12 days in a mitogen-reduced medium variant. The 4-methylumbelliferyl heptanoate fluorometric microassay and Ki-67 staining were used for assessing cell proliferation, and the immunophenotype was evaluated using various monoclonal antibodies. Melanocyte proliferation in complete medium was inhibited by rTNF-alpha (-24%), rTNF-beta (-17%), rIL-1a (-21%), rIL-1b (-18%) and rIL-6 (-29%); in contrast, rIL-2, rIL-3 and rIL-4 had no antiproliferative effect. Measurements of Ki-67-positive nuclei confirmed these results. In the reduced medium variant, none of the above cytokines inhibited melanocyte proliferation. Recombinant TNF-alpha and rTNF-beta markedly reduced the expression of the pigment cell-associated antigens HMB-45 and K.1.2, and they enhanced the expression of VLA-2, ICAM-1 and HLA class I antigens and strongly induced HLA-DR. Similar changes were induced by rIL-1a, rIL-b and rIL-6, and rIL-2 decreased the expression of HLA class I antigens and of ICAM-1. In conclusion, several cytokines inhibited the growth and modulated the phenotype of melanocytes in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Aberrant proliferation of tumor cells characterizes cancer growth. Investigations of cellular growth control mechanisms have contributed to our understanding of carcinogenesis and to the identification of compounds with specific antitumor activity. Many cytokines have been found to act on melanoma tumors, either produced by the tumor cells themselves or by infiltrating host cells. Purified cytokines allowed direct comparison of the growth response between normal human melanocytes and malignant melanoma cells. The present paper summarizes results of a series of our own experiments not yet published and data from a review of the recent literature. Proliferation of normal human melanocytes is enhanced by several cytokines, including basic fibroblast growth factor (bFGF), melanoma growth stimulatory activity (MGSA), hepatocyte growth factor (HGF), and mast cell growth factor (MGF). Melanoma cells are additionally stimulated by epidermal growth factor (EGF)/transforming growth factor alpha (TGF-alpha) and nerve growth factor (NGF). Tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta 1), and interleukin (IL)-6 are all potent inhibitors of melanocyte growth, but they are less effective on melanoma cells or even stimulate their growth. Interferon (IFN)-alpha and IFN-gamma inhibited proliferation of melanoma cells but not of melanocytes, whereas IFN-beta showed antiproliferative effects in both cell types. These findings suggest an alteration in growth control mechanisms during melanocyte transformation and possibly play a role in melanoma pathogenesis.

Experimental animal models have shown that various cytokines, depending of their specific properties, may support growth and metastasis of tumor cells or even lead to tumor rejection. The analysis of expression of cytokine genes by melanoma cell lines indicated that melanoma cells constitutively produce both autostimulatory and inhibitory cytokines. Using reverse transcriptase polymerase chain reaction analysis, simultaneous expression of several cytokines, including interleukin-1 beta (IL-1 beta), IL-6, IL-8, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor, by melanoma cells was found. The same cytokine transcripts were detected in melanocytes, suggesting that cells of the melanocytic lineage express a specific pattern of cytokines in vitro. All these cytokines are known to be able to stimulate effector cells of the host. Additionally, production of mRNA for IL-10, a cytokine with potential immunosuppressive properties, was detected in melanoma cells and melanocytes. These and other cytokines are likely to be involved in the immune response to cancer and at this time it is unknown what the net effects of multiple cytokines are on the outcome of the host response to tumor.

The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response.

