Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.

Although alpha-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-alpha-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9 x 10(-11) M and approximately 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10(-9) M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that alpha-MSH and its synthetic analogue Nle4DPhe7 alpha-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.

Purpose Chemotherapy-induced peripheral neuropathy (CIPN) with associated chronic pain is a common and disabling condition. Current treatments for neuropathic pain in CIPN are largely ineffective, with unfavorable side-effects. The capsaicin 8% patch (capsaicin 179 mg patch) is approved for the treatment of neuropathic pain: a single topical cutaneous application can produce effective pain relief for up to 12 weeks. We assessed the therapeutic potential of capsaicin 8% patch in patients with painful CIPN, and its mechanism of action. Patients and methods 16 patients with chronic painful CIPN (mean duration 2.5 years), in remission for cancer and not receiving chemotherapy, were treated with 30 min application of capsaicin 8% patch to the feet. Symptoms were monitored using the 11-point numerical pain rating scale (NPRS), and questionnaires. Investigations were performed at baseline and three months after patch application, including skin biopsies with a range of markers, and quantitative sensory testing (QST). Results Patients reported significant reduction in spontaneous pain (mean NPRS: −1.27; 95% CI 0.2409 to 2.301; p =0.02), touch-evoked pain (−1.823; p =0.03) and cold-evoked pain (−1.456; p =0.03). Short-Form McGill questionnaire showed a reduction in neuropathic ( p =0.0007), continuous ( p =0.01) and overall pain ( p =0.004); Patient Global Impression of Change showed improvement ( p =0.001). Baseline skin biopsies showed loss of intra-epidermal nerve fibers (IENF), and also of sub-epidermal nerve fibers quantified by image analysis. Post-patch application skin biopsies showed a significant increase towards normalization of intra-epidermal and sub-epidermal nerve fibers (for IENF: structural marker PGP9.5, p =0.009; heat receptor TRPV1, p =0.027; regenerating nerve marker GAP43, p =0.04). Epidermal levels of Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), and Langerhans cells were also normalized. QST remained unchanged and there were no systemic side-effects, as in previous studies. Conclusion Capsaicin 8% patch provides significant pain relief in CIPN, and may lead to regeneration and restoration of sensory nerve fibers ie, disease modification.

To Identify a broad sperm of melanosomal proteins, antisera were raised In rabbits against melanosomal protein fractions separated on the basi oftheir soubilit in the onic detergent Triton X-114. Antisera METHODS Subceflular Fractionation and Prparation of Antisera. Melanosomes were purified as described (7) from B16 cells grown as tumors subcutaneously in C57BL/6J mice. Stage III-IV melanosomes made up >90%6 ofthe organelles in the purified fraction as assessed by electron microscopy (7). To further fractionate melanosomes, they were solubilized in 0.1 M sodium phosphate buffer (pH 6.8) containing 1% (vol/vol) Triton X-114 at 40C for 10 min. Detergent-insoluble material was then removed by centrifugation at 10,000 x g for 10 min. The Triton X-114-soluble proteins were subjected to separation into aqueous and detergent phases [adapted from the method of Bordier (6)] as described (7). The protein content ofeach fraction was determined, and rabbits were immunized monthly with 0.2 mg of protein emulsified with complete Freund's adjuvant initially and subsequently in incomplete adjuvant as described (5). Rabbits were bled from the ear vein, the blood was allowed to coagulate at room temperature, and sera were isolated by centrifugation at 10,000 x g for 30 min. Final bleeds were obtained at 6 months. The production of antiserum to the Triton-insoluble melanosomal matrix has been described (5).The polyclonal antibody aPEP13 was produced in rabbits (8) using procedures as detailed (3). Briefly, a 15-amino-acid sequence representing the predicted carboxyl terminus ofthe cloned murine si-encoded protein (8) was synthesized (BioSynthesis, Denton, TX) and conjugated to bovine serum albumin SuperCarrier (Pierce). Immunization and sera collection from the rabbit and the titration and specificity of antibody production, as determined by ELISA, was as reported (3).Immunoblotting. Melanocyte lines were established from C57BL/6J mice, either wild type ("melan-a" cells), homozygous for the silver (si) mutation ("melan-si-l" cells), or homozygous for the albino (c) mutation ("melan-c" cells),

Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink-eyed-dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell-free melanosomal melanization assay. Upon incubation of a melanosome-rich fraction with the melanin precursor ~-3,4-dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinaserelated family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane-bound p locus protein may also interact closely with melanin.Keywords: melanosome ; tyrosinase ; pink-eyed dilution locus ; silver locus ; melanoma antigen.Melanocytes synthesize and deposit melanin pigments within a subcellular organelle, the melanosome. Melanosomes are highly specialized members of the endolysosomal lineage of organelles [l]. It is recognized that melanosomal biogenesis involves a bipartite pathway in which tyrosinase-containing coated vesicles fuse with premelanosomes containing a well-defined matrix (stage I1 melanosomes). This fusion is believed to initiate the complex process of melanization [2, 31. Although a family of membrane proteins involved in the synthesis of melanin and related to the prototypic enzyme tyrosinase has been well studied [4-61, little is known about other melanosomal proteins or their role in melanin synthesis and degradation. Recently, a melanosomal protein encoded by the pink-eyed-dilution (p) locus has been identified [7]. This 110-kDa integral membrane protein is unrelated to the tyrosinase-related protein (TRP) family, and its function remains unknown [7].The least well studied melanosomal proteins are those that comprise the internal structure of the melanosome, termed the matrix. It is upon this proteinaceous matrix that melanin polymers are deposited [l]. Even the partial disruption of the internal matrix of melanosomes requires extensive extraction with strongly chaotropic agents such as SDS [8, 91. Effective partial Abbreviafions. Dopa, ~...