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Several studies have demonstrated that dendritic cells can be generated in vitro from CD34+ hematopoietic progenitor cells. In vivo, dendritic cells are found in many tissues and reside in direct proximity to extracellular matrix proteins. Because extracellular matrix proteins affect differentiation and location of cells in tissues, this study was designed to investigate potential effects of extracellular matrix proteins on differentiation of dendritic cells. Dendritic cells were generated from CD34+ human cord blood cells in the presence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha for 6 d and subsequently cultured for an additional 6-d period on tissue culture plates coated with various extracellular matrix proteins. Among the extracellular matrix proteins tested, exposure to fibronectin stimulated dendritic cell/Langerhans cell differentiation as indicated by the 50% increase of the number of cells expressing the Birbeck granule-associated marker Lag and displaying numerous Birbeck granules. Adhesion on fibronectin was shown to be specifically mediated by the integrin alpha5beta1. Because laminin and collagen were unable to cause similar changes in Langerhans cell development, these results suggest that fibronectin may cause changes affecting cellular differentiation of progenitors. Hematopoietic progenitors may exhibit maturational regulated differences in response to both matrix molecules and cytokines. The influence of combined signals emanating from a supportive microenvironment, specific integrins, and particular cytokines in the differentiation of Langerhans cells is discussed.

The majority of in vitro reconstructed human epidermis is composed of keratinocytes only. Recently, the introduction of melanocytes into epidermal reconstructs has enlarged their field of application. The completion of reconstructed epidermis by introducing Langerhans cells remained an important challenge because Langerhans cells, unlike the other epidermal cell types, cannot be subcultured and expanded. To solve this problem, we used cord blood-derived CD34+ hematopoietic progenitors. Seeding these cells, after induction of their differentiation by granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha, onto a reconstructing epidermis, composed of keratinocytes and melanocytes, gives rise to a pigmented epidermis with melanocytes in the basal layer and resident epidermal Langerhans cells located suprabasally. Interestingly, the same result was obtained by co-seeding a mixture of keratinocytes, melanocytes, and nondifferentiated CD34+ hematopoietic progenitors on the dermal equivalent, indicating that keratinocytes provide the environmental conditions for hematopoietic progenitors to differentiate into resident epidermal Langerhans cells, expressing major histocompatibility complex class II molecules, CD1a antigen, and Birbeck granules.

In addition to T cell receptor triggering, activation of T cells requires costimulatory signals that have been shown to be mainly initiated through CD28. We analyzed the expression and function of the two ligands for CD28, B7-1 (CD80) and B7-2 (CD86), on human Langerhans cells (LC), the antigen-presenting cells from epidermis. Human LC freshly isolated from epidermis (fLC) expressed significant level of B7-2, which was increased upon a short culture in vitro. In contrast, B7-1 was undetectable on fLC but appeared at the cell surface after a 3-day culture in vitro. Pre-incubation of 18-h cultured LC with anti-B7-2 monoclonal antibodies (mAB) was sufficient to abrogate the binding of CTLA4-Ig fusion protein, while a combination of both mAB against B7-1 and B7-2 was necessary to obtain a complete inhibition of CTLA4-Ig binding on 3-day cultured LC, showing the absence of a third CTLA4 ligand. The function of B7-1 and B7-2 on human LC has been analyzed by adding mAb at the beginning of mixed epidermal cell lymphocyte reactions. Anti-B7-2 mAb and CTLA4-Ig, but not anti-B7-1 mAb, strongly inhibited allogenic. as well as recall antigen-induced T cell proliferation supported by fLC or 3-day cultured LC. Collectively, these results demonstrate that B7-2 is the major ligand for CD28/CTLA4 at the LC surface and that it plays a crucial role in human LC co-stimulatory function with little, if any, dependence of B7-1 expression.

Dendritic cells are characterized by their dendritic shape, by high level of class II MHC molecules expression, by their ability to induce primary antigen-specific T cell responses and by their motility. In the skin, the most well-characterized dendritic cells are Langerhans cells (LC). LC, which originate from bone marrow are mobile messenger cells which actually carry antigens from the skin to the lymph nodes. However, the driving and regulatory mechanisms for LC motility remain poorly understood. Studies in mice have revealed that epicutaneous hapten application induced a significant accumulation of antigen-bearing dendritic cells in draining lymph nodes. But signals in addition to simple antigen binding are probably necessary for the induction of migration. The production of specific cytokines by epidermal cells has to be considered. Indeed, TNF alpha has been shown to provide one signal for LC migration. In vitro studies showed that human epidermal LC interact with the surrounding extracellular matrix (ECM) through beta 1 integrin receptors and that interaction with the ECM environment may play a crucial role in the directed emigration of LC from the epidermis. In vitro hapten treatment resulted in a stimulation of LC migration through ECM, and fibronectin and type IV and type I collagen promoted the migration of hapten-modified LC. By contrast, contact of human LC with an irritant did not increase the number of migrating cells. ECM plays some important roles in LC migration from the epidermis to the dermis and this is regulated by some of the beta 1 integrins. These results demonstrate that the regulation of LC migration is a complex phenomenon, largely dependent on factors such as ECM proteins, cell adhesion molecules, cytokines.