It has been considered that the growth of human melanoma cells is positively and negatively regulated by transforming growth factors. To investigate further the role of transforming growth factor beta (TGF-beta) in melanoma biology, we analysed paracrine and autocrine growth regulatory properties of TGF-beta in normal human melanocytes and malignant melanoma cell lines in vitro. Exogenously added TGF-beta 1 potently inhibited normal melanocyte proliferation and DNA-synthesis in all cultures examined; in contrast, TGF-beta 1 inhibited only moderately or not at all the growth of cultured melanoma cells. Melanoma cell lines established from metastatic lesions were found to be less sensitive to TGF-beta 1 than those derived from primary melanomas. TGF-beta 1 resistance correlated with high levels of active TGF-beta secreted by metastatic cell lines. Inactivation of endogenously produced TGF-beta by neutralizing anti-TGF-beta antibody resulted in the stimulation of cell proliferation of a TGF-beta-sensitive primary melanoma cell line but not of a resistant metastatic one. These findings suggest that TGF-beta may function as a paracrine and autocrine growth inhibiting factor in the growth regulation of human melanocytic cells. The gradual loss of response of melanocytic cells to TGF-beta during malignant progression suggests that escape of melanoma cells from growth regulation by TGF-beta could be involved in melanoma oncogenesis.

Immunohistochemical and immunoserological evidence supports the involvement of both cell-mediated and humoral mechanisms in the pathogenesis of melanocyte destruction in vitiligo. Punch biopsies from depigmented vitiliginous skin (VS), normal-looking pigmented skin (PS), and marginal skin (MS) from patients with generalized vitiligo (n = 15) were labeled with K 1.2.58, OKM1 (CD11b), Leu 11b (CD16), Leu 19 (CD56), IFN-gamma receptor, IL-2 receptor (CD25), IgG, IgM, C3c, and C3d MoAbs. In addition, in vitro effects of vitiligo sera (n = 13) on human newborn melanocytes (HMel) under different culture conditions were studied. The immunohistochemical findings showed absence of K 1.2.58+ epidermal melanocytes in VS and abnormal morphology in MS. In these areas, a few CD11b+ cells in the dermis and epidermis could be detected but no significant numbers of CD16+ or CD56+ cells were seen among the mononuclear cellular infiltrate. IL-2 and IFN-gamma receptors were clearly expressed by the cellular infiltrate. No significant deposition of complement or immunoglobulin was seen. The addition of vitiligo sera to HMel cultures induced a significant cellular proliferation. The stimulation of cell proliferation occurred regardless whether the sera were added alone or when preheated (56 degrees C for 1 hr) and then supplemented with a complement source (P < 0.01 at 2%, P < 0.001 at 10%, and P < 0.01 at 20% for sera alone) (P > 0.05 at 2%, P < 0.05 at 10%, and P < 0.01 at 20% for decomplemented sera plus complement).(ABSTRACT TRUNCATED AT 250 WORDS)

Various cytokines are involved in growth regulation of human melanoma cells. Malignant melanoma cells express multiple growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-alpha, platelet-derived growth factor (PDGF)-alpha, and melanoma growth stimulatory activity (MGSA), substances which are not expressed in normal human melanocytes. The simultaneous synthesis of growth factors and expression of their receptors by melanoma cells, leading to permanent stimulation of cell proliferation, has been clearly shown for bFGF and MGSA. This phenomenon has been designated autocrine growth stimulation. Increased or altered expression of growth factor receptors has been described for nerve growth factor (NGF) receptor, for PDGF-beta receptor and for a truncated form of epidermal growth factor (EGF) receptor encoded by the c-erb-B2 oncogene. Lymphokines are mainly involved in growth control of melanoma cells. Interferons (IFN)-alpha, -beta and -gamma, Interleukins (IL)-1 and -6 as well as tumour necrosis factor (TNF)-alpha inhibited melanoma cell proliferation, with the strongest effects displayed by IFN. TGF-beta which was found to inhibit proliferation of normal human melanocytes exhibited marginal effects on melanoma cells, or even stimulated their growth. In conclusion, a complex network of cytokines is involved in the regulation of melanoma cell growth. Further insight into these mechanisms may contribute to the finding of new strategies in melanoma therapy.

