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Species delimitation remains a crucial issue for widespread plants occurring across forest-savanna ecotone such as Lophira (Ochnaceae). Most taxonomists recognize two parapatric African tree species, widely distributed and morphologically similar but occurring in contrasted habitats: L. lanceolata in the Sudanian dry forests and savannahs and L. alata in the dense Guineo-Congolian forests. Both species co-occur along a ca. 3000 km long forest-savanna mosaic belt, constituting ideal models for investigating hybridization patterns and the impact of past glacial periods on the genetic structures in two types of ecosystems. We genotyped 10 nuclear microsatellites for 803 individuals sampled across the distribution range of Lophira. Both species exhibit similar levels of genetic diversity [He = 0.52 (L. alata); 0.44 (L. lanceolata)] and are well differentiated, consistent with taxonomic delimitation (FST = 0.36; RST = 0.49), refuting the hypothesis that they might constitute ecotypes rather than distinct species. Furthermore, L. alata displayed two deeply differentiated clusters (FST = 0.37; RST = 0.53) distributed in parapatry, one endemic to Western Gabon while another cluster extended over the remaining species range, suggests that L. alata is made of two cryptic species. We showed that rare hybrids occur in some contact zones between these three species, leaving a weak signal of introgression between L. lanceolata and the northern cluster of L. alata. At the intra-specific level, the latter species also show weak genetic structuring between Upper and Lower Guinea and the intensity did not differ strikingly between rainforest and savanna ecosystems. The discovery of a new species of Lophira with a narrow distribution in West Gabon where it is intensively exploited for its timber requires to evaluate its conservation status., SCOPUS: ar.j, info:eu-repo/semantics/published

Odontoblasts, dental pulp fibroblasts and immature dendritic cells (DCs) have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process. How they regulate the inflammatory response to Gram-positive bacteria remains nevertheless largely unknown. In this study we investigated the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (CXCL8) in these three cell types upon stimulation with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria that activates the pattern recognition molecule Toll-like receptor 2 (TLR2). We observed that TNF-alpha gene expression was up-regulated in all LTA-stimulated cell types. IL-1beta gene expression was not or barely detectable in odontoblast-like cells and pulp fibroblasts when stimulated or not, but was expressed in immature DCs and increased upon stimulation. TNF-alpha and IL-1beta proteins were detected in DC culture supernatants but not in odontoblast-like cell and pulp fibroblast ones. CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation. These data indicate that LTA-dependent TLR2 activation in odontoblasts and pulp fibroblasts, in contrast to immature DCs, does not lead to significant TNF-alpha and IL-1beta production, but that all three cell types influence the pulp inflammatory/immune response through CXCL8 synthesis and secretion.

The known cross-reactivity of monoclonal antibodies prepared against CD1 and HLA-DR antigens with skin components prompted us to study the reactivity pattern of human eccrine sweat glands with a panel of monoclonal antibodies directed against CD1 antigens (OKT6, BL6, D-47) and against HLA-class II antigens (anti-DR, BL2, LEU-10, IV-D12, MAJA-7). The labelling pattern of eccrine glands with the panel of monoclonal antibodies used in this study permits to establish three different antigenic compartments on eccrine glands: 1) acrosyringium and distal part of dermal duct anti-DR+, BL2+, LEU-10+, IV-D12+; 2) proximal part of dermal duct MAJA-7+; 3) secretory part D-47+. The immunological markers used in this work provide a useful tool for investigation of eccrine gland differentiation and human eccrine glandular pathology.

Members of the TGF-beta/BMP family of growth factors induce odontoblast differentiation and reparative dentin synthesis, and their use has been proposed to stimulate pulp healing during dental therapeutics in human. However, factors that modulate TGF-beta and/or BMP signalling during odontoblast differentiation and physiology remain largely unknown. To identify them, we compared expression profiles of TGF-beta/BMP-related genes in pulp fibroblast- and odontoblast-like cells cultured from human dental pulp explants using cDNA gene arrays. We evidenced that the gene encoding ecotropic viral integration site-1 (EVI1), a transcription factor that inhibits TGF-beta/BMP signalling, was under-expressed in odontoblast-like cells. This result was verified by real-time PCR and, at the protein level, by immunohistochemistry. In vivo, real-time PCR analysis revealed that EVI1 was expressed in the dental pulp, at a level similar to brain, but lower than in lung, kidney or trachea. The protein was localized in dental pulp samples in pulp core and subodontoblast cells. Staining intensity progressively decreased from the radicular to the coronal pulp where EVI1 staining was almost undetectable in odontoblasts. Our data suggest that fine regulation of the EVI1 level in the human dental pulp might be important in the TGF-beta/BMP-induced modulation of dental pulp cell kinetics and/or odontoblast differentiation.

