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Spontaneous regression of primary melanoma lesions is regarded as the result of the recognition of melanoma-associated antigen (MAA)-derived peptides by cytotoxic T-lymphocytes and destruction of melanoma cells. The transporter associated with antigen processing (TAP1/2) is likely to play a crucial role in this process since it loads antigen peptides onto MHC class I molecules. To determine the impact of TAP defects on the spontaneous regression of melanoma lesions, we have compared the expression of TAP1 and TAP2 in 39 primary melanoma lesions exhibiting clinical and histological signs of tumour regression and in 35 primary melanoma lesions without regression phenomena. TAP1 expression was significantly associated with regression of melanoma lesions, since the staining pattern with anti-TAP1 antibody was positive in 38 of the 39 lesions exhibiting regression phenomena and in only 24 of the 35 lesions without histopathological signs of tumour regression. In the latter group, six lesions were stained with a heterogeneous pattern and five with a negative pattern. Furthermore, in lesions with a heterogeneous staining pattern, a clear association was found between TAP1 expression in melanoma cells and the presence of tumour-infiltrating lymphocytes. These results suggest that TAP1 plays an important role in the MAA-specific cytotoxic T-lymphocyte response, which has been suggested to underlie the spontaneous regression of primary melanoma.

Tumor cells with alterations in the MHC class I peptide-loading complex are unable to load peptide antigens onto MHC class I molecules. These alterations result in destabilization of MHC class I expression on the tumor cell surface and thus play a critical role in escape from immunological recognition by the acquired cellular immune system. By forming physical links between class I heavy chains and TAP molecules, a component of the class I peptide-loading complex, tapasin, plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. In the present study, we compared 104 human melanoma lesions representing different stages of tumor progression for their expression of tapasin in tumor cells by immunohistochemistry. Tapasin downregulation was significantly associated with tumor progression. Whereas 100% of melanomata in situ and 96.2% of primary melanomas with a Breslow index of 0.75 mm as well as in 21.1% metastatic melanoma lesions. The downregulation of tapasin in advanced stages of human melanoma may reflect the accumulation of alterations in the antigen-presenting/processing machinery associated with neoplastic progression. These alterations may lead to failure of the acquired cellular immune system to control progression and metastatic spread of melanoma cells in vivo, and thus contribute to the immune escape phenotype of human melanoma cells.

CD44 is a family of cell-surface receptors on human lymphocytes that act as co-stimulatory molecules leading to the induction of effector functions in T cells. We have analyzed primary cutaneous malignant melanomas with clinical and histologic signs of tumor regression using immunohistochemistry and observed the predominant expression of the CD44 variant isoform v10 on CD3 CD4/CD8 co-expressing tumor-infiltrating lymphocytes (TIL). We further analyzed the role of CD44v10 in adhesion of lymphocytes to human melanoma cells. In contrast to CD44- lymphatic cells, CD44v10+ lymphatic cells strongly bound to cultured human melanoma cells and to frozen tissue samples of melanomas. Antibody blocking studies revealed a hyaluronan-, integrin-, and selectin-independent pathway of adhesion. Furthermore, CD44v10+ lymphatic cells exhibited significantly higher invasiveness in three-dimensional collagen matrices as compared with CD44H+ and CD44-negative lymphocytes. These results indicate that expression of CD44v10 on TIL may mediate adhesion to melanoma cells and result in gain of novel invasive properties.

Molecular oxygen plays a central role in the pathogenesis and therapy of chronic wounds. When reactive oxygen species are overproduced, oxidative stress results, with detrimental cytotoxic effects causing delayed wound healing. Therefore, elimination of reactive oxygen species could be an important strategy to improve healing of chronic wounds. Currently first therapeutic strategies targeting reactive oxygen species by antioxidants are being introduced into the treatment of chronic wounds.

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.

Colonization with methicillin-resistant Staphylococcus aureus (MRSA) strains is an increasing problem in the treatment of wounds. Only a very limited repertoire of effective treatment strategies is available, especially for outpatient care. We successfully treated a chronic leg ulcer colonized with MRSA on an ambulatory basis, using larvae of the common greenbottle fly Lucilia sericata. Whereas the proteases secreted by Lucilia sericata may lead to efficient selective necrolysis, the phenylacetate and phenylacetataldehyde may exert antimicrobial effects. Treatment with Lucilia sericata represents an effective and inexpensive treatment strategy of chronic wounds, especially when colonized with MRSA Due to the low acceptance by patients and medical stuff, it is not often employed.