For serial cultivation of normal human melanocytes media supplemented with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) are largely employed. By using a culture medium that permits cultivation of melanocytes without TPA, the effects of TPA on melanocyte proliferation, phenotype, and susceptibility to lymphokine-activated killer cells were studied. Addition of 50 ng/ml TPA to the medium induced rapid dendrite formation and increased the cell proliferation rate by 16-63% in mitogen-rich media (four of seven cultures, p < 0.01), and by 237% in mitogen-reduced media (p < 0.001). Furthermore, several phenotypic changes indicating early stages of melanocyte transformation were induced by 50 ng/ml TPA. These included increased expression of melanoma progression-associated antigens such as A.1.43 and A.10.33, upregulation of nerve-growth factor receptor as well as of the melanocyte-activation marker HMB-45 and of histocompatibility class I antigens. In contrast, the expression of the differentiation marker K.1.2 and of intercellular adhesion molecule-1 was decreased in TPA-treated cultures. Most of these changes persisted even after removal of TPA from the culture medium (> or = 2 weeks). Staurosporine, a protein kinase C inhibitor, modulated melanocyte-antigen expression similar to TPA, suggesting that protein kinase C downmodulation rather than activation by TPA is involved. In addition to the antigenic alterations, the susceptibility of TPA-treated melanocytes to lymphokine-activated killer cell cytotoxicity decreased by 40% (p < 0.01), possibly due to their altered surface antigen expression. The presented data reveal that the tumor promoter TPA hitherto used as a supplement of melanocyte culture media induces profound phenotypic and functional changes of the cultured cells, indicating incipient transformation of normal human melanocytes in vitro.

In order to enable a direct comparison of gene expression in human melanoma cells and in normal human melanocytes, culture conditions were investigated under which both cell populations can be grown in vitro. Five of 13 melanoma cell lines tested could be cultured to confluence in a melanocyte medium containing fetal calf serum, 12-O-tetradecanoylphorbol-13-acetate and stimulators of cyclic adenosine monophosphate, whereas none could be grown to confluence in a serum-free hormone-supplemented melanocyte medium containing basic fibroblast growth factor and other mitogens. In both melanocyte media the melanoma cells showed signs of increased morphological differentiation with dendrite formation. Normal human melanocytes could be grown in melanoma cell medium for a few days, after which the cultured cells showed a less differentiated phenotype. Since morphological changes may reflect variations in gene expression, we suggest that comparative studies on gene expression of benign and malignant melanocytes should also include thorough investigation under identical culture conditions.

This review summarizes recent information on the effects of immunomodulatory cytokines on human melanoma cells. The action of interferon (IFN)-alpha, -beta, and -gamma has been extensively examined in melanoma and melanocyte cultures in vitro, and increasing information on the action of other cytokines is now available. All IFNs revealed a dose-dependent antiproliferative effect on melanoma cells with the highest growth inhibition caused by IFN-beta. Proliferation was also inhibited by interleukin (IL) 1-alpha and -beta, and tumor necrosis factor (TNF)-alpha. For IL-4, both growth-stimulatory and -inhibitory properties have been reported. Cellular differentiation in terms of melanin synthesis, formation of dendritelike structures, and antigenic changes was not affected by IFN-alpha or -beta. IFN-gamma, however, induced a more dedifferentiated and biologically more aggressive phenotype of melanoma cells. Histocompatibility antigen (HLA) class I molecules were found upregulated by all IFNs and by TNF-alpha, associated with a marked increase of melanoma cell lysis by tumor infiltrating lymphocytes in vitro. HLA class II molecules were de novo expressed or enhanced by IFN-gamma and TNF-alpha. The adhesion molecules ICAM-1, LFA-3, and VLA-2 were upregulated by IFN-gamma, TNF-alpha, and IL-1-beta, whereas melanoma-associated antigens were hardly affected by cytokines. It seems that both antiproliferative and immunomodulatory effects may contribute to the antitumoral activity of cytokines in vivo. In vivo application of cytokines as well as combinations with cytotoxic drugs, therefore, may be promising for future treatment strategies.