GB36, a mouse monoclonal antibody (mAb) raised against an epithelial antigen of the human trophoblast, reacts with the epithelial basement membrane of chorionic villi; it does not react with the invasive extravillous cytotrophoblast. Expression and characterization of the antigen of GB36 (designated GBA36) were investigated in normal keratinocytes by immunoprecipitation, immunofluorescence and immunoelectron microscopic studies. Immunoprecipitation experiments demonstrated that the proteins identified on keratinocytes by mAb GB36 and a rat mAb anti-integrin alpha 6 (GoH3) were the same. Using immunofluorescence and immunoelectron microscopic methods, GBA36 was localized on the cell membrane facing the epithelial basal lamina of basal keratinocytes. GBA36 distribution in benign and malignant skin tumours was evaluated by immunostaining methods (immunofluorescence and immunoperoxidase). Analysis of tumours revealed that whereas benign epithelial tumours and intradermal naevi displayed high levels of GBA36, the expression of this antigen decreased progressively in spinocellular and basal cell carcinomas, and in cutaneous melanomas in relation to invasiveness. During cell transformation, GBA36 undergoes quantitative alterations, and expression is down-regulated. Although the functional relevance of these changes remains unknown, the correlation of decreased GBA36 expression with tumour progression may indicate a role for altered integrin expression in tissue invasion by human skin carcinoma and melanoma.

To migrate from epidermis to regional lymph nodes, antigen-bearing epidermal Langerhans cells (LC) must move through extracellular matrix (ECM) of various composition. The present study was designed to contrast the ability of basement membrane (BM)- and dermis-ECM components to successively stimulate the adhesion of normal human epidermal LC, in vitro. For this purpose, we used highly enriched LC suspensions (70%-80%), allowed them to attach to one ECM substrate, and then studied the readhesion properties of these recovered ECM-attached LC to the same and different ECM substrates. Each of four ECM molecules (laminin (LM), fibronectin (FN), type I and type IV collagen) was tested in pairs. Readhesion of recovered LM and type IV collagen-attached cells did not affect readhesion to FN and type I collagen, indicating that the interaction of LC with the BM components can be normally followed by interaction with the dermis-ECM molecules. In contrast, readhesion of recovered FN-, type I collagen-, and type IV collagen-attached cells to LM was significantly reduced. The findings indicate that following contact to BM components, epidermal LC are able to attach to ECM proteins present in the dermis, whereas once they have made contact with ECM present in the dermis, they reduce their binding capacity to the BM laminin, suggesting the contact with the dermal components could prevent LC from reentering the epidermal compartment. Binding to LM and FN was also shown to induce a decline in the expression of CD1a, known as a specific marker restricted to epidermal LC.(ABSTRACT TRUNCATED AT 250 WORDS)

Three counter-receptors for LFA-1 of the immunoglobulin family have been discovered: ICAM-1, ICAM-2, and ICAM-3. Despite their homologies, their patterns of expression suggest specialized roles. The finding that ICAM-3 is much better expressed than other LFA-1 ligands on monocytes and resting T cells, and that this discovery may be important in the initiation of immune responses prompted us to search for the expression of ICAM-3 by human epidermal Langerhans cells (LC). Six out of eight different ICAM-3 monoclonal antibodies were found to be reactive with epidermal LC. Immunoelectron-microscopy staining revealed that 100% of freshly-isolated, typical Birbeck granules containing LC expressed ICAM-3. After one day and three days of culture, 100% of LC still expressed ICAM-3, but the staining intensity was decreased by 58% and 76% respectively. Immunoprecipitation of 125I surface-labeled LC with anti-ICAM-3 antibodies revealed a polypeptide with apparent M(r) of 122,000-125,000. To determine whether ICAM-3 was involved in LC function, mixed epidermal cell-lymphocyte reactions were performed with freshly isolated LC in the presence of various concentrations of different anti-ICAM-3 antibodies. Among the different antibodies tested, HP2/19 and CBR-IC3/1 were found to partially block the reaction in a dose-dependent manner, suggesting that ICAM-3 represents a new molecule involved in the initiation of the immune response driven by epidermal LC.

To study the migration capacity of Langerhans cells in vitro, a reconstituted basement membrane invasion assay (Matrigel assay) was performed. Partially enriched human epidermal Langerhans cell suspensions (6-20%) were incubated with or without haptens [2,4,6-/trinitrobenzenesulfonic acid (TNBS) and fluorescein isothiocyanate (FITC)] or an irritant sodium lauryl sulfate (SLS) in Hanks' balanced salt solution for 10 min at 37 degrees C, and were then used for the invasion assay. Fibroblast-conditioned medium prepared from human dermal fibroblasts was used as a source of chemoattractants. Non-treated cells showed a gradual increase in migration of HLA-DR-positive cells through the Matrigel-coated filters in a time-dependent manner. In vitro TNBS and FITC treatment resulted in a significant stimulation of this migration. By contrast, SLS treatment did not increase the number of migrating cells. The migration capacity of hapten-treated cells decreased both in the absence of human fibroblast-conditioned medium and in the presence of this medium in the upper compartment of the chamber, indicating that this medium actually functioned as chemoattractant. Our findings suggest that the contact between hapten and Langerhans cells could be one of the triggers to initiate and/or stimulate Langerhans cell migration from the epidermis to the dermis. In addition, some factors derived from the dermal fibroblasts may promote and regulate the directional migration of Langerhans cells.