Inherited mutations of Patched (PTCH) in the nevoid basal cell carcinoma syndrome (NBCCS) lead to several developmental defects and contribute to tumor formation in a variety of tissues. PTCH mutations have been also identified in sporadic tumors associated with NBCCS including basal cell carcinoma (BCC) and medulloblastoma. Mice heterozygous for Ptch recapitulate the typical developmental symptoms of NBCCS and develop rhabdomyosarcoma (RMS) and medulloblastoma. PTCH is assumed to act as a tumor suppressor gene although inactivation of both alleles has been demonstrated only in a fraction of tumors. We have investigated the status of Ptch in RMS of heterozygous Ptch neo67/+ mice. Although the wild-type Ptch allele was retained in tumor tissue, the high levels of Ptch mRNA in these tumors result from overexpression of the mutant Ptch transcript. Our results suggest that the wild-type Ptch allele might be selectively silenced in RMS tissue or, alternatively, that haploinsufficiency of Ptch is sufficient to promote RMS formation in mice.

Epithelioid sarcoma is a rare soft tissue tumor that, due to its clinically unspecific features, is frequently mistaken for other benign and malignant entities. We report on a 63-year-old male presenting with a relapse of an epithelioid sarcoma. At clinical inspection, the tumor presented as an erythematous nodule with ulceration in the center. Despite its slow growth, epithelioid sarcoma should be regarded as an aggressive neoplasm exhibiting frequent local recurrences and subsequent lymphatic and visceral spreading in about half of the patients. Histopathological findings are the presence of large epithelioid and spindle cells with immunoreactivity for cytokeratin and vimentin.

We have recently shown the CD44 variant isoform 10 (CD44v10) to be expressed on reactive as well as malignant cutaneous lymphocytes; however, the functional consequences of CD44v10 expression on lymphocytes are not elucidated. By using appropriately transfected lymphatic cells we analyzed the role of CD44v10 on lymphocytes in cell-matrix adhesion and homotypic and heterotypic cell-cell adhesion assays. Despite a low binding affinity to hyaluronan, CD44v10-expressing lymphocytes exhibited heterotypic cell-cell adhesion to inflamed dermal microvascular endothelium and keratinocytes, as indicated by Stamper-Woodruff assays on tissue sections of delayed type hypersensitivity reactions and adhesion assays with cultured keratinocytes and cytokine-stimulated human dermal microvascular endothelial cells. Antibody-blocking assays excluded interaction of CD44v10 with the principal CD44 ligand hyaluronan as well as involvement of selectins or integrins in these heterotypic cell-cell adhesion assays. In contrast, cellular aggregation assays with fluorescence-labeled CD44v10- and CD44H-expressing lymphocytes revealed homotypic CD44v10/CD44v10 binding as well as binding of CD44v10 with CD44H. Heterotypic cell-cell adhesion assays with ultraviolet-A-irradiated CD44v-negative cytokine-stimulated endothelial cells demonstrated binding kinetics of CD44v10-expressing lymphocytes paralleling those of endothelial CD44H expression. These results imply that a hyaluronan-independent CD44v10/CD44H-mediated pathway is involved in lymphocyte infiltration into the dermis and epidermis of inflamed skin and suggest modulation of CD44H expression on inflamed dermal microvascular endothelium as a mechanism of ultraviolet-A-induced therapeutic effects on the skin.

Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumour cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1alpha and CCL21/6Ckine exhibit peak levels of expression in organs representing the first destinations of breast cancer metastasis. In breast cancer cells, signalling through CXCR4 or CCR7 mediates actin polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. In vivo, neutralizing the interactions of CXCL12/CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung. Malignant melanoma, which has a similar metastatic pattern as breast cancer but also a high incidence of skin metastases, shows high expression levels of CCR10 in addition to CXCR4 and CCR7. Our findings indicate that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000

Thus far, the use of classical anti-cancer treatment modalities had only rarely a beneficial impact on the prognosis of patients with metastatic melanoma. We as physicians have therefore the obligation as well as the chance to develop and test new therapeutic strategies. Our growing knowledge about the genetic basis of melanoma provides one platform to fulfil this task. Another one comes from our increasing understanding of the molecular and cellular mechanisms involved in the induction/modulation of immune responses, as well as the progress made in the field of identification of melanoma antigens, and allows for the development of a new generation of vaccines. The aim of this article is to discuss several of these new concepts towards the use of immune and gene therapy of melanoma.