The effects of human recombinant interferon-alpha-2a (rIFN-alpha), natural interferon-beta (nIFN-beta) and recombinant interferon-gamma (rIFN-gamma) on the proliferation, morphology and antigen expression of cultured human melanocytes were studied in vitro. The investigations were performed in 12-O-tetradecanoylphorbol-13-acetate (TPA)- and serum-containing melanocyte growth medium (MGM), in TPA- and serum-free complete melanocyte medium (CMM) and its mitogen reduced variant (RMM). In MGM, none of these interferons inhibited the growth of normal melanocytes at concentrations 1-10,000 international units (IU)/ml over a period of 5 d. Only nIFN-beta, dose dependently, inhibited melanocyte proliferation in CMM and RMM in a 6- and 12-d assay (growth inhibition at 10,000 IU/ml; 77-80% of the controls, p less than 0.001). In contrast, rIFN-alpha and rIFN-gamma exerted no (RMM), or minor effects (CMM) on melanocyte proliferation (only in 12-d assays at 10,000 IU/ml: 24% and 21% of the controls respectively, p less than 0.01). In parallel experiments performed on melanoma cells, all three interferons were potent inhibitors of proliferation in a 5-d serum-free assay (growth inhibition at 10,000 IU/ml; rIFN-alpha 59%, nIFN-beta 78%, rIFN-gamma 56%, all p less than 0.001). In addition, nIFN-beta and also rIFN-gamma caused striking morphologic changes of normal melanocytes in vitro. Especially under greater than or equal to 10 IU/ml rIFN-gamma cytoplasmic spreading and flattening of the cultured melanocytes and their nuclei were seen, thus resembling melanoma cells in vitro. Untreated human melanocytes grown in MGM showed high expression of the melanoma-associated antigens HMB-45 (95-100%) and K.1.2 (40-100%), whereas the progression marker A.1.43 was present only on less than 5% of the cells. Cultured melanocytes were 95-100% positive for histocompatibility antigen class I (HLA-I), 30-75% were positive for ICAM-1, whereas they were negative for HLA-DR. After treatment with rIFN-alpha, increased expression of HLA-I antigens was found; nIFN-beta and rIFN-gamma decreased the labeling with HMB-45 (75-100%) and with K.1.2 (25-80%), whereby the expression of A.1.43 was found slightly increased (5-15%). The HLA class I antigens were upregulated by both nIFN-beta and rIFN-gamma, nIFN-beta being the most potent agent. Also, both nIFN-beta and rIFN-gamma increased the expression of ICAM-1 (nIFN-beta, 75-90%; rIFN-gamma, 90-95%) and induced de novo expression of HLA-DR antigen (nIFN-beta, 15-20%; rIFN-gamma, 65-95%).(ABSTRACT TRUNCATED AT 400 WORDS)

A simple, rapid and reproducible assay for the determination of melanoma cell proliferation in vitro is described, based on the hydrolysis of a fluorogenic substrate by cell esterases in the cytoplasm of living cells. Human melanoma cells were cultured at several densities in 96-well culture plates for 24 h and were then incubated with 4-methylumbelliferyl heptanoate. The generated fluorescence showed a strong correlation with the cell numbers, similar to those assessed by determining the [3H]thymidine incorporation into cellular DNA and by quantifying the fluorescence obtained after DNA labelling with Hoechst 33258. The latter, however, was less sensitive and exhibited higher standard deviations. In addition, the method reliably detected the anti-proliferative effects of the anti-cancer compounds cisplatin and vindesine. It is, therefore, suggested that the fluorometric assay with 4-methylumbelliferyl heptanoate as substrate could prove useful for the screening of potential anti-cancer agents with anti-proliferative activity.

