Influence of neutralizing antibodies to adalimumab and infliximab on the treatment of psoriasis.

BACKGROUND: Current treatment with biologics has produced dramatic therapeutic effects in patients with psoriasis, although these agents occasionally decrease in efficacy. One of the main factors responsible for this attenuation is attributed to the development of antidrug antibodies (ADAs). OBJECTIVES: To analyse the relationship between serum drug concentrations, the presence of ADAs and treatment efficacy of adalimumab and infliximab, and to determine the optimal use of these biologics. METHODS: This was a 1-year prospective study in the dermatology departments of Kobe University Hospital and collaborating hospitals. All patients starting a regimen of adalimumab and infliximab for psoriasis were included. We measured the serum concentration of the drugs and titres of antibodies to adalimumab and infliximab, as well as the Psoriasis Area and Severity Index scores at weeks 0, 4, 12, 24 and 48 during the first year of treatment. RESULTS: We observed a 50% positive rate of ADAs to adalimumab, and a 41% positive rate of ADAs to infliximab. The titres of ADAs showed a wide range from low to high titres. In the high-titre groups, the patients exhibited a decreased clinical response, and demonstrated a negative correlation between titre and clinical response. However, an equivalent therapeutic effect was observed between the low-titre group and the group with no antibodies detected for adalimumab. For infliximab, the patients with ADAs showed decreased clinical response. An apparent negative correlation between antibody production and reduced clinical response was observed. CONCLUSIONS: Two biologics, adalimumab and infliximab, showed different therapeutic behaviour. The measurement of ADAs and drug concentrations has important implications for treatment with biologics.

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial-mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT. METHODS: We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells. RESULTS: In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells. CONCLUSIONS: This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC.

Soybean beta-conglycinin as the main allergen in a patient with food-dependent exercise-induced anaphylaxis by tofu: food processing alters pepsin resistance.

BACKGROUND: Food-dependent exercise-induced anaphylaxis (FDEIA) due to soybeans is a rare disorder. The allergen responsible for FDEIA due to soybeans has not yet been determined. OBJECTIVE: We characterized the clinical features of a patient with FDEIA due to tofu, who was well tolerant to drinking soy milk. We then sought to identify the responsible soybean allergen(s) in that patient. We further studied whether different stabilities of the allergen(s) to pepsin digestion between two soybean products are related to their clinical allergenicity. METHODS: Skin prick tests and provocation tests using soybean products were performed to detect the responsible food and other factors that induced the allergic symptoms. Specific IgE to various soybean allergens were examined by ImmunoCAP, ELISA and protein microarray assays. Immunoblotting for soybeans and soybean products using the patient's serum was also performed. Soybean products were serially digested by pepsin to disclose the stability of the allergens. RESULTS: Provocation with ingestion of tofu and exercise induced the allergic symptoms, while ingestion of soy milk and exercise did not. Immunoblot analysis, ELISA and protein microarray assay revealed that beta-conglycinin mainly reacts with IgE antibodies in the patient's serum. By immunoblot analysis, beta-conglycinin in soy milk completely disappeared after pepsin digestion within 20 min, whereas beta-conglycinin in tofu was almost intact after more than 120 min of pepsin digestion. CONCLUSION: We identified beta-conglycinin as the causative allergen in a patient with FDEIA induced by tofu. The difference in resistance to pepsin digestion between tofu and soy milk suggests that the presence of undigested allergens in the digestive tract is a prerequisite for the development of FDEIA.

Association of T-cell receptor Vbeta haplotypes with dry skin in DS-Nh mice.

BACKGROUND: Although dry skin and T cell-dependent disease exacerbation are characteristic features of atopic dermatitis (AD), the involvement of T cells in the development of dry skin remains unclear. AIMS: We aimed to elucidate the role of T cells in the development of dry skin in DS-Nh mice as a model for AD, and to evaluate this skin condition pharmacologically. METHODS: We prepared DS-Nh mice harbouring a T-cell receptor (TCR)Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, and mice harbouring a TCRVbeta(b) haplotype without any deletion. We analysed the TCRVbeta chain usage and cytokine response to antimouse CD3 monoclonal antibodies in the splenocytes from the two mouse substrains. Transepidermal water loss (TEWL) was measured, and histochemical examination of these mice was carried out. Finally, a pharmacological analysis using loratadine was also performed to evaluate the features of spontaneous dry skin in DS-Nh mice as a model of AD. RESULTS: Although the deletion of TCRBV gene segments in the TCRVbeta(a) haplotype yielded different representations of each TCRVbeta mRNA, this deletion did not evoke distinct cytokine profiles in the splenocytes compared with those of mice with the TCRVbeta(b) haplotype. Furthermore, our results indicated that the onset of dry skin occurred earlier in mice with TCRVbeta(b) than in those with TCRVbeta(a). Pharmacologically, AD-like dry skin in DS-Nh with TCRVbeta(b) mice is susceptible to an H1 blocker. CONCLUSIONS: A specific lymphocyte subpopulation bearing T-cell receptors may be responsible for loratadine-responsive dermatitis in DS-Nh mice.

