Association with clinical outcome of expression of VLA-4 in primary cutaneous malignant melanoma as well as P-selectin and E-selectin on intratumoral vessels.

BACKGROUND: The process of tumor growth and metastasis is a complex multistep cascade. The ability of tumor cells to adhere to and detach from extracellular matrix and endothelial cells may be crucial in the metastatic process and may dramatically alter the clinical prognosis and outcome for patients with certain cancers. A number of adhesion molecules have been detected on human melanoma cells and have been associated with various properties in vitro including invasiveness. Recent findings from our laboratory have indicated an ordered change in integrin expression during the process of tumor progression. PURPOSE: This study was designed to identify molecular markers present on human melanoma cells and in intratumoral vessels that have prognostic significance regarding disease-free interval and survival time. METHODS: Specimens of primary cutaneous malignant melanoma were obtained from 60 patients who had been followed for at least 36 months, with development of metastases in 29 patients during that period of time, and were analyzed for their expression of VLA-4, VLA-6, ICAM-1, ELAM-1 (E-selectin), CD62 (P-selectin), and CD44v6 molecules on tumor and endothelial cells by immunostaining. Light microscopy was used to evaluate and categorize the number of positively stained cells. Statistical analyses were done to determine the relationship of the expression of individual adhesion molecules with time to disease progression (i.e., disease-free interval) and overall survival time. RESULTS: In each case, positive staining for ELAM-1 and CD62 on intratumoral vessels and for VLA-4 on human melanoma cells was negatively associated with disease-free interval (P < .01) and overall survival time (P < .01). The presence of VLA-6, CD44v6, and ICAM-1 on melanoma cells was not associated with clinical outcome. CONCLUSIONS: Immunohistochemical screening and detection of ELAM-1, CD62, and VLA-4 may help to define a subgroup of melanoma patients at risk of developing metastases.

Human c-myb is expressed in cervical carcinomas and transactivates the HPV-16 promoter.

Human c-myb is normally involved in the regulation of proliferation and differentiation of hematopoietic cells. Until now, only a few reports have described elevated c-myb gene expression in epithelial tissue, suggesting that under certain circumstances, c-Myb protein might play a role during the process of malignant transformation of epithelial cells. To investigate a possible role of c-myb during papillomavirus-associated carcinogenesis, we investigated the c-myb mRNA expression in human papillomavirus (HPV)-associated tumors and tumor cell lines. Seven of nine cervical carcinomas and two of three carcinoma cell lines exhibited elevated c-myb transcriptional activity. In contrast to malignant cervical neoplasias, only 3 of 15 condylomata acuminata expressed a sparse signal for c-myb mRNA. Since the c-Myb protein has been described as a potent transcriptional regulator, we investigated the transactivating properties of c-Myb on the HPV-16 promoter/enhancer. Cotransfection of a chloramphenicol acetyltransferase-reporter plasmid containing the HPV-16 enhancer/promoter element with a full-length c-Myb-expressing plasmid resulted in a significant induction (4.3-fold) of the HPV-16 promoter, whereas expression of a carboxy-terminally deleted c-Myb protein led to no effects. Gel shift experiments showed a specific binding of recombinant c-Myb protein on the HPV-16 P97 enhancer. These data indicate that elevated c-myb expression occurs with HPV-associated cell transformation. Since c-Myb has been shown to stimulate the HPV-derived oncoprotein expression via transcriptional activation, it may play a role in the process of HPV-associated cervical carcinogenesis.

Gangliosides enhance IgE receptor-dependent histamine and LTC4 release from human mast cells.

Releasability of mast cells and basophils to an IgE-dependent stimulus is regulated by extra- and intracellular factors which are only partly understood. As gangliosides are known to modulate receptor-dependent processes in various cell types, we have evaluated the effect of these molecules on mast cell mediator release. Human skin mast cells and the human mast cell line HMC1 were pretreated with the gangliosides GM2, GM3 and GD1a as well as with asialo-GM3, heparin and buffer alone (controls). After washing, the cells were stimulated with anti-IgE, calcium ionophore A 23187, N-FMLP or substance P. All gangliosides but not asialo-GM3 and heparin augmented anti-IgE-induced mediator release in a dose-dependent fashion, whereas the release to A 23187, N-FMLP and substance P remained unaffected. Only sequential but not simultaneous addition of ganglioside and anti-IgE showed an enhancement in mediator release compared to controls. Mediator release in both ganglioside-pretreated cells and controls was calcium-dependent and could be inhibited by pretreatment of cells with staurosporine or dibutyryl cAMP, indicating an unchanged signal transduction. Gangliosides appear to specifically optimize IgE-receptor-ligand interaction and alterations in cellular gangliosides could thus induce enhanced releasability as observed in atopics.

