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Integrin alphabeta heterodimers mediate adhesion to the extracellular matrix and at cell-cell contacts and initiate intracellular signalling cascades in response to a variety of inductive factors. Apart from the expression of alphavbeta3 that we have previously reported, little is known about the expression of integrins in odontoblasts. Here, we investigated the expression of alphav-binding beta integrin subunits in healthy human dental pulp in vivo and in odontoblasts differentiated in vitro. Reverse transcription/polymerase chain reaction analysis revealed the expression of alphav, beta1, beta5 and beta8 integrin mRNA, but not beta6, in whole pulp cells. Flow cytometry showed that the alphav and beta1 subunits were the most intensely expressed. Immunohistochemistry demonstrated that the beta1 subunit was localised in newly differentiated odontoblasts in the root and in mature odontoblasts in the crown, including their intradentinal cell processes. The alphav chain was predominantly expressed by mature odontoblasts and alphavbeta5 was only observed in mature odontoblasts. In vitro differentiated odontoblasts expressed genes for alphav, beta1 and beta5, but not for beta6 and beta8. A comparison of integrin profiles between cultured pulp cells and in vitro differentiated odontoblasts revealed that odontoblast maturation was characterised by a significant increase in the expression of alphav and beta1 subunits and alphavbeta5 integrin. The beta8 subunit was detected in nerve cells only. Histological analysis of teeth from alphav knockout mice showed no obvious structural modification in the odontoblast layer. Thus, human mature odontoblasts express alphavbeta3, alphavbeta5 and perhaps alphavbeta1 integrins, with the possible presence of alpha-beta1 pairs. The roles that these molecules play in the exchange of information throughout the odontoblast layer remain to be determined.

The events occurring during the penetration of melanoma cells through the dermal-epidermal junction, which is the first crucial step in the process of metastasis, are poorly understood, partly because no suitable tissue models exist. In the in vitro model reported here, two melanoma clones (T1C3, which generates lung metastases in experimental animals, and IC8, which does not) derived from a single parental cell line were co-seeded with normal allogenic keratinocytes onto acellular human de-epidermized dermis with preserved intact basement membrane and cultured for up to 1 month at an air-liquid interface. Histological, immunohistochemical and ultrastructural studies showed that melanoma cells from the metastatic clone (T1C3), but not from the non-metastatic clone (IC8), penetrated the dermal-epidermal junction to invade the dermis after 3 weeks of culture. Local invasion was associated with the dissolution of the native epidermal basement membrane collagens type IV and VII. Confocal laser scanning microscopy analysis demonstrated that numerous T1C3 cells were able to colonize the interstitial dermis and to rapidly penetrate empty dermal cavities. Our model represents a significant technical advance over others currently available since: (i) the organized three-dimensional architecture of the native dermal-epidermal junction is preserved; (ii) the active invasion process coincides with the dissolution of native components of the epidermal basement membrane, i.e. collagen types IV and VII; and (iii) the ability of melanoma cells to cross the dermal-epidermal junction correlates with their metastatic potential. This model provides a valuable tool for the study of the time-course of the cellular and molecular events that occur during the earliest steps of invasion in cutaneous melanoma. It also offers new opportunities to study the possible role of the keratinocyte environment in melanoma invasion.

Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) generate a heterogeneous population of dendritic cells (DC), including Langerhans cells (LC). This combination of cytokines has been shown to be crucial for differentiation into LC. After day 5 of culture, TNF-alpha has been maintained in the medium in most studies despite the observation of spontaneous maturation of LC after day 12. Five-day samples of in vitro differentiated LC were cultured in parallel with or without TNF-alpha. The absence of TNF-alpha was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogate the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-alpha abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-alpha-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-alpha. These data indicate that the suppression of TNF-alpha after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.

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.

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)

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.

The keratin polypeptide composition of different types of warts was studied by means of SDS polyacrylamide gel electrophoresis. It was composed of five main polypeptides, similar to those found in normal epidermis (mol. wt. 67K to 55K) and an additional polypeptide (72K) was often detected. The distribution of each polypeptide was expressed as percentage of total keratins in the different samples. The most characteristic feature observed in warts was the marked decrease of the 67K polypeptide. There were increased amounts of the 67K-63K proteins and some difference was observed in the distribution of these proteins in the different types of warts. Thus, the viral infection seems mainly to modify the proteins which are only involved in the latest stages of the differentiation of keratinocytes.

SUMMARY 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.