Skew in T cell receptor usage with polyclonal expansion in lesions of oral lichen planus without hepatitis C virus infection.

Oral lichen planus (OLP) is a refractory disorder of the oral mucosa. Its predominant symptoms are pain and haphalgesia that impair the quality of life of patients. OLP develops via a T cell-mediated immune process. Here, we examined the characteristics of the infiltrating T cells in terms of the T cell receptor (TCR) repertoires, T cell clonality, T cell phenotypes and cytokine production profiles. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of OLP biopsies from 12 patients. The cytokine expression profiles and T cell phenotypes were measured by real-time quantitative polymerase chain reaction. We observed that there were skewed TCR repertoires in the tissue samples (TCRVA8-1, VA22-1, VB2-1, VB3-1 and VB5-1) and PBMCs (TCRVA8-1, VB2-1, VB3-1 and VB5-1) from OLP patients. Furthermore, the CDR3 distributions in the skewed TCR subfamilies exhibited polyclonal patterns. We observed increases in CD4(+) T lymphocytes, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and human leucocyte antigen D-related in the OLP tissue specimens. Taken together, the present results suggest that T cells bearing these TCRs are involved in the pathogenesis of OLP, and that IL-5 and TNF-alpha may participate in its inflammatory process.

Comparison of ultrasonic pachymetry and Fourier-domain optical coherence tomography for measurement of corneal thickness in dogs with and without corneal disease.

Several ultrasonic and Fourier-domain optical coherence tomography (FD-OCT) pachymeters are used to measure corneal thickness in canine patients and research subjects. This study assessed the reliability of and consistency between two ultrasonic pachymetry (USP) devices, Pachette 3 and Accupach VI, as well as automated and manual measurements obtained using FD-OCT in dogs with and without corneal disease. Corneal thickness measurements were compiled from 108 dogs and analyzed using mixed effects linear regression, with Bonferonni adjustments for post-hoc comparisons, to determine the effects of age, weight and disease state. Data are presented as predicted mean±standard error. Canine corneal disease can result in marked increases in thickness that frequently exceed the upper limits of measurement of some pachymetry devices developed for human use. In this study, the corneas of dogs with endothelial disease or injury frequently exceeded the upper limits of quantitation of 999 and 800µm for the Accupach VI and automated FD-OCT pachymeters, respectively. Using values <800µm, the Pachette 3 generated significantly greater values for central corneal thickness (CCT) than the Accupach VI, manual FD-OCT and automated FD-OCT at 625±7.0, 615±7.2, 613±7.2, and 606±7.4µm respectively (P<0.001). Of the two devices where measurements >1000µm were obtained, manual FD-OCT demonstrated less variability than the Pachette 3. Corneal thickness increased linearly with age and weight with an increase of 6.9±1.8µm/year and 1.6±0.8µm/kg body weight (P<0.005 and P=0.038, respectively).

Inhibition of the motility and growth of B16F10 mouse melanoma cells by dominant negative mutants of Dok-1.

Dok-1 (p62(Dok)) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.

Roles for the protein tyrosine phosphatase SHP-2 in cytoskeletal organization, cell adhesion and cell migration revealed by overexpression of a dominant negative mutant.