To perfuse or not to perfuse? A retrospective comparative study to evaluate the effect of adjuvant isolated regional perfusion in patients with stage I extremity melanoma with a thickness of 1.5 mm or greater.

The use of isolated regional perfusion in an adjuvant setting for stage I melanoma of the extremity continues to be controversial. The present retrospective study evaluates the past 20 years' experience by comparing 227 perfused patients from Groningen with 238 matched controls from five hospitals in The Netherlands and Westphalia (a region of West Germany bordering the Netherlands). All patients underwent wide local excision for a primary extremity melanoma of 1.5 mm or greater in thickness. A proportional hazards regression analysis for recurrence of disease and survival identified the significant prognostic factors, of which tumor thickness was the most important. Corrected for these factors, it was not possible to demonstrate a statistically significant effect for perfusion in terms of time to limb recurrence (P = .61), time to regional lymph node metastasis (P = .11), time to distant metastasis (P = .73), disease-free interval (P = .42), and survival (P = .90). No statistically significant differences were seen for adjuvant perfusion in any of the subgroups.

Inhibitory effects of shale oils (Ichthyols) on the secretion of chemotactic leukotrienes from human leukocytes and on leukocyte migration.

Ichthyols are sulfated shale oils with well-known anti-inflammatory effects in dermatologic diseases. Their possible mechanisms of action were studied by measuring chemotactic factor release from peripheral human leukocytes in vitro. Ichthyols caused no release of such factors by themselves but inhibited ionophore-induced release. After elution of the cell supernatants on reverse-phase high-pressure liquid chromatography, followed by analysis of the fractions in the chemotaxis assay and the radioimmunoassay, Ichthyols caused a reduction of lipids at marker positions for leukotriene B4 (LTB4) and 20-COOH-LTB4. The inhibition was also evident in the LTB4 radioimmunoassay, was dose- and time-dependent, and occurred in noncytotoxic concentrations of the agents. Ichthyols also inhibit the chemotactic response of neutrophils toward LTB4 and the unstimulated migration of cells. These inhibitory effects of Ichthyols on secretion of chemotactic arachidonate metabolites from leukocytes and on cell migration provide a plausible explanation for their anti-inflammatory activity.

Gene amplification by polymerase chain reaction in dermatology.

The polymerase chain reaction (PCR) is another new powerful technique in molecular biology that has begun to open new perspectives in modern science and also in dermatology. This brief report will therefore elucidate the general principles of the polymerase chain reaction, as well as its limitations and possible pitfalls. Furthermore an overview will be provided on the impact of PCR on molecular biologic approaches in oncology, immunology, and human genetics. The use of the method as a tool to detect microorganisms particularly viruses and bacteria, in cutaneous tissue and it potential other future applications are described as well. Because PCR is automated and is being more and more established in routine laboratories, physicians and scientists should be familiar with the basic principles and potential uses of this methodology.

Comparative studies on eosinophil chemotactic factors during leukocyte migration under agarose.

Several known eosinophil chemotactic factors are compared with regard to their biological behavior during in vitro migration under agarose: zymosan activated serum (Zas), containing the chemotactic fragment C5a, the lymphokine eosinophil stimulation promoter (ESP), neutrophil-derived eosinophil chemotactic factor (ECF) and the N-formyl-methionyl-phenylalanine (NFMP) peptide induced chemotaxis and chemokinesis of granulocytes. All factors except NFMP attracts eosinophils and neutrophils. ECF alone selectively enhances eosinophil migration, and NFMP is inactive towards eosinophils. ESP is the least potent factor but affects eosinophil migration over a prolonged period of time. These different biological properties of the chemotactic factors may help to explain the differential influx of eosinophil and neutrophil leukocytes to tissue sites.

Surfactants cause in vitro chemotaxis and chemokinesis of human neutrophils.

The surfactant sodium lauryl sulfate (SLS) and alkyldimethylbenzylammonium chloride (ADB) cause erythema and leukocyte infiltration on epicutaneous application. To elucidate the mechanism of this inflammatory response, the in vitro effect of the same agents was studied on human neutrophil migration, basophil histamine release, and leukocyte lysosomal enzyme (beta-glucuronidase) release. At concentrations of greater than 0.02%, both surfactants were cytotoxic, as was evident by decreased eosin exclusion, massive histamine and beta-glucuronidase-release, and absent migration of cells. At dilutions of less than 0.002% of both surfactants, viability of cells was normal, and small amounts of histamine and beta-glucuronidase were released at a dilution of 0.001%. The most striking finding was a dose-dependent chemotactic and chemokinetic response at dilutions from 10(-3) to 10(-8)%. These observations offer a possible explanation for the pathomechanisms of irritant dermatitis due to surfactants.