The remarkable functional diversity of the cell surface receptor CD44 may be due to expression of multiple variant isoforms generated by alternative splicing of variant exons. Functional and correlative data implicate a role of CD44 variant isoforms in adhesion dependent processes such as lymphocyte recirculation and tumor progression and metastasis. We have analyzed 25 primary cutaneous lymphomas and 35 reactive lymphoid cell skin infiltrates or T cell-mediated skin diseases for the expression of CD44 variant isoforms. Irrespective of histologic typing, staging, and grading, cutaneous lymphomas as well as nonmalignant skin-infiltrating CD3+ CD4+ and CD8+ T and CD19+ B lymphocytes exhibited a strong expression of CD44v10 and a moderate expression of CD44v3 as determined by immunohistochemistry, immunofluorescence microscopy, and mRNA analysis. Expression of v5, v6, v7, and v9-containing CD44 variant isoforms was not detected. Furthermore, flow cytometry revealed expression of CD44v10 on a significant proportion of peripheral blood lymphocytes from Sézary's syndrome patients and a remarkable co-expression with cutaneous lymphocyte antigen. These results indicate a distinct CD44 variant isoform expression pattern associated with skin-homing lymphocytes different to lymphatic cells at noncutaneous sites. This differential expression pattern of CD44 variant isoforms may contribute to the development of lymphocyte skin infiltrates and/or the unique biologic behavior of cutaneous lymphomas.

Spontaneous regression of melanoma lesions is thought to be the result of an efficient immune response against melanoma cells in vivo. The outcome of immune responses is critically influenced by a complex network of interacting cytokines present in the local microenvironment. Analysis of cytokine gene transcription in melanoma lesions exhibiting or lacking a sufficient anti-tumor immune response thus may help to define cytokines or cytokine combinations critical to the development of this immune response. In the present study, we have investigated an extended panel of cytokine and cytokine receptor genes by reverse transcription-PCR and in situ hybridization in regressive and progressive primary human cutaneous melanoma samples. Whereas the presence of a lymphocyte infiltrate in tissue samples was associated with a TH1 cytokine mRNA profile (TNF-alpha, INF-gamma, IL12p35, IL12p40, IL2Rbeta, and IL2Rgamma), clinically and histologically regressive samples exhibited additionally increased transcript levels for GM-CSF, IL2, and IL15. mRNAs of TH2 cytokines IL4 and IL5 were detected only in a minor portion of progressive melanoma samples and regressive melanoma lesions. These results were further supported by comparison of progressive with regressive regions in three melanoma samples. Again, regressive regions contained higher transcript levels for GM-CSF, IL2, and IL15. In comparison to cutaneous metastatic melanoma lesions, regressive melanomas also overexpressed the same cytokine mRNA profile. These results provide evidence for an association of spontaneous regression with increased transcript levels for the cytokine combination GM-CSF, IL2, and IL15 in malignant melanoma. This cytokine combination could be relevant for experimental anti-tumor immune response studies and for immunotherapeutic and gene transfer studies in the treatment of melanoma patients.

DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.

Chromosome 9p21 is frequently deleted in malignant melanoma, and one familial malignant melanoma gene has been linked to 9p21-22. Recently, the cyclin D-dependent kinase inhibitors (CDKIs) p16INK4a and p15INK4b have been localized within chromosome 9p21, and the presence of p16INK4a point mutations has been demonstrated in familial melanoma and melanoma cell lines in vitro. To analyse the role of these CDKIs in sporadic human cutaneous non-metastatic malignant melanoma, we examined 36 primary tumour specimens representing different stages of melanoma progression and their corresponding normal skin samples for the three mechanisms of CDKI inactivation described so far. Homozygous codeletion of the p16INK4a and the p15INK4b gene was detected by Southern blot analysis in two tumour samples. By direct sequencing of polymerase chain reaction (PCR)-amplified microdissected genomic DNA; no somatic or germline p16INK4a point mutations or small deletions were detected in the remaining 34 tumour samples; one individual exhibited the previously described germline codon 148 (Ala-->Thr) polymorphism. In these tumour specimens, comparative semiquantitative reverse PCR analysis of p16INK4a transcript levels revealed no evidence for repression of p16INK4a gene transcription and thus for p16INK4a promoter inactivation by DNA methylation. These results indicate homozygous p16INK4a and p15INK4b loss to occur in a subset of cutaneous melanomas and suggest, in view of the frequent loss of heterozygosity on chromosome 9p21, the presence of another tumour suppressor gene within this chromosomal region.