Monolayer cultures of the human melanoma cell lines StML-12, StML-11, StML-14 (third, respectively, twenty-fifth subculture), and SKMel-28 derived from specimens representing different stages of tumor progression were treated with 10-10,000 U/ml rTNF-alpha applied for 72 h. The effects of rTNF-alpha on cell proliferation, DNA synthesis, cell viability, cloning efficiency, cell division, cell morphology, and the immunophenotype were studied in triplicate experiments. The cell line StML-14(3) revealed a significantly dose-dependent reduction of growth due to both cytostatic and cytotoxic activities of rTNF-alpha as well as a decrease of CE. Increased numbers of cells in prophase were observed 24 h after addition of r-TNF-alpha. In addition, dislocation of chromosomes in the metaphase, formation of micronuclei, and dose-dependent increases of cells exhibiting micronuclei and the DNA amount per cell were detected at the end of treatment. On the other hand, only a slight sensitivity to the anti-proliferative effect of rTNF-alpha was observed with StML-14(25) and SKMel-28, whereas StML-12 and StML-11 were significantly resistant. The last four cell lines were serially subcultivated and presented common phenotypic patterns with more malignant characteristics than the cell line StML-14(3) before treatment. Overall, rTNF-alpha enhanced the malignant immunophenotype of the cell lines tested. It increased the expression of the "late" melanoma progression markers A.10.33 and A.1.43, and Ki67, and it decreased the expression of the "early" progression marker K.1.2. The expression of HLA-I, HLA-DR, and ICAM-1 was also enhanced after rTNF-alpha treatment, whereas in contrast to other cytokines, rTNF-alpha did not induce the de novo expression of HLA-DR in HLA-DR-negative melanoma cell lines. These findings indicate that rTNF-alpha induces cytostasis and decreases cell viability of certain rTNF-alpha-sensitive melanoma cells. These effects may result in selection of rTNF-alpha-non-sensitive human melanoma cell populations with higher proliferation rates and a more aggressive immunophenotype in vitro.

The antitumor activities of human interferon (IFN) alpha, beta, and gamma alone or in combination were studied on four human melanoma cell lines (StML-11, StML-12, StML-14, and SKMel-28) in various concentrations (1-50,000 IU/ml IFN alpha, 0.1-1000 IU/ml IFN beta, 1-10,000 IU/ml IFN gamma) in vitro. In all experiments IFN beta exhibited the most potent antiproliferative effect of all IFN tested. After 3 d of incubation a 50% growth inhibition was achieved with 20-40 IU/ml for natural IFN beta and with 600-1200 U/ml for recombinant IFN gamma. Substantially higher doses (7,000 to more than 50,000 IU/ml) of recombinant IFN alpha 2a were required to achieve a 50% growth inhibition. A strong synergistic antiproliferative activity resulted from the combination of IFN alpha with IFN gamma and IFN beta with IFN gamma. None of the IFN tested induced terminal differentiation of melanoma cells in vitro. The formation of dendrites was inhibited, and the portion of differentiated cells in vitro was reduced after treatment with IFN in comparison to the untreated controls (untreated controls: 100%; portion of differentiated cells after treatment with IFN alpha: 58%-74%, IFN beta: 48%-96%, IFN gamma: 10%-33%). The melanin synthesis was slightly elevated after treatment with IFN alpha (untreated controls: 100%; after treatment with IFN alpha: 103%-157%, ns.) and decreased significantly after treatment with IFN beta (49%-71%, p less than 0.05) as well as with IFN gamma (80%-88%, ns.). Cell surface markers were modulated varyingly by the IFN: HLA-I antigens were enhanced by all IFN, with IFN beta emerging as the most potent inducer. Only IFN gamma, however, induced a de novo expression of HLA-DR and -DQ antigens and increased the expression of the ICAM-1 molecule and of the melanoma progression marker A.1.43. Possibly, these findings indicate a biologically more aggressive phenotype of melanoma cells.