In vitro migratory response of rat peritoneal macrophages and mast cells towards chemotactic factors and growth factors.

In order to elucidate the mechanisms of mast cell accumulation at tissue sites, mast cell-containing and -depleted rat peritoneal exudates were tested for their migratory ability in the in vitro microdroplet assay. In some experiments, peritoneal cells from ascaris sensitized rats and immature, cultured mast cells were studied as well. Mature mast cells never responded to any stimulus tested, and immature cells exhibited far less responsiveness than their precursors. Macrophages migrated towards LTB4, PAF, formylated peptide, activated and sensitized serum, spleen cell supernatants and less so towards fibronectin, growth factors (EGF, FGF) and growth factor containing media. In general, cells from sensitized animals and young elicited cells were more active than resting macrophages. The data suggest that increased numbers of mast cells at tissue sites are due to in situ maturation of immigrated, macrophage-like precursors.

Leukotriene B4 induces in vitro fibroblast chemotaxis.

In vitro fibroblast chemotaxis was studied in the presence of synthetic leukotriene B4 (LTB4) and of supernatants from ionophore-stimulated lymphocytes, monocytes, and basophils (LMB-S). LTB4 and LMB-S induced a dose-related, directed migration of human and rat embryonic fibroblasts. By checkerboard analysis, this response was chemotactic and not chemokinetic. Optimal migration toward LTB4 occurred at 10(-8) M concentrations, while higher concentrations were inhibitory. The LMB-S contained two types of fibroblast chemotactic factors, one that corresponded to LTB4 and one additional, heat labile, non-dialyzable substance of greater than 10 kilodaltons. LTB4 and LMB-S were inactive against transformed human connective tissue cells (HT 1080, McCoy) while these cells moved well in response to conditioned medium derived from confluent fibroblast monolayer cultures. These results suggest that LTB4 is a specific chemoattractant for fibroblasts and that it acts in concert with other chemotactic factors derived from inflammatory leukocytes to regulate the influx of fibroblasts to tissue sites.

In vitro generation of chemotactic leukotrienes from unfractionated murine epidermal cells.

Single cell suspensions of murine epidermal cells were studied for the generation of leukotrienes (LTs), using in vitro bioassays for chemotaxis, reverse-phase high-pressure liquid chromatography (HPLC), and radioimmunoassays (RIAs). A combination of arachidonic acid (AA) at 10(-3)-10(-4) M with the calcium ionophore A 23187 at 5 X 10(-6) M was the most potent stimulus, causing release of LTs within 10-30 min. Other stimuli, like the N-formyl-methionyl-leucyl-phenylalanine, at 10(-7) M and bradykinin at 10(-3) M, were less effective, and the tumor promotor phorbol-myristate-acetate (10(-5)-10(-8) M) caused no release at all. AA induced release at cytotoxic concentrations, but the other stimuli did not, and keratinocytes from different body regions were equally good sources of the LTs. In vivo or in vitro pretreatment of keratinocytes with UV radiation did not alter spontaneous or stimulated secretion of LTs, while pretreatment of cells with Ia, but not with Thy-1, monoclonal antibodies caused a moderate decrease of release. Analyses by HPLC indicated the release of 20-OH-LTB4 in addition to LTB4 in cell supernatants. Murine keratinocytes and epidermal dendritic cells serve therefore as a source of chemotactic leukotrienes after appropriate in vitro stimulation with agents that are known to play a role in cutaneous inflammation.

Production of LTB4-like chemotactic arachidonate metabolites from human keratinocytes.

Keratinocytes have recently been recognized as a source of mediators of cellular immune function. We present here further data on the production of 5-lipoxygenase-dependent arachidonate metabolites from freshly isolated human epidermal cells. Stimulation of cells with arachidonic acid or the calcium ionophore A 23187 alone or together caused a dose- and time-dependent release of chemotactic activity which was maximal during the first 10 min and which continued for up to 18 h. Indomethacin (10(-6) M) enhanced and compound BW 755C (20 micrograms/ml), a lipoxygenase inhibitor, decreased release. The chemotactic activity was heat stable for 30 min at 56 degrees C and was extractable into ether at pH 3.0. Analysis of 15- and 30-min supernatants showed coelution of biologic activity and of leukotriene B4 (LTB4), as measured by radioimmunoassay, at marker positions of LTB4 and of 20-OH-LTB4. Elimination of Langerhans cells did not alter the secretion of chemotactic lipids, suggesting that keratinocytes are the main source of potent, biologically active, lipoxygenase-dependent arachidonate metabolites.