Pmel17/gp100-encoded tumor-associated antigens are recognized by cytotoxic T lymphocytes in melanoma patients and may represent attractive target antigens for immuno- and gene-therapeutic strategies. An important prerequisite for identification and monitoring of melanoma patients that could potentially benefit from Pmel17/gp100-based immuno- and gene-therapies is the detailed knowledge of Pmel17/gp100 expression in vivo. Immunophenotyping is considerably hampered by the different immunoreactivities of Pmel17/gp100-reactive antibodies. Therefore, we analyzed an extended series of different primary normal and malignant human tumor specimens for Pmel17/gp100 expression at the mRNA level. Transcripts were detectable in all malignant melanoma tissue specimens representing all stages of tumor progression, with significant levels even in early and amelanotic melanoma lesions. In contrast, normal melanocytes exhibited significantly less Pmel17/gp100 mRNA in vivo, as determined by comparative in situ hybridization. Tissue specimens from the retina and substantia nigra also contained Pmel17/gp100 mRNA, whereas other normal and malignant human tissues were negative. As determined by comparative in situ hybridisation and HMB-45 immunostaining, even tumor tissue lacking Pmel17/gp100 immunoreactivity contained Pmel17/gp100 transcripts. Our results indicate a melanocytic-cell-lineage-restricted expression of Pmel17/gp100 with significant transcript levels in all stages of melanoma progression, including early and amelanotic melanoma lesions, and a significantly differential expression between melanoma cells and normal melanocytes in vivo. Owing to its higher sensitivity, phenotyping of individual tumor specimens by mRNA expression analysis seems to be more valuable than phenotyping by immunostaining.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

In experimental models, plasminogen activator-mediated degradation of the extracellular matrix is inhibited by type-1 plasminogen activator inhibitor (PAI-1). PAI-1 has also been shown to protect tumour stromal tissue from autoproteolytic activities and may thus substantially promote tumour growth and metastasis formation. Human renal cell carcinoma (RCC) cells express significant amounts of plasminogen activator activity. In the present study, the expression of its specific inhibitor PAI-1 has been investigated in 32 cases of RCC and compared with adjacent non-tumour renal tissues. RCC tissue exhibited higher levels of PAI-1, determined at both the antigen and the mRNA level by ELISA and Northern blot analysis respectively. Immunohistochemical analysis showed that PAI-1 antigen was primarily confined to tumour cells and vascular endothelium, a distribution similar to that previously reported for plasminogen activator activity in RCC. The close co-localization with endogenous plasminogen activator activity may be important in the regulation of RCC-associated proteolysis. The increased expression of PAI-1 and its predominant localization within the tumour may help to conserve tumour tissue integrity and may thus promote RCC progression and metastasis formation.

The predominant cutaneous side effect of lithium is the exacerbation or aggravation of psoriasis, but the pathogenesis is still unclear. The hyperproliferation of keratinocytes and a dense lesional infiltrate of mononuclear cells are the hallmarks of psoriatic skin lesions. Interactions between keratinocytes and T cells are thought to be one reason for an increased secretion of proinflammatory cytokines and growth factors. To investigate whether lithium influences cytokines of the "psoriatic cytokine network', we established a coculture model with keratinocytes from psoriatic patients and from healthy controls cultured with HUT 78 lymphocytes and measured the cytokine levels of Il-2, Il-6, Il-8, IFN gamma and TGF alpha in the culture supernatants after treatment with lithium. Il-6 levels were slightly elevated in the supernatants obtained from psoriatic and control keratinocyte cultures after lithium treatment, but IFN gamma and Il-2 levels were elevated only in the lithium-treated cocultures with psoriatic keratinocytes. In contrast, these two cytokines were not affected by lithium in HUT 78 monocultures or in cocultures with normal epidermal cells. We also found slightly elevated TGF alpha levels in lithium-treated psoriatic cocultures but not in control cultures. We therefore demonstrated that lithium influences the cell communication of psoriatic keratinocytes with HUT 78 lymphocytes by triggering the secretion of TGF alpha, Il-2 and, massively, IFN gamma. It seems possible that lithium also influences similar parts of the psoriatic cytokine network in vivo.