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.

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.

Migration and maturation of human dendritic cells derived from CD34+ progenitor cells (DC) infected by Toxoplasma gondii were studied in an in vitro model. We demonstrated that infection with virulent type I strains RH and ENT or type II low virulent strains PRU and CAL induced DC migration towards MIP-3β. However, type II strains induced a higher percentage of migrating cells than that induced by type I strains or positive controls (chemical allergen or lipopolysaccharides). Type II strains produced soluble factors responsible of the high migration whereas heat killed tachyzoites did not induced a migration higher than positive controls. We also demonstrated that infection by virulent strains and not by type II stains or heat killed tachyzoites triggers DC maturation. A soluble factor released by type II strains was responsible of the absence of DC maturation. Taken together, these results demonstrated that the interference of T. gondii in the behaviour of DC functions is related to the strain types and can be supported by secretion of soluble factors by the parasite.

In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-beta1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry. Results showed that a 2-day treatment with the representative allergens DNCB and NiSO(4) induced significant changes of most antigens while other chemicals such as balm of Peru (strong allergen), kathon (moderate allergen), cinnamic aldehyde (mild allergen) or the irritant SLS had no significant effect. In contrast, the 4-day treatment with allergens substantially improved the results. Indeed, despite a large variability according to the donors, the number of modified antigens was significantly higher with all the tested chemicals, except kathon, as compared to that observed with the irritant SLS. The present study indicates that, in this model, the screening of mild or moderate allergens requires both the consideration of many antigens and a prolonged time of incubation with the chemicals.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Background: Migration and maturation of epidermal dendritic cells, the Langerhans cells (LC), are central events in the initiation of the cutaneous immune response. LC migration from skin to draining lymph nodes is regarded as an indispensable step for the early phase of antigen-specific sensitization. Among the several agents which influence the ability of LC to migrate, previous studies have revealed that matrix metalloproteinases (MMPs) and protein kinase C (PKC) contribute to promoting LC migration. In this work, we studied the effect of two recently developed PKC and MMPs inhibitors of vegetable origin on the migration of in vitro activated LC. Methods: The migratory capacity of epidermal and in vitro generated LC was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to the lymph nodes. Results: Contact with chemical allergens, Bandrowski’s base or 2,4-dinitrobenzenesulfonic acid (DNBS), triggered migration. In the presence of PKC inhibitors, D-erythro-sphingosine and OX100, or an inhibitor of MMPs, LU105, allergen-induced migration of LC was strongly decreased. The association between OX100 and LU105 was more efficient in modulating the migration of activated LC compared to each molecule tested separately. Conclusions: These results showed that PKC and MMPs inhibitors act in synergy to inhibit the migration of activated epidermal dendritic cells in vitro. They underscore the role of PKC and MMPs inhibitors and suggest they may be of relevance for therapeutically regulating epidermal dendritic cell migration in inflammatory dermatoses.

Summary Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3β (MIP-3β) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.

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.

Interleukin (IL)-9 is known to regulate many cell types involved in T-helper type 2 responses classically associated with asthma, including B- and T-lymphocytes, mast cells, eosinophils and epithelial cells. In contrast, target cells mediating the effects of IL-9 in the lower respiratory tract remain to be identified. Therefore, the authors evaluated the activity of IL-9 on human alveolar macrophages (AM) from healthy volunteers. AM preincubated with IL-9 before lipopolysaccharide (LPS) stimulation exhibited a decreased oxidative burst, as previously shown with IL-4. The inhibitory effect of IL-9 was abolished by anti-hIL-9R alpha monoclonal antibody, and presence of IL-9 receptors on AM was demonstrated by immunofluorescence. Both IL-4 and IL-9 failed to modulate tumour necrosis factor-alpha, IL-8 and IL-10 release by LPS-stimulated AM. However, several observations suggested that IL-9 and IL-4 act through different mechanisms: 1) interferon-gamma antagonised the IL4- but not the IL-9-mediated inhibition of AM oxidative burst; 2) expression of CD14 was downregulated by IL-4 but not by IL-9 and 3) production of tumour growth factor-beta by activated AM was potentiated by IL-9 and not by IL4, and was required for the IL-9-mediated inhibition of AM oxidative burst. These observations provide additional information concerning the activity of interleukin-9 in the lung, related to inflammatory or fibrosing lung processes.