Defective release of eosinophil chemotactic factor from peripheral leukocytes in patients with chronic urticaria.

Release of eosinophil chemotactic factor (ECF) and histamine was studied peripheral leukocytes of 16 normal and 12 ragweed allergic volunteers and compared to 15 patients with chronic urticaria. Cells were exposed to varying concentrations of anti-IgE or to ragweed antigen E in the case of allergic donors. Twelve of 15 patients with chronic urticaria showed defective release of ECF as well as histamine, while almost all normal and all allergic donors were able to release larger quantities of both mediators. Since the eosinophil is thought to have a modulating effect at sites of inflammation induced by anaphylactic mechanisms, the defective release of a factor attracting these cells may explain the persistence of symptoms in chronic urticaria.

Serum eosinophil chemotactic factor levels in patients with bullous pemphigoid, drug reactions and atopic eczema.

The sera of 4 patients with bullous pemphigoid, of 5 patients with drug reactions and of 13 patients with atopic eczema were examined for the occurrence of low molecular weight eosinophil chemotactic factor (ECF) by fractionation on a Sephadex G 25 column. Almost all patients had a peripheral eosinophilia, and many had raised total serum IgE levels. ECF was demonstrated in the sera of all 4 patients with bullous pemphigoid and in 4 of 5 patients with systemic drug reactions. In contrast, the sera of the 13 patients with atopic eczema did not contain any ECF activity, nor did the 13 control sera. These findings suggest that the ECF from phagocytosing polymorphonuclear leukocytes (PMN) and/or the mast cell derived ECF-A contribute to the elevated serum ECF levels in patients with bullous pemphigoid and drug reactions. A correlation between serum ECF and IgE levels and peripheral eosinophilia could not be established.

Response of cutaneous mast cells to PUVA in patients with urticaria pigmentosa: histomorphometric, ultrastructural, and biochemical investigations.

In six patients with urticaria pigmentosa, population density and ultrastructure of the cutaneous mast cells and histamine levels of the lesional skin were studied before, immediately after, and again 5-8 months after photochemotherapy (PUVA). Immediately after PUVA, the total mast cell number was not reduced, but on separate analysis of the intradermal distribution, significantly fewer mast cells were found in the papillary dermis and correspondingly more mast cells in the adjacent upper dermis. On electron microscopic examination, 4% of the mast cell granules were immature before and 27% after PUVA therapy, based on the lower electron density of the granular matrix. This was associated with a markedly lower histamine content of the lesional skin. Five to eight months after recovery from PUVA, the morphologic changes and the histamine levels had all returned to the pre-PUVA status. These findings were paralleled by a reversal of all clinical beneficial effects that had been observed with PUVA.

Detection of human papillomaviruses in paraffin-embedded condylomata acuminata--comparison of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

The polymerase chain reaction (PCR) is used for detection of human papillomavirus (HPV) DNA in paraffin-embedded tissue sections of condylomata acuminata. Incorporation of biotinylated nucleotides during the amplification process allows a highly sensitive, fast, and non-isotopic detection of viral DNA in a subsequent Southern dot blot. In 100% (13 of 13) of histologically confirmed condylomata, HPV-6 or -11 could be detected by polymerase chain reaction. By in situ hybridization 77% (10 of 13) and by immunohistochemistry (IHC) 69% (nine of 13) positive results were obtained. Because HPV genital infection is linked to penile and cervical dysplasia, polymerase chain reaction provides a powerful and highly sensitive tool for epidemiologic studies on sexual transmission of HPV.

Glutathione and related enzymes in tumor progression and metastases of human melanoma.