Members of the beta 1 or very late antigen (VLA) integrin family represent the predominant class of integrin extracellular matrix receptors. Adhesion assays were developed for the identification of the beta 1 integrins involved in the adhesive interactions between Langerhans cells (which mainly express alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1) and extracellular matrix proteins. For this purpose, binding assays were performed on fibronectin-, laminin-, collagen type IV-, and collagen type I-coated plates. 59% +/- 21% of Langerhans cells (LC) specifically attached to fibronectin. Using as inhibitory probes monoclonal antibodies against the beta 1, alpha 5, and alpha 3 chains and the synthetic peptide GRGDSP resulted in a decrease of 43%, 41%, 15%, and 42% respectively of LC binding to fibronectin. 76% +/- 20% of LC specifically adhered to laminin. Anti-alpha 6 monoclonal antibody potently inhibited this adhesion, which dropped to 36%, whereas the synthetic peptide GRGDSP was ineffective. A low number of LC adhered to type I and type IV collagen (13-15%). These results indicate that alpha 5 beta 1 and alpha 6 beta 1 were the major beta 1 integrins involved in LC adhesion to fibronectin and laminin. Ultrastructural cell morphology of adherent cells was examined and showed that LC were largely spread on laminin and became tightly bound to the substrate on a large portion of membrane. On fibronectin surface, the contact between LC and substrate was smaller, thus cells could conserve their general round aspect. Moreover, LC binding to fibronectin and laminin induced a significative decrease of the Birbeck granule number. The finding that LC attach to LM and FN in vitro suggests they exist similarly in vivo. By mediating a passage through basement membrane and migration throughout the fibronectin network of the dermis, alpha 5 beta 1 and alpha 6 beta 1 could contribute to the ability of LC to migrate into and out of the epidermis.

Human epidermal Langerhans cells are dendritic cells that can capture, process, and present antigens to T cells. It was previously shown that these Langerhans cells express the very late activation antigen (VLA) protein family of beta 1 integrins. beta 1 integrins mainly mediate the adhesion of cells to a number of extracellular components, such as laminin, fibronectin, and collagen, which are present in the skin. In this report, we demonstrate that a large percentage of the Langerhans cell population was able in vitro to attach to laminin and fibronectin but not to collagen. An ultrastructural study of adherent Langerhans cells showed that they were spread largely on laminin, with a loss of their round shape, and partially on fibronectin. Langerhans cell binding to laminin or fibronectin induced a decrease of the Birbeck granule number. Specific inhibitions of cell adhesion were performed, and it was demonstrated that VLA-6 was the major receptor involved in Langerhans cell adhesion to laminin. This adhesion was not RGD dependent and was slightly enhanced by Mn2+. VLA-3 and especially VLA-5 mediated Langerhans cell binding to fibronectin through the RGDS sequence of the protein. Mn2+ sharply increased the Langerhans cell adhesion to fibronectin. VLA-6 mediated in vitro Langerhans cell adhesion to laminin, which suggests that in vivo VLA-6 could permit Langerhans cells to attach and migrate through the basement membrane. Moreover, VLA-5 and VLA-3 take part in the in vitro Langerhans cell binding to fibronectin, suggesting that in vivo these fibronectin receptors could facilitate Langerhans cell passage throughout the fibronectin network of the dermis before migration to lymph nodes.

The integrin alpha 6 beta 4 complex is a protein of the membrane of basal keratinocytes, localized at the surface of cells in contact with the basement membrane zone in normal skin. The expression of alpha 6 beta 4 was investigated in several autoimmune blistering skin diseases including bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), bullous systemic lupus erythematosus (BSLE), and pemphigus vulgaris (PV) by an indirect immunofluorescence technique. In lesional bullous skin of BP, alpha 6 beta 4 expression was either absent, or in some cases represented an unusual irregular patchy staining. In contrast, in lesional bullous skin from EBA, BSLE, and PV, alpha 6 beta 4 expression was comparable to that observed in normal skin, i.e., a linear staining of the BMZ. Thus, analysis of the alpha 6 beta 4 integrin reactivity on lesional skin, in conjunction with the typical localization of collagen IV, allows a rapid and accurate distinction between BP and EBA.

Histiocytic cells infiltrating the lesions in eosinophilic granuloma of bone as well as in cutaneous histiocytosis X were studied using a murine monoclonal antibody (MA) produced with proliferating cells from an eosinophilic granuloma of bone. This MA reacts with Langerhans cells (LC) of normal human skin or mucous membranes and with proliferating cells of eosinophilic granuloma of bone and skin lesions of Letter-Siwe disease, as shown by immunohistochemistry and immunogold labelling. As other murine MA's obtained after immunization with human cortical thymocytes, this MA immunoprecipitates the 49-kDa CD1a antigen found on human LC and thymic-cell surfaces but not its breakdown product after treatment with trypsin, as demonstrated by analysis of immunoelectron labelling, cytofluorometry and gel electrophoresis. This first production of a CD1a MA from an eosinophilic granuloma supports the concept of Langerhans-cell histiocytosis.

The Very Late Activation (VLA) antigen family is involved in cell-extracellular matrix interactions and consists of six heterodimeric cell surface receptors with a common beta 1 and a variable alpha subunit. Using a panel of specific antibodies, we showed that human epidermal basal cells expressed VLA-2, VLA-3 and VLA-6 but failed to express VLA-4. Their functional roles were investigated and VLA-2 appeared as a specific receptor for type IV collagen and also as a laminin receptor. VLA-3 appeared as a receptor for fibronectin and laminin and to a lesser extent as a type I collagen receptor. VLA-6 appeared as a specific receptor for laminin. It also appeared that the VLA-alpha subunit specifically mediates the recognition of ligand but the beta 1 subunit is also involved in adhesion and that both subunits have a synergistic influence. Immunoprecipitation analyses confirmed that VLA-2, VLA-3 and VLA-6 were expressed by basal keratinocytes. Endocytosis of VLA-2 and VLA-3 was observed involving coated vesicles and endosomes that are structures characteristic of a receptor-mediated pathway. These findings provide first evidence that normal human basal keratinocytes are able of endocytosis mediated by receptors. Taken together, these results indicate that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix.