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and beta-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and beta-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

Signals regulating the traffic of Langerhans cell precursors from blood to the epidermis are not yet fully understood. The observations that TGF-beta1 is of unique importance in Langerhans cells (LC) ontogeny and that macrophage inflammatory protein-3alpha (MIP-3alpha) is able to attract LC within the epidermis, prompted us to study the effect of MIP-3alpha and TGF-beta1 on the migration of LC precursors. The migratory capacity of immature dendritic cells (DC) was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way into the epidermis. DC differentiated from cord blood CD34 cells in the presence of GM-CSF plus TNF-alpha were subjected to migration using modified Boyden chambers. Day-6 DC progenitors migrated in a dose-dependent fashion in response to MIP-3alpha, and CD1alpha+ LC precursors responded preferentially to the chemokine. Immature DC did not respond strongly to TGF-beta1 alone in migration assays, but up to 68% of the cells migrated in response to MIP-3alpha plus TGF-beta1. Among them, at least 50% expressed CD1a and E-cadherin and can be considered LC precursors. The allostimulatory function of these cells was significantly more potent than that which migrated in response to MIP-3alpha alone. Our results show that a significant proportion of immature DC is able to migrate through a dermal-epidermal basement membrane equivalent. In the presence of TGF-beta1, the DC which respond to MIP-3alpha have the phenotype and the functional capacity of epidermal LC. Our findings underline the role of MIP-3alpha and TGF-beta1 in attraction and localization of immature LC within the epidermis under normal conditions.

BACKGROUND: Megakaryocyte polyploidization results from the lack of cytoplasmic separation while the nucleus keeps dividing. METHODS: To investigate the role of actin in the megakaryocyte polyploidization, three human cell lines with megakaryocytic properties (DAMI, HEL and K562) were incubated in the presence of cytochalasin B, an inhibitor of actin polymerization. These data were then compared with normal megakaryocytes. RESULTS: Compared with control conditions, cells cultured in the presence of cytochalasin B revealed an augmentation of cell size and ploidy and an arrest of cell proliferation. The expression of platelet membrane glycoproteins Ib, IIb/IIIa, IIIa and thrombospondin and transferrin receptors was augmented after treatment with cytochalasin B. Physiologically, the role of actin in inducing polyploidization could be related to an imbalance between G- and F-actins. To test this hypothesis, we measured G-, F- and total actin in cytochalasin B-treated cells. Actin was found to be increased significantly in cytochalasin B-treated DAMI and HEL cell lines. In contrast, the G/F-actin ratio was not affected by cytochalasin B. To confirm these actin changes in physiological megakaryocytopoiesis, G- and F-actin contents were then estimated in normal megakaryocytes. The G- and F-actin contents of megakaryocytes from eight normal patients exponentially decreased from 2 to 128n, whereas the total actin content per cell kept increasing. The G/F ratio was unaffected. CONCLUSION: Polyploidization of human megakaryocytes results from either a diminution of actin synthesis or an increased actin turnover, which in turn possibly abrogates the formation of the actin cleavage furrow in telophasis.