We have shown previously that overexpression of p-170 glycoprotein-mediated multidrug resistance plays only a minor role in conferring chemoresistance to human melanoma cells. In addition to membrane transporters like p-170, metabolizing enzyme systems have been implicated in altered drug sensitivity. Recently, glutathione and associated enzymes have been associated with resistance to alkylating substances, particularly in gastrointestinal and gynecologic cancers. In this study, we investigated whether increased levels of glutathione and related enzymes may play a role in chemoresistance in melanoma. Levels of glutathione, glutathione S-transferase (GST), glutathione reductase, and gamma-glutamyl transpeptidase were analyzed in melanoma and non-melanoma cell lines. In addition, 18 melanoma metastases derived from skin and lymph nodes were examined. Levels of gamma-glutamyl transpeptidase were statistically different in cells derived from melanocytic tumors compared with non-melanoma cell lines and normal cells. In addition, GST levels in metastases derived from skin or lymph nodes were significantly lower than those in permanent cell lines. However, levels of glutathione and related enzymes in metastases and cell lines fluctuated over a wide range, up to 40-fold, regardless of treatment status or origin of metastases. In a second part of the study, the expression of GST isoenzymes alpha, mu, and pi was studied by immunohistology in 10 benign nevi, 29 primary melanomas, and 39 melanoma metastases before and during chemotherapy. Expression of GST isoenzymes was increased with tumor progression, and GST pi was the strongest isoform expressed. However, no correlation was found between GST levels by immunohistochemistry and the course of tumor progression, between GST levels in metastases obtained before or during chemotherapy, or between GST levels and clinical response. These data suggest that alterations in glutathione metabolism and the expression of GST do not play a major role in resistance to chemotherapeutic drugs in melanoma.

A murine model for inducing and manipulating hair follicle regression (catagen): effects of dexamethasone and cyclosporin A.

Most cases of hair loss are based on premature induction of follicle regression (catagen). Deciphering the unknown regulation of catagen is therefore clinically important, but catagen is also an excellent model for organ involution by rapid terminal differentiation and for epithelial cell death (apoptosis). We here report an assay for the controlled pharmacologic induction and manipulation of catagen follicles. Dexamethasone-21-acetate (0.1%) was applied once daily to depilation-induced, growing follicles (anagen VI) on the backs of C57 B1-6 mice. Characteristic catagen-associated changes in skin color were photodocumented and assessed by morphometric histology. Topical dexamethasone induced catagen-like follicles significantly earlier, more homogeneously, and also more extensively than vehicle. This process was inhibited by high intraperitoneal doses of cyclosporin A. In addition to its clinical relevance as a screening assay for catagen-blocking drugs, this simple murine model is an attractive tool for dissecting the molecular, cellular, and developmental biology of catagen.

Interleukin-7 induces differential lymphokine-activated killer cell activity against human melanoma cells, keratinocytes, and endothelial cells.

To assess the potential role of interleukin (IL)-7 in immunotherapy of human malignant melanoma, we have examined the lymphokine-activated killer (LAK) cell sensitivity of four human melanoma cell lines against LAK cells generated by IL-7 or IL-2. Lysis was determined by a 24-h cytotoxicity test using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). All melanoma cell lines were susceptible to IL-7- and IL-2-generated LAK cells. The sensitivity of melanoma cells to IL-2-induced LAK cells was higher compared to IL-7-induced LAK cells. At an effector target ratio of 20:1, the lysis by IL-7-induced LAK cells ranged between 41% and 52%, whereas IL-2-induced lysis ranged between 80% and 94% (p < 0.01). IL-7-induced LAK cells, however, showed almost no cytotoxicity towards HaCat keratinocytes and human umbilical vein endothelial cells (HUVECs). Immunophenotyping revealed a higher expression of the tac antigen (CD 25) on IL-7-generated LAK cells, particularly those cells that were CD 56 negative or CD 3 positive compared to IL-2-induced LAK cells. In contrast, IL-2-generated LAK cells killed 62% of the HaCat keratinocytes and 60% of the HUVECs. Secretion of tumor necrosis factor-alpha into culture supernatants was significantly higher in IL-2-generated LAK cells compared to IL-7-stimulated LAK cells (p < 0.01), whereas TNF-alpha levels of IL-7-induced LAK cells were in the range of unstimulated lymphocytes. Because nonspecific cytotoxicity against other normal cells such as keratinocytes and endothelial cells contributes to the dose-limiting side effects of immunotherapy with IL-2, immunotherapy using IL-7 might be a better tolerated future alternative.

Effect of neutrophil-derived eosinophil chemotactic factor (ECF) in human and guinea pig skin.

The in vivo effectiveness of eosinophil chemotactic factor (ECF), secreted in vitro by human neutrophils (PMN) during phagocytosis, is tested in 2 model systems. Injection of ECF into guinea pig ears causes a preferential attraction of eosinophils with time that is more marked in animals with preexisting eosinophilia. In the same model system, activated serum attracts fewer eosinophils and more PMN. In human skin windows, ECF and activated serum are equally effective on a quantitative basis, but ECF is more selective for eosinophils in patients with eosinophilia. With normal controls, ECF and activated serum attract PMN equally well. The studies confirm previous in vitro observations on the properties of ECF and suggest a potentially significant role of this factor during pathological processes.