The expression of VLA integrins on human epidermal Langerhans cells (LC) was investigated by indirect immunogold labeling on transmission electron microscopy, followed by a quantitative analysis. Labelings on suspensions enriched in freshly isolated LC were carried out with an antibody recognizing the beta 1 subunit, common to all members of the VLA family, and with antibodies specific for the six different alpha subunits, alpha 1 to alpha 6. Normal human epidermal LC were all beta 1 positive, of which 60% were highly positive. By labelings with different VLA-alpha-chain MoAb, there appeared two subpopulations of LC: one positive and one negative. On average 40% of LC bore small amounts of VLA-1 and VLA-3, 53% and 77% of LC expressed moderate amounts of VLA-2 and VLA-5, respectively, and VLA-4 and VLA-6 were expressed by 67% and 90% of LC, respectively. VLA proteins are mainly extracellular matrix protein receptors. VLA-6 (laminin receptor) and VLA-5 (fibronectin receptor) are expressed mainly by LC, and in this way could subserve LC to leave the epidermal compartment through the basement membrane, to migrate throughout the fibronectin network of the dermis before migrating via the afferent lymphatics to the regional lymph nodes, where they present antigen to T cells. VLA proteins such as VLA-4, VLA-3, or VLA-2, which are found involved in cell-cell contacts, could contribute to the promotion of T-cell activation by facilitating adherence between LC and T cells.

The cortical thymocytes expressed at least three distinct cell-surface differentiation antigens. CD1a (Mr 49,000), CD1b (Mr 45,000) and CD1c (Mr 43,000) which are non-covalently attached to beta 2-microglobulin. In the present study, we confirm the presence of two out of the three CD1 molecules on epidermal Langerhans cells by biochemical analysis. Furthermore some CD1a monoclonal antibodies immunoprecipitated an additional molecule with an apparent relative mass of 27,000 from Langerhans cell-enriched epidermal cell lysates and not from fresh iodinated thymocyte lysates. From trypsin-treated thymocyte lysates, this low molecular weight protein was considered as a cleavage product of Mr 49,000 molecule (CD1a molecule) by this enzyme which is used to obtain epidermal cell suspensions. This Mr 27,000 was found to content one N-linked oligosaccharide residue by endoglycosidase F treatment. On CD1-expressing cells (thymocytes and Langerhans cells) it would be tempting to take advantage of the sensitivity of CD1a molecule to trypsin in order to precise the structure/function relationship of CD1a antigen.

Many biological events involve an initial stage of adhesion. The molecules that are responsible for the process of adhesion have been studied extensively in recent years. The most widespread receptors taking part in cell-external matrix and cell-cell reactions are from the families of integrins and immunogloblins. In normal skin, the keratinocytes and fibroblasts essentially express beta 1 (VLA) receptors and the cells of Langerhans the beta 2 receptors of the integrin family. Their pathological modification is under study. In contrast, involvement of the ICAM-1 molecule (ligand of receptor LFA-1) in pathology seems to be considerable. ICAM-1 is expressed by keratinocytes in many benign and malignant skin diseases and this makes possible interaction with LFA-1 lymphocytes. The passage of lymphocytes towards the epidermis is the basis of all inflammatory and immune reactions and is dependent on adhesion molecules. It will doubtless, in the future, be possible to explain the physiopathology of inflammatory skin reactions in terms of excessive expression or insufficient ICAM-1 by pathological keratinocytes.

Cell adhesion to extracellular matrix is mediated by a set of heterodimeric cell surface receptors called integrins. We have examined the expression of the very late antigens or alpha beta 1 group of integrins in human epithelial cells. The six known members of this group share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward collagen, fibronectin, and laminin essentially. Using a panel of specific antibodies we showed that freshly harvested human epidermal basal cells express VLA-2 and VLA-3 receptors, a low amount of VLA-5, but fail to express VLA-4. The findings reveal that these receptors are characterized by the alpha subunits which associate with a beta subunit different in weight (Mr 110,000 reduced) from that normally seen (Mr 130,000). Moreover, immunoprecipitates of VLA-2 contained additional proteins of Mr 80,000 and Mr 40,000 and immunoprecipitates of VLA-3 contained an additional protein of Mr 90,000. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to extracellular matrix revealed that cell attachment to type IV collagen was completely inhibited by antibodies to VLA-2 alpha chain, that antibody to VLA-3 alpha chain significantly blocked attachment to fibronectin while antibodies to both VLA-2 and VLA-3 partially inhibited attachment to type I collagen. Cell attachment to types I and IV collagen and to fibronectin was not affected by antibodies to VLA-4 and VLA-6. These results show that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix. This cooperation function of multiple VLA receptors and their differential expression could be considered to be one of the controlling points in the localization of epithelial basal cells in the epidermis.

Experimental anti-keratin polypeptide sera (KPS) which were prepared by immunizing guinea pigs with the P1 polypeptide (molecular weight: 67 0000 dalton) of alpha-keratin of normal human stratum corneum, were shown to react in immunofluorescence only to cell cytoplasmic antigen of the upper layers of the epidermis. No staining was detected in the basal layer. An immunolabeling performed on free epidermal cells obtained after trypsinization demonstrated by electron microscopy that receptors for KPS were tonofilaments. Minor proportions of negative cells containing tonofilaments and numerous melanosomes detected might correspond to the basal cells. An IF pattern similar to that seen with KPS was observed with some human epidermal cytoplasmic sera (ECS). However, reciprocal blocking tests performed on both rabbit lip and normal human skin with the different sera showed no inhibition. No cross-reaction was detected by immunodiffusion test between whole purified alpha-keratin and human ECS. In electron microscopy, the receptors for ECS appeared not to be keratin-like, but were located on a granular part of the peripheral keratinocyte cytoplasm. These findings confirmed two steps (basal and malphighian layers) in epidermal differentiation defined by antigenic markers and especially by the alpha-keratin component of MW 67 000 d present in normal stratum corneum.