SHP-2, a SRC homology 2 domain-containing protein tyrosine phosphatase, mediates activation of Ras and mitogen-activated protein kinase by various mitogens and cell adhesion. Inhibition of endogenous SHP-2 by overexpression of a catalytically inactive (dominant negative) mutant in Chinese hamster ovary cells or Rat-1 fibroblasts has now been shown to induce a marked change in cell morphology (from elongated to less polarized) that is accompanied by substantial increases in the numbers of actin stress fibers and focal adhesion contacts. Overexpression of the SHP-2 mutant also increased the strength of cell-substratum adhesion and resulted in hyperphosphorylation of SHPS-1, a substrate of SHP-2 that contributes to cell adhesion-induced signaling. Inhibition of SHP-2 also markedly increased the rate of cell attachment to and cell spreading on extracellular matrix proteins such as fibronectin and vitronectin, effects that were accompanied by enhancement of adhesion-induced tyrosine phosphorylation of paxillin and p130Cas. In addition, cell migration mediated by fibronectin or vitronectin, but not that induced by insulin, was impaired by overexpression of the SHP-2 mutant. These results suggest that SHP-2 plays an important role in the control of cell shape by contributing to cytoskeletal organization, and that it is an important regulator of integrin-mediated cell adhesion, spreading, and migration as well as of tyrosine phosphorylation of focal adhesion contact-associated proteins.

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Etsl in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes.

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single-channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.

Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma.

PURPOSE: The EBV latent membrane protein-1 (LMP-1) is a multifunctional protein. Recently, the contribution of LMP-1 to the metastasis of nasopharyngeal carcinoma (NPC) has been suggested. Angiogenesis is a key step for metastasis. Thus, the association of LMP-1 to neovascularization of NPC was examined in this study. EXPERIMENTAL DESIGN: The association of LMP-1 to angiogenesis in 39 patients with NPC was evaluated by immunohistochemical study, and then induction of angiogenic factors by LMP-1 was examined by ELISA and luciferase reporter assay. RESULTS: In an immunohistochemical study, the expression of LMP-1 was significantly correlated to microvessel counts (P = 0.0003), suggesting that LMP-1 may induce some angiogenic factors. Therefore, we studied the relationship between LMP-1 expression and interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) expression by immunohistochemical analysis. IL-8, VEGF, and bFGF expression were correlated to microvessel counts, but only IL-8 expression was significantly correlated to LMP-1 expression (P < 0.0001). Transfection with LMP-1 expression plasmid induced IL-8 protein expression in C33A cells. The expression of LMP-1 transactivated IL-8 promoter, as demonstrated by IL-8 promoter luciferase reporter assay. Mutation of the nuclear factor kappaB responsive element in the IL-8 promoter region completely abolished transactivation by LMP-1, whereas mutation of the activator protein responsive element did not affect promoter activity. CONCLUSION: These results suggested that LMP-1 induces expression of IL-8 through the nuclear factor kappaB binding site, which may contribute in part to angiogenesis in NPC.

Production of granulocyte-macrophage colony-stimulating factor or GM-CSF-like growth factor by rat thymic epithelial cell line.

An epithelial cell line, IT-45R1, has been established from a thymus of normal Wistar rat and was found to produce growth factor which stimulated the proliferation of bone marrow cells. This growth factor induced the formation of colonies composed of macrophages, granulocytes, or both, in semi-solid medium and stimulated the proliferation of an interleukin 3 (IL3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent clone. Neither IL3-dependent clone nor IL6-dependent clone responded to IT-45R1 factor. Additionally, IT-45R1 factor acted on both rat and mouse bone marrow cells but not on human bone marrow cells. Molecular weight of IT-45R1 factor was 30 kD and its isoelectric point was 4.5. To determine whether IT-45R1 factor is GM-CSF or not, Northern blot analysis and neutralization with anti-mouse GM-CSF antibody were carried out. IT-45R1 expressed GM-CSF mRNA, but neither M-CSF nor IL6 transcripts. However, antiserum specific for mouse GM-CSF did not neutralize IT-45R1 factor. Taken together, a rat thymic epithelial cell line, IT-45R1, constitutively produces GM-CSF or GM-CSF-like growth factor.

Preparation and characterization of high-specific-surface-area activated carbons from K2CO3-treated waste polyurethane.

An activated carbon with high specific surface area was prepared from polyurethane foam by chemical activation with K2CO3 and the influences of carbonization temperature and impregnation ratio on the pore structure of the prepared activated carbon were investigated. It was found that the specific surface area of the activated carbon was at a maximum value (about 2800 m(2)/g) at a carbonization temperature of 1073 K and at an impregnation ratio of 1.0. It was concluded that the polyurethane foam structure was modified during impregnation by K2CO3, K2CO3 promoted charring during carbonization, and then the weight loss behavior was changed below 700 and above 1000 K, carbon in the char was consumed by K2CO3 reduction, and this led to the high specific surface area. The prepared activated carbon had a very sharp micropore size distribution, compared with the commercial activated carbon having high specific surface area. The amounts of three organic vapors (benzene, acetone, and octane) adsorbed on the prepared activated carbons was much larger than those on the traditional coconut shell AC and the same as those on the commercial activated carbon except for octane. We surmised that the high specific surface area was due to the modification of the carbonization behavior of polyurethane foam by K2CO3.