Megakaryocyte polyploidization is an advantageous and regulated mechanism that leads to an increase in platelet production. In megakaryocytic cell lines, polyploidization can be obtained by using colchicine, an inhibitor of the tubulin spindle. The nucleolar organizer regions (AgNORs) are parts of nucleolar DNA transcribed into ribosomal RNA and are detected by the silver-staining technique. Their number is proportional to protein synthesis.To estimate protein synthesis in polyploid megakaryocytes, AgNORs are measured in three cell lines with megakaryocyte properties (DAMI, HEL and K562) after a 4-day culture in the presence or absence of colchicine. The mean number of AgNORs per cell was 16+/-4 (mean+/-SEM), 24+/-3 and 14+/-3 for DAMI, HEL and K-562 cell lines respectively. The addition of colchicine (10 ng mL[-1]) significantly increased the number of AgNORs per cell (DAMI 556%, HEL 338% and K-562 300% of controls, P0.05 using the t-test). Moreover, the number of nucleoles per cell after the addition of colchicine was augmented significantly (DAMI 246%; HEL 237% and K-562 148% of controls, P0.05 using the t-test). The total protein content estimated by Bradford's method increased significantly to 226%, 215% and 304% of controls in DAMI, HEL and K562 respectively (P005 using the t-test). After treatment with colchicine, the endomitotic index (EI) [mean of (log2 DNA content expressed in N)-1] measured by flow cytometry (and reflecting ploidy) increased to 234%, 255% and 301%, respectively, in DAMI, HEL and K-562 cell lines (P0.05 using the t-test). Concomitantly, the number of AgNORs per unit of DNA increased in the DAMI and HEL cell lines (P0.05 using the t-test) from 48+/-8 and 39+/-5, respectively, to 79+/-11 and 61+/-10. In contrast, the number of nucleoles and the total protein content per endomitotic index were not affected by colchicine (P0.05 using the t-test), but the number of nucleoles per endomitotic index of DAMI cells was affected.The increase in the number of the NORs induced by an agent known to stimulate polyploidization of megakaryocytic cell lines suggests that polyploidization occurs by a proportional increase in protein synthesis per DNA unit.

In addition to T cell receptor triggering, activation of T cells requires co-stimulatory 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 allogeneic, 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 on B7-1 expression.

International audience; Introduction: Previous studies have suggested that odontoblasts sense gram-positive bacteria components through Toll-like receptor 2 (TLR2) and trigger dental pulp immunity by producing proinflammatory cytokines. Currently, the factors that modulate odontoblast TLR2 activation are unknown. Our aim was to investigate lipopolysaccharide-binding protein (LBP) effects on the TLR2-mediated odontoblast response. Methods: Human odontoblast-like cells were stimulated with lipoteichoic acid (LTA) (a TLR2 ligand), LBP, CD14 (a TLR2 cofactor), or various combinations of LTA/LBP, LTA/CD14, or LTA/CD14/LBP. CXCL8, IL6, and TLR2 gene expression was assessed by real-time polymerase chain reaction. CXCL8. and interleukin (IL)-6 production was determined by enzyme-linked immunosorbent assay in culture supernatants of cells stimulated with LTA, LTA/CD14, or LTA/CD14/LBP. LBP effects on nuclear factor kappa B (NF-kappa B), p38, JNK, ERK, STAT3, and p70S6 signaling pathways were determined in LTA-stimulated odontoblast-like cells with a multiplex biometric immunoassay. LBP effects were compared with specific inhibitors of these signaling pathways. LBP transcript and protein were investigated in vivo in healthy and inflamed dental pulps by real-time polymerase chain reaction and immunohistochemistry. Results: Activation of CXCL8, IL6, and TLR2 gene expression and CXCL8 and IL-6 secretion in LTA- and LTA/CD14-stimulated odontoblast-like cells was significantly decreased by LBP. LBP inhibited NF-kappa B and p38 signaling pathways in LTA-stimulated cells in a similar way to NF-kappa B and p38 inhibitors. LBP transcript and protein were detected in vivo in inflamed dental pulps but not in healthy ones. Conclusions: These results demonstrate that LBP reduces TLR2-dependent production of inflammatory cytokines by odontoblast-like cells. We suggest that in this way it could modulate host defense in human dental pulp.

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 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.

Initial sensing of infection is mediated by germline-encoded pattern-recognition receptors (PRRs), the activation of which leads to the expression of inflammatory mediators responsible for the elimination of pathogens and infected cells. PRRs act as immune sensors that provide immediate cell responses to pathogen invasion or tissue injury. Here, we review the expression of PRRs in human dental pulp cells, namely, receptors from the Toll-like (TLR) and Nod-like NLR families, by which cells recognize bacteria. Particular attention is given to odontoblasts, which are the first cells encountered by pathogens and represent, in the tooth, the first line of defense for the host. Understanding cellular and molecular mechanisms associated with the recognition of bacterial pathogens by odontoblasts is critical for the development of therapeutic strategies that aim at preventing excessive pulp inflammation and related deleterious effects.