The influence of the immunosuppressants, cyclosporin A (CsA) and FK506, on cAMP formation was studied in T cells from healthy controls and patients with psoriasis. While basal cAMP levels were not affected, CsA (1 microM) and FK506 (2 nM) prevented the isoprenaline (0.1 microM)-induced increase in cAMP formation. Half-maximal inhibition by FK506 and CsA was observed at about 0.2 nM and 20 nM, respectively. In addition, both agents significantly reduced (by about 50%) the forskolin (8 microM)-stimulated cAMP formation. No differences were noted in cAMP responses (basal, stimulation by isoprenaline, inhibition by CsA and FK506) of T cells from healthy controls and psoriatic patients. We conclude that CsA and FK506 potently and efficiently interfere with the stimulatory adenylyl cyclase pathway in T cells and that regulation of T cell cAMP formation is apparently not altered in psoriasis.

Tumor cell invasion and metastasis are considered to represent a multistep process leading to the degradation of the extracellular matrix by proteolytic enzymes. The functional activity of matrix metalloproteinases (MMPs) is controlled by tissue inhibitor of metalloproteinases-2 (TIMP-2), which has been shown to inhibit tumor cell invasion and metastasis in vitro and in vivo. To assess the role of TIMP-2 in skin-derived epithelial tumors, we have analyzed the expression of TIMP-2 mRNA in primary tissue samples from human cutaneous basal (BCC) and squamous cell carcinoma (SCC) for a correlation with their different invasive and metastatic potential. Comparative quantitative analysis of TIMP-2 mRNA levels by Northern blot hybridization demonstrated significantly higher TIMP-2 tissue levels in BCC than in SCC, indicating an inverse correlation between TIMP-2 expression and the metastatic capacity of these tumors in vivo. By in situ hybridization, tumor stromal cells were identified as the principal source of TIMP-2 mRNA in both BCC and SCC. A comparable distribution has been reported previously for several matrix metalloproteinases in cutaneous BCC and SCC, indicating co-localization of metalloproteinases with their respective inhibitor. These results may suggest that TIMP-2 substantially contributes to the biological behavior of epithelium-derived skin tumors by significantly inhibiting tumor cell metastasis.

The urokinase-type plasminogen activator (uPA) is secreted into the urine at high concentrations and both the uPA protein and mRNA are present in human renal tissue. Normal kidney tissue also expresses the receptor for uPA. Neither the precise sites of uPA mRNA expression, nor the distribution of the uPA-receptor antigen, have been elucidated in the human kidney. In the present study, the sites of uPA mRNA expression were identified by in situ hybridization, and the cellular localization of both uPA and uPA-receptor was determined by immunohistochemical analysis. High-level uPA mRNA expression was restricted to epithelial cells of the convoluted proximal tubules and the thick ascending limb of Henle's loop (the straight part of the distal tubule). However, uPA immunoreactivity was not confined to sites of uPA mRNA expression, but was present in all segments of the tubular epithelium. Tubular epithelial cells also exhibited a consistent immunoreactivity with uPA-receptor antibody, indicative of a co-localization of the uPA antigen and its receptor in the uriniferous epithelium. We propose that the uPA antigen expression in nephron segments lacking demonstrable endogenous uPA synthesis may be the result of a uPA-receptor-mediated uptake of uPA.

FK 506 and cyclosporin A (CyA) are two immunosuppressive drugs which are known to be effective in the treatment of psoriasis by inhibiting the activation of T cells. In contrast, their influence on the proliferation of keratinocytes is discussed controversially. The second messenger cyclic adenosine monophosphate (cAMP) has been regarded as a regulator for cell growth and proliferation for 20 years. Hyperproliferation of many cells and particularly of psoriatic keratinocytes was speculated to be due to a decrease in cAMP levels in the psoriatic epidermis, whereas new findings could not confirm these observations. To clarify this discussion we determined the intracellular cAMP content in isoprenaline-stimulated keratinocytes from psoriatics and controls after treatment with CyA or FK 506. Ethanol and the beta-blocking drug propranolol served as controls. The basal level of cAMP and the response to isoprenaline in psoriatic keratinocytes did not differ from those of controls. CyA dramatically reduced the cAMP level and FK 506 just slightly diminished it in a dose-dependent manner. Both drugs diminished the cAMP level more effectively in the keratinocytes from lesional psoriatic skin than in keratinocytes from controls. These data provide evidence that CyA influences early signal transduction pathways by depressing the intracellular cAMP in keratinocytes. This supports the view of other groups that CyA and perhaps also FK 506 influence not only immuno-competent cells but also keratinocytes in the treatment of psoriasis. Furthermore, it is doubtful that a low cAMP level is a positive regulator for cell growth and the hyperproliferation of psoriatic keratinocytes.