Development of a simple method to evaluate medical staff radiation dose and its application to a software system supporting PET facility operation.

In recent years, positron-emitting labelled radiopharmaceuticals have come to be used in conjunction with positron emission tomography (PET) in various clinical diagnoses. Radiation exposure of the medical staff is a key issue in the design of PET facilities intended to handle large numbers of persons for PET diagnosis. As a first step, the radiation dose to individuals who received radiopharmaceuticals was calculated using a mathematical phantom model and the EGS4 electromagnetic cascade Monte Carlo code and MCNP Monte Carlo code. Dose rate behind a lead shield was also calculated for various lead thicknesses. The radiation dose distribution around a syringe containing a positron emitter was calculated. The calculated dose distributions were fitted to polynomial equations. These calculations were evaluated against measurements. The second step was to evaluate medical staff dose at a specified time by superimposing dose distribution from each person who received radioisotopes taking into account radioactive decay. In this way, we developed software to support PET facility operation, namely, planning, prediction, control of medical staff dose and facility operation. This system was also designed to schedule daily radiopharmaceuticals production and to manage radioactive wastes by taking decay time into account.

Elimination of CD4(+) T cells enhances anti-tumor effect of locally secreted interleukin-12 on B16 mouse melanoma and induces vitiligo-like coat color alteration.

CD4(+) T cells have been reported to suppress immunity against cancer in certain animal models. In this study, we investigated the role of CD4(+) T cells in the anti-tumor immune response when interleukin-12-producing melanoma cells are inoculated in mice. We found that interleukin-12-transfected B16 melanoma showed retarded tumor growth in syngeneic mice; however, all the mice developed tumors eventually. In vivo depletion of CD4(+) T cells led to complete regression of B16/interleukin-12 tumors in 12 of 20 mice (60%). Immunohistochemical analyses revealed that a number of CD8(+) T cells accumulated in close proximity to the B16/interleukin-12 tumors in the CD4(+) T cell-depleted mice, whereas CD8(+) T cells were only scarcely observed at the periphery of the tumors in control immunocompetent mice. Furthermore, 10 of 20 mice treated with both B16/interleukin-12 inoculation and CD4(+) T cell depletion exhibited vitiligo-like coat color alteration. B16/interleukin-12 tumors completely regressed in all the mice with vitiligo. Histologic examination showed that CD8(+) lymphocytes accumulated around the hair bulbs of mice with vitiligo, but not in those without vitiligo. These results suggest that CD4(+) T cells have an inhibitory effect on tumor rejection by suppressing cytotoxic CD8(+) T cells in this melanoma loading model with local interleukin-12 secretion. To investigate the mechanism of enhanced anti-tumor effects by CD4(+) T cell depletion, we examined the T helper type 1/2 cytokine profile in the tumor draining lymph nodes of B16/interleukin-12-bearing mice with or without CD4(+) T cell depletion using the reverse transcription-polymerase chain reaction method. We found that CD4(+) T cell depletion eliminated T helper type 2 cells and resulted in a T helper type 1-dominant cytokine profile in tumor draining lymph nodes. We emphasize that this T helper type 1-dominant cytokine profile may generate further activated CD8(+) T cells against B16 melanoma cells, lead B16/interleukin-12 to regress, and result in the destruction of the melanocytes in hair bulbs due to cross-antigenicity between both cell types. This mouse model not only demonstrates the depletion of CD4(+) T cells as a useful strategy for cancer gene therapy with interleukin-12 but also provides a model for human melanoma-associated vitiligo.J Invest Dermatol 115:1059-1064 2000

Melanocyte mitogens induce both melanocyte chemokinesis and chemotaxis.