Immune sera against total keratin, as well as three keratin polypeptide subunits, were raised in guinea pig with different bands of stratum corneum fibrous proteins derived from normal human epidermis and isolated by SDS polyacrylamide gel electrophoresis. The staining pattern of the different polypeptide antisera using the indirect immunofluorescence and immunoperoxidase methods in biopsy specimens of normal human epidermis confirmed recently published results. Biopsy specimens from 10 lesions of lichen planus were studied by indirect immunofluorescence and immunoperoxidase methods utilizing the same four different specific antikeratin immune sera. Most often the staining pattern with the different immune sera was similar to that seen in normal keratinized epidermis. In all specimens, intense staining of the colloid bodies was detected with the different immune sera. This finding favors the concept that colloid bodies originate from keratin filaments. Control staining with pre-immune serum was always negative. In one case of bullous lichen planus, colloid bodies were not labeled by 67 K and 62 K immune sera, but were intensely stained with 55 K and total keratin antisera. Moreover, with 55 K immune serum there was more intense staining of certain basal cells. The correlation between these findings and previously published studies by electron microscopy is discussed.

The epidermis, composed mainly of keratinocytes, undergoes continuous proliferation and differentiation characterized by a series of morphological and biochemical changes as cells progress from the basal to the outer cornified layer. One approach to the study of keratinocyte differentiation is to separate the keratinocytes at their successive stages of maturation. Previous attempts to isolate the histologically defined keratinocytes of newborn mouse epidermis have mainly involved the action of trypsin or EDTA combined with separation on density gradation [1, 2, 11]. Fractions enriched in various cell types of human epidermis have been obtained either by serial sectioning parallel to the skin surface [5] or sequential release of cells from the tissue [12]. In the present report, we describe the keratin composition of spinous and granular cell fractions selected by flow cytometric sorting. The specific marker used in this study is the KM48 monoclonal antibody. KM 48 (IgM class) was obtained by immunization of Balb/c mice using a sonicated normal human epidermal cell suspension the which has been shown to recognize a keratinocyte membrane antigen localized at desmosomal regions. Quantitative electron microscopy studies using the immunogold technique have revealed that the expression of the KM48 antigen by keratinocytes increases in parallel with cell differentiation; basal cells were negative, spinous and granular cells were, respectively, mildly and strongly KM48 positive. Corneocytes remained unstained [7].

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.

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.

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.

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.

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.

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.

Bullous pemphigoid (BP) is an autoimmune disease characterized, at histology, by subepidermal bullous separations. Antibodies (Ab) and complement are present at the dermal-epidermal junction, and most patient with the disease have serum antibodies directed against a normal constituent of stratified epithelia (10, 25). During the past few years, the immunotransfer technique has been used to study autoimmune bullous diseases of the skin. The antigen (Ag) of bullous pemphigoid has been described by Stanley et al. (16, 17) as the polypeptidic doublet 220-240 KD. However, different results in favour of an heterogeneous antigen have been obtained by Labib et al. (11) who reported the presence of 5 separate polypeptides: 240, 200, 180, 97 and 77 KD. The purpose of the present study was to confirm on infirm these findings. We recorded the antigens recognized at immunotransfer by the circulating antibodies of BP patients, then extended this study to sera from BP patients in whom no antibody had been detected by indirect immunofluorescence (IIF). We report the results of our immunotransfer study in 9 seropositive and 9 seronegative BP patients and in 11 seronegative controls. The antigenic extract we used was prepared from suspensions of epidermal cells and represented the intracellular BP antigen. Epidermal cell suspensions were obtained by trypsinization of human abdominal skin. The cells were homogenized in a Tris-HCl 10 mM pH 7.8 buffer with SDS 2 p. 100 and B-mercaptoethanol 5 p. 100. The soluble proteins were analyzed in a polyacrylamide gel in the presence of SDS.(ABSTRACT TRUNCATED AT 250 WORDS)

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 study of specific immune response in guinea-pigs after immunization with human papilloma viruses (HPV) purified from plantar warts, hand warts and condyloma acuminata is reported. The respective viruses induced the production of high antibody titres as well as specific cellular immunity as demonstrated by intradermal tests in all the animals. Formalin-inactivated HPV gave similar results. The antigenic cross reactions between the different viruses were evaluated by immunofluorescence and immunodiffusion tests and delayed hypersentitivity reactions. Serologically the results showed viral heterogeneity between condylomata and hand warts, and partial heterogeneity between condylomata plantar warts, and between hand and plantar warts. In skin-hypersensitivity reactions, however, the different viruses appeared as homologous antigens. This experimental study in guinea-pigs showed marked variance of specificity between delayed hypersensitivity reactions and humoral immunity for HPV.

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.

The identification of mononuclear cells extracted from various skin disorders has been investigated and the relative amount of B and T cells determined according to various assays such as E-rosette test, FITC and peroxidase anti-immunoglobulin conjugates. It has been found that: (a) there were two different patterns of distribution of the cells in the infiltrates; one with a T/B ratio above 3 (closest to a delayed type reaction), the other with a T/B ratio below 2. (b) The morphological appearance of tissue lymphocytes differs from that of circulating lymphocytes by the presence of both membrane and cytoplasmic immunoglobulins.