Background: mAb HMB-45 recognizes a melanocyte lineage-associated Ag present in "activated melanocytes" and malignant melanoma cells. Despite its important practical significance in diagnostic pathology and its potential role as an "activation marker" of melanocytic cells, the HMB-45-reactive Ag remained undefined. Molecular characterization of the HMB-45-reactive Ag may help in analyzing underlying mechanisms of melanocyte activation and melanoma tumor progression., Experimental Design: A cDNA library constructed from the HMB-45-immunoreactive human melanoma cell line SK-MEL-28 was screened for expression of the cDNA encoding the HMB-45-reactive protein. Screening was performed by expression in COS-7 cells and subsequent immunocytochemical screening. Correlation between HMB-45 immunoreactivity and mRNA expression of the cloned cDNA was analyzed by antisense mRNA expression in HMB-45-immunoreactive SK-Mel-28 cells, by Northern blot hybridization, and by comparative immunohistochemistry and in situ hybridization., Results: A cDNA clone encoding the HMB-45-reactive Ag was isolated and shown to be homologous to the Pmel 17 cDNA. Expression of either HMB-45 or Pmel 17 cDNA in COS-7 cells induced cytoplasmic HMB-45 immunoreactivity as described for melanoma cells. Conversely, constitutional HMB-45 immunoreaction in SK-MEL-28 cells could be markedly reduced by expression of antisense Pmel 17 RNA. In several tissues, Pmel 17 mRNA content conformed to the known expression pattern of HMB-45-reactive Ag. Comparative in situ hybridization and immunohistochemistry demonstrated a consistent co-localization of Pmel 17 transcripts and HMB-45-reactive Ag at the cellular level, with strong expression in "activated" melanocytes and melanoma cells and significantly less expression in normal adult melanocytes., Conclusions: We conclude that Pmel 17 cDNA encodes the HMB-45-reactive Ag. Pmel 17 has been postulated to be involved in melanin synthesis and expression of melanoma-peptide epitopes recognized by CTLs. Differential expression of Pmel 17/HMB-45 may have considerable effects in the stepwise process of melanoma progression by aberrant pigment formation and expression of CTL-reactive epitopes.

In vivo and in vitro experimental models have suggested a major role for the urokinase-type plasminogen activator (uPA) in tumor cell invasion and metastasis. The uPA proteolytic activity of tumor cells has been shown to be largely determined by the extent of the expression and saturation of the uPA receptor. We have analyzed the expression and cellular localization of both uPA and uPA receptor at the protein and mRNA levels in 33 paired samples of renal cell carcinoma (RCC) and non-tumorous kidney tissue. In comparison with adjacent normal non-tumorous kidney tissues RCC tumor cells modestly overexpressed uPA-receptor mRNA and showed significantly decreased uPA mRNA expression. However, the immunoreactive uPA content of tumor cells was comparable to that of the surrounding normal non-tumorous kidney tissue. Assuming constancy of the uPA-receptor affinity for uPA this indicates that a proportion of the RCC-associated uPA may be derived from an exogenous source and subsequently concentrated at the tumor cell surface via uPA receptor expression. The modest increase in uPA receptor expression may lead to a normalization of uPA antigen content in RCC; however, it is not sufficient to substantially increase tumor tissue-uPA content over the level of normal non-tumorous kidney tissue.