A statistical method for interpreting data on the efficacy of anticancer agents is proposed. Tables were prepared for the retrospective analysis of studies evaluating the activity of anticancer agents against individual tumor types. The number of responding patients and the number of patients entered in the study were used to more objectively and formally determine the future disposition of the drug. Several examples of clinical studies show that use of our tables allows a more satisfactory and reliable classification than is often currently reported.

A rapid and convenient immunocytochemical method is presented for the simultaneous detection of surface antigen expression and S-phase cells using monoclonal antibodies and cells in suspension. The first reagent permits the nature of the cells to be determined while the second immunostaining stage reveals which of these cells are proliferating (BrdU-positive) cells. Staining and fixation conditions have been defined for the detection of cell surface markers by red fluorescence and BrdU labelling by green fluorescence. All the steps were carried out on cells in suspension and the membrane antigen distribution was not affected by the BrdU staining protocol. The method has been applied to two human epidermal cell subpopulations: Langerhans' cells and basal keratinocytes. This technique should have general applicability to the study of the cytokinetic characteristics of phenotypically defined cells in heterogeneous populations.

Human epidermal Langerhans cells express two (CD1a and CD1c) of the three human thymic cell surface differentiation antigens (CD1a, CD1b, and CD1c). The first cluster of differentiation antigens (CD1) is defined by a group of monoclonal antibodies (MCA). All these MCA were obtained after immunization of mice or rats with human cortical thymocytes. OKT6 MCA (a CD1a MCA) was the first to be described as reactive with human epidermal Langerhans cells. We produced a murine MCA, called DMC1, after immunization with proliferating Langerhans cells of Eosinophilic Granuloma of the bone (Histiocytosis X). In tissues DMC1 MCA reacted with epidermal dendritic cells (Langerhans cells) in the skin and cortical thymocytes in the thymus as observed on indirect immunofluorescence. At the ultrastructural level, DMC1 MCA was specific for Birbeck granule-containing Langerhans cells and did not react with melanocyte and keratinocyte populations. The quantitative analysis of immunoelectron labeling and the cytofluorometric study showed that the intensity of labeling was inversely correlated with the concentration of trypsin used in the preparation of epidermal cell from skin samples. DMC1 MCA precipitated a protein with a relative mass of 49,000 (CD1a molecule) from lysates of iodinated epidermal Langerhans cells under reducing conditions. It recognized the original CD1a molecule (Mr 49,000) but not the membrane breakdown product of CD1a (Mr 27,000) brought about by trypsin.

We compared some morphological and biochemical aspects of the epidermal changes occurring in hyperkeratotic scabies in humans and domestic pigs. Clinically and histologically, pig skin alterations look very similar to those observed in humans, i.e., brittle hyperkeratosis with parakeratotic crusting and thickening of the epidermis. Parakeratosis seems to correspond to previous passage of scabies mites through the incompletely differentiated layers of the epidermis. Indeed, by serial sectioning we observed beneath the stratum corneum cellular lysis some distance ahead of the mouth parts of the parasites. Epidermal cells surrounding this initial epidermolytic focus finally underwent disturbed terminal differentiation and appeared as parakeratotic cells. In pigs we observed intraepithelial microabscesses, but we rarely observed these in our human subjects.

The purpose of this work is to analyze the strategy used for the discovery of potentially clinically effective drugs by means of transplantable tumor systems in mice. With the present strategy used by the National Cancer Institute and the European Organization for Research on Treatment of Cancer, most new compounds are initially tested in the P388 leukemia model. Drugs showing activity in this prescreen are subsequently evaluated in a panel of eight screens (B16, CD8, Co38, L1210, LL, CX-1, LX-1, and MX-1). Positivity in any one of these screens qualifies a new drug for further preclinical studies leading to phase I-II clinical trial. Our analyses of the experimental screening data for 1949 compounds, and the comparison of results in humans and mice for 69 drugs that have been clinically evaluated against solid tumors, have indicated that the correlation of screening and clinical results is low and that a modified approach using four systems (P388, B16, L1210, and MX-1) would have been sufficient to uncover most of the drugs presently shown to be clinically active. In order to take advantage also of the information provided by other screens of the panel, a three-stage strategy can be used. In the first stage, compounds would be tested in the P388 prescreen, with a slightly higher level of response being required (T/C% greater than or equal to 125 instead of 120). In the second stage, prescreen-positive drugs would be tested in the B16, L1210, and MX-1 systems. Drugs positive in any one of these screens may then be tested in Co38, LL, and CX-1 to determine priorities for clinical study. For new chemical structures of particular promise, this third stage could be dropped. LX-1 and CD8 are not useful in this context. Such a strategy would be much more economical and faster than the automatic use of nine systems, and the resultant savings should permit the discovery of an increased number of potentially active compounds.

We have used tritiated thymidine (3HT) labelling and immunoperoxidase staining of 67 K polypeptide in human epidermis to study normal skin before and after irritation induced by application of croton oil (20% and 50% in olive oil). By this double labelling method, it was possible to identify, on the same skin section, DNA-synthesizing nuclei and epidermal cells which contained 67 K polypeptide. Our results clearly indicate that the germinative compartment of normal epidermis is a mixed population comprising basal and adjacent suprabasal cells; as a rule, absence of expression of 67 K polypeptide, which characterizes all basal cells, could not be regarded as a good marker to distinguish the germinative from the differentiating compartments. In mild primary irritation, the ratio of 3HT-labelled undifferentiated cells to keratinizing ones was similar to that observed in normal epidermis. In severe irritation, this homeostatic process was altered, as 3HT-labelled cells were mainly 67 K negative.

