Reverse transcription (RT) of the tyrosinase mRNA and specific cDNA amplification by nested polymerase chain reactin (PCR) have been reported to facilitate the early detection of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. In the current study we analyzed peripheral blood samples of 65 melanoma patients of different clinical stages for the presence of tyrosinase mRNA by RT-PCR using nested oligonucleotide primers specific for tyrosinase cDNA. Furthermore, blood samples were evaluated for tumor cell growth by cell culture assays in vitro. No tyrosinase mRNA was detectable in blood samples of 26 patients with primary melanoma and 16 patients with regional lymph node metastases. In five of 13 patients with visceral metastases we found at least one blood sample positive for tyrosinase mRNA during a 2-to 4-mo interval. Analyses of different blood samples of patients with visceral metastases taken in a 2-h interval, furthermore, indicate that tumor cells only transiently persist in the peripheral blood. We obtained in vitro proliferating melanoma cells from two blood samples derived from different patients with visceral melanoma metastases. This demonstrates that viable melanoma cells indeed circulate in the peripheral blood with retained proliferative capacity in vitro. The analysis of blood samples by RT-PCR for tyrosinase mRNA, however, is not suitable for the early detection of tumor progression in melanoma patients.
Electrophysiological evidence from cutaneous nociceptors suggested a synergism between excitatory actions of inflammatory mediators (IM) and low pH. In human skin it is possible to induce constant ongoing pain with continuous infusion of acid buffer. This method was used to study the interaction with mediators of inflammation psychophysiologically. A skin area on the palmar forearm of 6 subjects (either gender, age 22-35 years) was continuously infiltrated with a phosphate buffered electrolyte solution (pH 5.2) using a motorized syringe pump that was adjusted so as to produce constant pain of about 20% on a visual analog scale (VAS; extending from 'no' to 'unbearable pain'). Pain was assessed on the VAS at 10-sec intervals; the rating was called up by means of an acoustic signal. An additional cannula was placed in the skin before the infusion of acidic buffer started. Injections of an acidic combination of IM (BK, 5-HT, HIS, PGE2) 0.2 ml were then given through the cannula at intervals of 10 min in a randomized double blind order of concentrations. The other arm was used for negative control, i.e. IM in neutral solution were injected into normal skin continuously infiltrated with a buffer solution at pH 7.4. The IM induced dose-dependent, transient burning pain on both arms-markedly more intense and prolonged, however, in the acidotic skin (P < 0.004, U-test). The difference corresponded to a 10-fold increase in algogenic potency with 10(-7) M IM, being smaller with 10(-6) and 10(-5) M concentration. The interaction between low pH and IM was mutual: additional injections of plain phosphate buffer (pH 5.2) into the acidotic skin were significantly more painful (20-fold) after application of IM than under control conditions. Thus, we tend to conclude that it is the inflammatory mediators that potentiate the algogenic effect of low pH rather than vice versa. Tissue acidosis appears as a dominant factor in inflammatory pain.
SUMMARYSkin-infiltralitig lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In ^H-thymidine incorporation assays, SIL showed proliferation in response to lL-2, IL-3, IL-4, ionomycin (Io)+ll-o-tetradecanoyl-phorbol-Hacetate (TPA) and OKT3 -\-TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-a) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-a upon stimulation with mitogens but failed to secrete IFN-y. Io in combination with phorbolester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-a and low amounts of I FN-y. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-y but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.
Vinculin and beta-catenin are intracellular attachment proteins linking transmembrane adhesion molecules (E-cadherin) to the actin microfilament cytoskeleton, thus participating in formation of cell-cell adherens junctions, or zonulae adherentes. This type of junction was only recently described in human epidermis due to the imprecise morphological criteria for its recognition. In this study, we investigated the relationship between the expression of the zonula adherens-associated proteins vinculin, beta-catenin, E-cadherin, and actin, on the one hand, and the presence of electron microscopically discernable structures in normal human epidermis on the other. Mouse jejunal epithelium with its orderly arrangement of various junctional structures served as a positive control. Simple and dual post-embedding immunogold labeling was performed on ultrathin sections of Lowicryl K4M and Lowicryl K11M embedded tissues. The overall distribution of the antigens in human epidermis was evaluated on frozen tissue sections using immunofluorescence and laser confocal scanning microscopy. Antibodies against proteins associated with desmosomes (i.e., keratins, desmoglein 1, and plakoglobin) were used as controls. Vinculin and beta-catenin were localized to junctional structures distinct from desmosomes, thus defining the presence of zonulae adherentes. Labeling of actin and E-cadherin was less clearly restricted to the junctions, but these two proteins were also co-expressed at zonulae adherentes and not at desmosomes. In human epidermis, zonula adherens-associated labeling was consistently detected near desmosomes, indicating the possibility of a functional relationship between the two types of junctions.
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