Ras gene mutations have been implicated in the pathogenesis of a variety of human tumors. Mutated ras genes have been isolated from human melanoma cell lines, but subsequent studies indicated that ras gene mutations may be a rare event in melanocytic lesions. Recently, a study reported a high frequency of ras mutations correlated with increasing invasion level. To address this inconsistency in the published data, we analyzed 50 primary melanomas to correlate invasion level, tumor thickness, histologic typing, and body localization with point mutations around codons 12/13/61 of the three ras genes. After micro-dissection of paraffin-embedded tumor tissue, ras gene mutations were analyzed by direct sequencing of tumor DNA amplified by polymerase chain reaction. Only two melanomas exhibited ras gene mutations, one sample containing a transition from A to G at position 2 of N-ras codon 61 and the other exhibiting a transversion from C to A at position 1 and a transition from A to G at position 2 of N-ras codon 61. Both tumors were classified as Clark level IV, with a tumor thickness of 2.5 and 1.2 mm, respectively. Both were typed as superficial spreading melanoma and localized to intermittently sun-exposed body sites. The low frequency of ras mutations in malignant melanoma and the lack of ras mutations in melanoma samples from constantly sun-exposed body sites argue against the hypothesis of ras mutations as a marker of progression in malignant melanoma and the suggestion that ras mutations occur predominantly in melanomas from constantly sun-exposed body sites.

Background: The induction of protein tyrosine kinases (PTKs) is known to be a key element in the activation of lymphocytes., Objective: Because immunologic mechanisms are important in the pathogenesis of psoriasis, we examined the time course of tyrosine-phosphorylated proteins (p-tyr) as a marker for cellular PTK activity in phytohemagglutinin (PHA)-stimulated T cells of psoriatic patients and healthy controls., Methods and Results: PHA-stimulated T cells from both groups expressed peaks of p-tyr after 15 min and 4 h. In T cells from psoriatics, the 15-min peak was smaller but the 4-hour peak reached an enormous maximum, which was 270% higher than the basic p-tyr value. PHA-stimulated T cells were additionally treated with psoriasis-provoking drugs (lithium, chloroquine, propranolol and ethanol) and the two immunosuppressive drugs cyclosporin A and FK 506. Lithium and propranolol were able to increase the p-tyr level after 15 min in PHA-stimulated T cells from psoriatics in contrast to controls. Chloroquine and ethanol did not have a significant effect on T cells of both groups. CsA markedly diminished the phosphorylation of intracellular tyrosines in T cells of psoriatics and controls, whereas FK 506 diminished the p-tyr level in controls only slightly., Conclusion: We have characterized important differences in p-tyr phosphorylation activities of psoriatic T cells compared to controls. This could be a hint to explain the known abnormalities of psoriatic T cells.

The most prominent abnormality of ras proto-oncogenes in human lung tumours has involved point mutations at codon 12 of the Ki-ras gene. We have analysed 35 tumour samples of neuroendocrine lung neoplasms (ten carcinoid tumours, ten well-differentiated neuroendocrine carcinomas, and 15 intermediate/small cell neuroendocrine carcinomas) for a point mutation at this site. For this purpose, formalin-fixed and paraffin-embedded tissue sections were microdissected to remove non-tumours areas. DNA in the remaining tumour tissue was amplified in vitro by the polymerase chain reaction (PCR) and double-stranded PCR products were subjected to sequence analysis. Neither point mutations at codon 12 nor additional structural alterations at codons 1-32 were detected in Ki-ras gene. Our results suggest that point mutations at codon 12 of the Ki-ras gene do not seem to be involved in the pathogenesis of pulmonary neuroendocrine neoplasms.

In basal cell carcinoma, release of proteolytic activity is implicated in extracellular matrix degradation and tumor infiltration. The stromelysin metalloproteinase family is a major candidate for the matrix proteolytic activity in infiltrative tumors. However, in murine models of basal cell carcinoma, neither stromelysin 1 nor 2 appears to play a role in tumor infiltration. We have analyzed the expression of the newly described stromelysin 3 in human basal cell carcinoma using Northern blot analysis and in situ hybridization. In 12 of 14 cases, levels of stromelysin 3 expression were more than tenfold above those observed in normal skin. In one of five cases of squamous cell carcinoma, stromelysin 3 expression was tenfold above levels seen in normal skin. Stromelysin 3 expression was either undetectable or extremely weak in all five cases of infiltrative malignant melanoma. In basal cell carcinoma, stromelysin 3 transcripts were localized by in situ hybridization to the stromal tissue immediately adjacent to basal cell carcinoma, the tumor cells themselves being negative. Therefore, expression of stromelysin 3 in stromal cells may be expected to play a significant role in destruction of the basal membrane zone and extracellular matrix in basal cell carcinoma invasion.