Anti-keratin polypeptide sera were obtained against the different bands of polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum derived from normal human epidermis. The sera were tested by indirect immunofluorescence of immunoperoxidase techniques. It was demonstrated that antibodies against P1 and P2 polypeptides, of MW 67,000 and 62,000 daltons respectively, were directed towards cytoplasmic antigens of keratinocytes of the upper Malpighian layers, while no labelling could be detected in the basal cell layer. Anti-P3 polypeptide sera and the anti-whole keratin serum labelled the whole epidermis, including the basal cell layer. Ultrastructural immuno-labelling performed on free epidermal cells obtained after trypsinization demonstrated that receptors for anti-P1 polypeptide sera were tonofilaments. These results showed that some keratin components (P1 and P2 polypeptides) might be absent in basal cell tonofilaments. This in in favour of various differentiation stages of the keratinizing cells. Keratin polypeptide 1 could be a useful marker of keratinization. According to preliminary studies, the expression of this keratin antigens was markedly disturbed in tumors such as basal and squamous cell carcinoma, in warts and in ichthyosis.

The expression of an epidermal keratin subunit and a specific antigen of the keratinocyte membrane, two differentiation antigens in normal human epidermis, was studied in benign and malignant epidermal lesions by use of monoclonal antibodies KL1 (anti 55-57 Kd keratins) and KL3 (anti keratinocyte membrane antigen). In normal human epidermis, KL1 labelled all keratinocytes from the suprabasal layers, KL3 stained the intercellular spaces in all epidermal layers with a fluorescence intensity increasing from the basal to the more upper layers and recognized a keratinocyte membrane antigen as demonstrated in electron microscopy. Frozen or deparaffinized sections of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) malignant melanomas, warts, and skin biopsies from benign lesions (psoriasis, lichen planus, bullous pemphigoid, lupus erythematodes, pemphigus, vasculitis) were tested with either KL1 or KL3 by indirect immunofluorescence and/or immunoperoxidase. Benign and malignant lesions in which modifications of the keratinization process and cell differentiation are known to occur (BCC, SCC, warts, psoriasis) showed the most severe alterations as compared to normal epidermis. With KL1 we observed an irregular staining of basal cells; a reorganization of keratin filaments and variable staining intensities within tumoral cells which did not express high MW keratins. With KL3 drastic alterations in the epidermal intercellular patterns and loss of reactivity of tumoral cells were noted. Conversely, the positivity of epidermal basal cells with KL1, in some cases, was the only modification noted in other skin lesions.

Previous studies revealed that the filaments of cytokeratin express different antigenic sites in the basal and suprabasal cell compartment of the skin and oral mucosa. It has been demonstrated that the keratin polypeptide subunit of molecular weight 67,000 dalton (67K) is a valuable marker of the suprabasal cells, since the normal basal cells remain unlabeled by antibodies against this large polypeptide. In the present study, the distribution of the 67K polypeptide has been investigated on human and guinea pig keratinocyte suspensions, purified basal cells of guinea pigs, normal human abdominal skin and normal human buccal mucosa, using the indirect immunoperoxidase method. The results of this investigation confirmed ultrastructurally that the 67K-antigen expression is lacking in the epidermal basal cell compartment. However, it could be shown that 67K polypeptide is a component of the tonofilaments in the upper Malpighian cell layers of the skin and oral mucosa. These observations suggest that keratinocyte differentiation is accompanied by modifications of antigenic determinants of the keratin filaments.

The error probabilities alpha and beta are widely used to compute sample sizes and to analyze results of clinical trials. These errors are, however, not the only probabilities to consider when assessing results of clinical studies. The rate of false positive (delta) and false negative (epsilon) results allows one to determine if an experimental finding is likely to reflect the true situation in the population of interest. The delta error is generally high in randomized phase III and in early phase II clinical trials in cancer patients, whereas the epsilon error is relatively low in these settings. This is essentially due to the small probability of detecting a more effective treatment or a new chemotherapeutic agent active in cancer. The delta error could be considerably reduced by increasing the sample sizes and by restricting the allowance made for the alpha error, which should be set at a 1% level as a minimum requirement.

Human wart virus (HPV) was isolated from a pool of genital warts. The electrophoretic mobility of virion proteins was studied by SDS polyacrylamide gel electrophoresis and showed the same pattern as that obtained with HPV-1. The analysis of DNA after restriction enzyme digestion with the endonucleases Hind III and Hae III, and nucleic acid hybridization did not show any difference with HPV-1. The viral particles were agglutinated by anti-HPV-1 serum, as shown by electron microscopic particle agglutination test. Furthermore, the immunological properties of this virus were investigated with guinea pig antiserum. Serologically, no antigenic cross-reaction between common and genital wart viruses were shown by immunodiffusion and immunofluorescence tests, whereas cross-reactions were detected between plantar and genital wart viruses. These results allow to think that HPV-1 can induce plantar warts as well as genital warts.

We studied the relation between an institution's degree of participation in a multicenter clinical trial and the quality of its participation. The quality of participation was judged on the basis of the percentage of patients entered into the trial who were in fact eligible, who were treated in accordance with the protocol, and for whom the appropriate forms were received. Those who satisfied these criteria were termed "valid" patients. Institutions were divided into two groups, "major participants" and "minor participants," according to the number of patients they entered in the trial. In a detailed analysis of a multicenter trial of chemotherapy for soft-tissue sarcoma, we found that major participants had a significantly higher percentage of valid patients than did minor participants (81 per cent vs. 39 per cent) and that the minor participants were actually detrimental to the study from both scientific and administrative viewpoints. These results suggest that institutions should not participate in multicenter studies unless they can enter some predetermined minimal number of patients per year.