It is believed that during repigmentation of vitiligo, inactive melanocytes in the outer root sheath of the hair follicle become activated, proliferate, and migrate into the depigmented skin. However, the mechanisms controlling melanocyte migration remain to be elucidated. In this study, we investigated the effects of well-described melanocyte growth factors on melanocyte migration. Using time-lapse photography, we demonstrated that melanocyte chemokinetic movement was induced by basic fibroblast growth factor, stem cell factor, and endothelin-1, with the greatest effect noted using 100 nM endothelin-1. Similar results were reported previously with leukotriene C4. When surrounded by these stimuli, melanocytes moved in a random, nonlinear fashion and showed no desensitization at the concentrations studied. In Boyden chamber checkerboard analysis, basic fibroblast growth factor, leukotriene C4 and endothelin-1 were chemotactic. They produced directional migration and showed desensitization at higher concentrations. The greatest effect again was seen with 100 nM endothelin-1. Stem cell factor showed no effect in this assay system at the concentrations tested. The four melanocyte mitogens--leukotriene C4, endothelin-1, basic fibroblast growth factor, and stem cell factor--stimulate melanocyte migration, and this migration may be either chemokinetic (activated random movement) or chemotactic (requiring a gradient, directional, and showing desensitization), depending on the conditions used. We believe that these factors may be effective in stimulating vitiligo repigmentation by inducing proliferation and migration of hair-follicle outer-root-sheath melanocytes into the depigmented epidermis.

Expression of E and P-cadherin by melanoma cells decreases in progressive melanomas and following ultraviolet radiation.

The purpose of our study was to determine whether the degree of E- and P-cadherin expression in melanomas correlates with the invasive behavior of the clinical lesions from which the cell lines were derived. Cadherins comprise a family of calcium-dependent cellular adhesion molecules expressed on most cell types that form solid tissues. In the human epidermis, melanocyte cadherin expression may function to maintain the integrity of the epidermal-melanin unit. Employing both immunofluorescence microscopy and fluorescence-activated cell sorter analysis, we localized and quantitated E- and P-cadherin expression on melanoma cell lines derived from primary or metastatic lesions using the monoclonal antibodies HECD-1 and NNC-CAD-299, respectively. Human epidermal melanocytes isolated from neonatal foreskin were evaluated by similar techniques and served as a biologic control. Melanoma cell lines were isolated from primary or metastatic lesions of patients described as having "early," "intermediate," or "advanced disease." Melanoma E- and P-cadherin immunofluorescence, as quantified by fluorescence-activated cell sorter, varied inversely with disease progression. Selected log mean ratios of E-cadherin fluorescence, as compared to human epidermal melanocytes (arbitrarily = 1), ranged from 1.04 in the WM 35 melanoma cell line (low invasive potential) to 0.1 and 0.02 in the WM 983A and 1361A melanoma cell lines (derived from primary lesions with metastases), respectively. Although values for P-cadherin fluorescence were less, the trend of decreasing cadherin amounts with more advanced disease was observed. Melanoma cells appear to express E- and P-cadherin levels inversely related to disease progression. Ultraviolet radiation significantly decreased E- and P-cadherin expression in the human epidermal melanocytes and P-cadherin expression in the WM 35 melanoma cell line (p < 0.05). Although not statistically significant, E-cadherin expression in the WM 35 melanoma cell line decreased substantially. Thus, ultraviolet radiation may have a direct effect on human epidermal melanocytes and melanoma cell attachment through cadherins within the epidermis or tumor nodules.

DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2.

It is believed that DOPA-negative melanocytes in the outer root sheath of the human hair follicle are activated, become identifiable by DOPA staining, and migrate into the epidermis during the repigmenting phase of vitiligo. These cells are difficult to identify, however, and otherwise have not been characterized. These cells are readily identified by immunofluorescence, immunohistochemistry, and immunoelectronmicroscopy using the antibodies NKI/beteb and A4F11, which recognize premelanosome-related antigens. The majority of the outer root sheath melanocytes were found in the mid to the upper portion of the hair follicle. Double staining revealed that these cells were distinct from HLA-DR-bearing dendritic cells. Further immunohistochemical investigation using alpha-PEP-7, alpha-PEP-1, or TMH-1 and alpha-PEP-8 antibodies revealed that outer root sheath melanocytes cannot be identified by antibodies to tyrosinase, TRP-1, or TRP-2, respectively. These cells also did not react with HMB45 antibody, which recognizes a melanosome-associated cytoplasmic antigen. We believe that the inactive outer root sheath melanocytes contain some of the early structural proteins but not any of the enzymatic proteins necessary for melanogenesis. Therefore, activation is the process whereby outer root sheath melanocytes acquire all of the structural and enzymatic proteins necessary for melanogenesis.