An application of decision theory to the design of phase II clinical trials in cancer is presented. A risk function which depends on the prior distribution of the response rate, the costs of treating a patient, and the gains or losses resulting from the decision taken upon completion of the study is derived and optimal restricted sampling plans for a response rate of 20% are provided.

A murine monoclonal antibody, designated K20, was raised by immunization with a human malignant T-cell line. It reacted specifically with membrane glycoprotein complexes on early haematopoietic cells, T cells, and monocytes. In epidermis, K20 specifically reacted with Langerhans cells and basal keratinocytes, as demonstrated by double labeling experiments. Membrane immunoprecipitation analysis demonstrated that the antigen identified by K20 on lymphoid cells and epidermal cells was different. While on lymphoid cells, K20 recognized glycoprotein complexes made of a constant 130-kD subunit associated with subunits of higher molecular weight ranging from 150 to 200 kD, a complex of 105-145 kD was precipitated from Langerhans and basal cells. Metabolic labeling studies demonstrated that these proteins were synthesized by the basal cells. The antigen identified by K20 was thought to belong to the integrins, a family of cell surface receptors that play a role in cell adhesion, cell interactions, wound healing, and immune defense mechanisms. K20 is the first monoclonal antibody that specifically recognizes a membrane antigen common to Langerhans and basal cells. Additionally, K20 is the first of five reported monoclonal antibodies to have been characterized on the epidermal cells that detect antigens shared by lymphoid subpopulations and normal basal keratinocytes.

Comparative studies on the specificity of the so-called antiepidermal antibodies (Abs) found in human sera were performed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM). After a screening test by indirect immunofluorescence (IF), sera obtained from patients with various diseases and controls could be classified in 5 different groups according to the IF patterns on the epidermis: sera reactive with: (1) the stratum corneum (SC); (2) the upper layer (U-Cyt); (3) the whole epidermis (G-Cyt); (4) basal cells (B-Cyt); and (5) negative ones. By immunoblotting, all the 23 IF-positive sera were found to bind to one or more keratin bands, and did not show any reactivity with epidermal Nonidet P-40 soluble proteins. SC-Abs were mainly directed against a 67 kD Keratin band, whereas U-Cyt- and G-Cyt-Abs bound to both 58-56 kD and 67-63 kD keratins. B-Cyt-Abs reacted strongly with 63 kD Keratins and slightly with a 50 kD band. Antikeratin Abs were detected by immunoblotting even in the IF-negative sera. The ELISA study showed that sera with high IF titers contained high levels of antikeratin Abs. In the IEM study using sera containing U-Cyt- or B-Cyt-Abs, 2 distinct reaction patterns were demonstrated: U-Cyt-Abs stained tonofilaments of suprabasal keratinocytes, while B-Cyt-Abs characteristically reacted with those of basal cells. Moreover, SC-, U-Cyt-, and G-Cyt-Abs were absorbed out by insoluble epidermal proteins, and B-Cyt-Abs were decreased in titer after the absorption test. The present study provides strong evidence that most, though not all, human antiepidermal Abs are directed against different keratin polypeptides, and that antikeratin Abs commonly occur in almost all human sera.

A monoclonal antibody against keratins (KL1) from normal human stratum corneum was obtained using hybridoma techniques. Spleen cells from immunized BALB/c mice were fused with NS1, a mouse myeloma cell line, to produce hybrids. Antibody activity to epidermal keratins was tested using an indirect immunofluorescence test on cryostat sections of human epidermis and rabbit lip. A stable clone producing antikeratin antibody was isolated and an ascitic fluid was produced and used as a source of antibody (IgG1 kappa). KL1 was characterized by its immunohistochemical staining of various epithelia and by its recognition of 55-57 kilodalton (kd) keratin polypeptide from normal epidermis using the immunoblot technique. Frozen and deparaffinized sections of normal human epidermis, mucosa, and esophagus were stained by this antibody only in the upper cell layers as demonstrated by both indirect immunofluorescence and immunoperoxidase techniques. Approximatively 80% of normal keratinocytes isolated after trypsinization were labeled by KL1 whereas most negative cells showed basement membrane zone antigens. This confirmed differences in the expression of medium-sized polypeptides between basal and supra-basal cells during the course of human epidermal differentiation. All epithelial cells from other human epithelia (thymus, thyroid, bronchial mucosa, stomach, intestines) were positive with KL1 whereas nonepithelial cells and tissues did not show any staining. In view of these results KL1 promises to be a useful tool in the exploration of human epithelial differentiation.

The analgesic efficacy of ketorolac tromethamine was compared to placebo in 126 patients suffering moderate or severe chronic pain due to cancer in a double-blind parallel randomized study. Ketorolac was administered intramuscularly in doses of 10, 30 or 90 mg. Pain intensity and pain relief were assessed for 6 hours by scoring standard verbal scales and an overall assessment of the medication was given by the patients and the observer on completion of the study. Each dose of ketorolac was statistically superior to placebo for the sum of pain intensity difference (SPID) but no difference was seen between the three ketorolac regimens. When the ketorolac groups are combined, there was a significantly better pain relief as compared to placebo. The global evaluation scores were also statistically superior in the ketorolac groups combined than in the placebo group. A total of 15 patients reported minor adverse events, 10 being after ketorolac doses. This study shows that single intramuscular doses of ketorolac of 10 mg and above are effective in the relief of cancer pain, and are associated with a low incidence of side-effects.

We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase. After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h. Studies of BrdU labeled cells included the determination of their cell surface phenotype. Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase. We observed 6% of the epidermal LC in S-phase. This is a proof that LC are able to proliferate in the epidermal microenvironment. This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.

A method is described for flow cytometric sorting of epidermal basal cells, based on the cellular keratin content. KL1, a monoclonal antikeratin antibody specific for the 56.5 kD polypeptide present in suprabasal cells was used to distinguish suprabasal from basal cells. After the action of Triton X-100, epidermal cells were stained in suspensions with KL1 antibody, and KL1-positive cells were separated by flow cytometry from KL1-negative cells (basal cells). This makes it possible to sort and analyze pure fractions of human epidermal basal cells since no less than 99% of the KL1 sorted cells were bearing bullous pemphigoid antigen. Therefore, the use of specific antibodies to cytoplasmic antigens can be of help in sorting pure fractions of cells of a particular tissue for further studies.

A murine hybridoma secreting an IgM monoclonal antibody (KL3) was produced by cell fusion of mouse myeloma cells with spleen cells from mice immunized with human epidermal keratins. On normal human epidermis KL3 stained the intercellular spaces from the stratum germinatum to the stratum granulosum with a fluorescence intensity increasing from the basal layer to the upper layers. Basal cells were not stained on the side facing the basement membrane. About 90% of free keratinocytes isolated after trypsinization were labelled by KL3 in a punctate staining. Immunoelectron microscopy allowed us to show that the antigen recognized by KL3 was exclusively localized on the keratinocyte membrane especially in the desmosomal plaques. KL3 reactivity was not modified by preincubation of skin sections with lectins showing a selective intercellular labelling of upper layers of epidermis or pemphigus antisera, nor by adsorption of the antibody on NP40 soluble proteins of the epidermis. Though KL3 reactivity was completely abolished after adsorption of purified keratins, no immunological reactivity of KL3 was detected with epidermal keratin polypeptides blotted on nitrocellulose paper. In psoriatic epidemis and epidermal tumors KL3 reactivity was drastically modified. These results suggest that KL3 recognized a keratinocyte membrane antigen implied in the epidermal differentiation process.

The epidermal changes occurring in various lesions of atopic dermatitis are reported. Quantitative evaluation or epidermal thickening suggests that both differentiated and undifferentiated compartments are increased in lichenified lesions. In dry skin (xerosis) of atopic dermatitis moderate but obvious inflammatory changes are observed. There is a mild increase of the epidermal volume without folding of the dermo-epidermal interface. The latter is most prominent in exudative and lichenified lesions where a fourfold overall increase of epidermal volume is observed.

This analysis was carried out to assess quantitative and qualitative relationships between animal and human toxicology data with anticancer drugs. Among 21 chemotherapeutic agents, one-sixth LD10 in the mouse or one-third toxic dose low (TDL) in the dog corresponded to acceptable doses in man when experimental and clinical data were obtained at identical schedules and compared on a mg/m2 basis. The mouse and the dog largely differed in their tolerance to individual drugs. One-tenth LD10 in the mouse seemed always tolerated in the dog. On the average, these species were equally relevant for establishing the initial dose in man. A similar number of dose escalation steps would have been required in phase I clinical trials if the starting dose had been based on one-tenth LD10 in the mouse or the lowest value of one-sixth LD10 in the mouse and one-third TDL in the dog. These observations indicate that the starting dose in phase I clinical trials could be safely and efficiently based on one-tenth LD10 in the mouse. Prior verification that the resulting dose is not lethal or life-threatening in the dog could add further safety to this procedure. The predictive value (PV+) in man of organ system toxicity in animals depends upon the prevalence of this toxicity in man. In our study, PV+ was high (greater than 0.85) for common toxic effects in man, i.e., gastrointestinal intolerance and myelosuppression. PV+ declined dramatically (0.05 to 0.54) with rarer toxic manifestations. There was no clear superiority of animal findings over the mere knowledge of the prevalence of these findings in man. Thus, it would appear that routine and undiscerned investigation of organ system toxicity in animals is of questionable usefulness for the clinician experienced in early clinical trials with chemotherapeutic agents.

We have investigated on human skin the reactivity of a panel of 42 anti-thymus monoclonal antibodies (MCA) supplied by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens, Oxford, 1986. MCA of the first cluster of differentiation (CD1) define a group of surface molecules expressed by cortical thymocytes. Some of them (OKT6, M241 and Na1/34) have been shown to react in normal human skin with the epidermal Langerhans cells (LC). Twenty-two CD1 MCA were investigated in the present study. On normal human skin, 13 MCA reacted with LC in situ. This result suggests and confirms the heterogeneity of CD1 MCA. Recently, some of them were shown to recognize biochemically different molecules and/or epitopes of thymocytes. In addition, 20 anti-thymic epithelium MCA were tested on human skin. The MCA which only reacted with the thymic epithelial cell network (except Hassall's corpuscles) decorated only the epidermal basal cell layer. The MCA which reacted with all the thymic epithelial cells (including Hassall's corpuscles) decorated all the epidermal cell layers. These results confirm the heterogeneity of the thymic epithelial microenvironment and underline the antigenic similarities between the thymic epithelial structures and the different epidermal cell layers. The existence of bone-marrow-derived CD1-positive cells (thymocytes or LC) in an epithelial cell network (the thymus and the epidermis) focus the speculation around the immunological role of the epidermal basal cell layer in the T cell education and the exact lineage of the epidermal LC.