Human odontoblasts trigger immune response s to oral bacteria that invade dental tissues during the caries process. To date, their ability to regulate the expression of the nucleotide-binding domain leucine-rich repeat containing receptor NOD2 when challenged by Gram-positive bacteria is unknown. In this study, we investigated NOD2 expression in healthy and inflamed human dental pulps challenged by bacteria, and in cultured odontoblast-like cells stimulated with lipoteichoic acid (LTA), a Toll-like receptor (TLR) 2 agonist which is specific for Gram-positive bacteria. We found that NOD2 gene expression was significantly up-regulated in pulps with acute inflammation compared to healthy ones. In vitro, LTA augmented NOD2 gene expression and protein level in odontoblast-like cells. The increase was more pronounced in odontoblast-like cells compared to dental pulp fibroblasts. Blocking experiments in odontoblast-like cells with anti-TLR2 antibody strongly reduced the NOD2 gene expression increase, whereas stimulation with the synthetic TLR2 ligand Pam2CSK4 confirmed NOD2 gene up-regulation following TLR2 engagement. These data suggest that NOD2 up-regulation is part of the odontoblast immune response to Gram-positive bacteria and might be important in protecting human dental pulp from the deleterious effects of cariogenic pathogens.

Following autologous bone marrow transplantation (ABMT), both impaired T cell activation and defective production of the principal T cell growth factor, interleukin-2 (IL-2), has been observed. These processes are dependent on a rise of intracellular calcium ([Ca2+]i), a step which follows binding of T cell receptor (TCR) and transduction of signal via the generation of cytoplasmic second messengers. In order to better understand the nature of defective cellular immunity in ABMT, in the present study we investigated the rise of [Ca2+]i in T cells of recipients of ABMT. By concomitant labelling lymphocytes with anti-CD4 antibody and addition of fluo-3 as fluorescent calcium indicator, we have selected for the T cell subset which is the principal source of IL-2. Short-term (less than 1 year post-transplantation) recipients of ABMT show a statistically significant blunted rise in [Ca2+]i in response to concanavalin A as compared to normal controls not accounted for solely by a decreased percentage of CD4+ cells in these patients. The [Ca2+]i response of CD4+ cells from long-term (greater than 1 year post-transplant) recipients was lower than that of the normal group although not to a statistically significant level. These findings suggest that following ABMT is a defect in the early stages of T cell activation involving either T cell receptor binding or early signal transduction ultimately resulting in depressed transcription of IL-2 mRNA. These defects are analogous to findings in both allogeneic transplantation where factors of histoincompatibility and graft-versus-host disease (GVHD) come into play, as well as in the defective T cell activation of the normal ageing process.

International audience; Recent studies have suggested that odontoblasts are involved in the dental pulp immune response to oral pathogens that invade human dentin during the caries process. How odontoblasts regulate the early inflammatory and immune pulp response to Gram-positive bacteria, which predominate in shallow and moderate dentin caries, is still poorly understood. In this study, we investigated the production of pro-and anti-inflammatory cytokines by odontoblast-like cells upon engagement of Toll-like receptor (TLR) 2, a pattern recognition molecule activated by Gram-positive bacteria components. We used a highly sensitive Milliplex (R) kit for detecting cytokines released by cells stimulated with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, or with the potent TLR2 synthetic agonist Pam2CSK4. We found that odontoblasts produce the pro-inflammatory cytokines interleukin (IL)-6 and CXCL8, as well as the immunosuppressive cytokine IL-10 in response to TLR2 agonists. GM-CSF, IFN gamma, IL-1 beta, IL-2, IL-4, IL-5, IL-7, IL-12(p70), IL-13 and TNF-alpha were not detected. These data indicate that TLR2 activation in human odontoblasts selectively induces production of mediators known to influence positively or negatively inflammatory and immune responses in pathogen-challenged tissues. We suggest that these molecules might be important in regulating the fine tuning of the pulp response to Gram-positive bacteria which enter dentin during the caries process. (C) 2010 Elsevier GmbH. All rights reserved.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

[Introduction]: Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubule-associated protein highly expressed during the development of the nervous system. Its expression, regulated by the fragile X mental retardation protein (FMRP), is essential to stabilize microtubules during the elongation of dendrites and neurites. Other microtubules-associated molecules such as tau or MAP2 seem to act similarly. The aim of this work was to identify the MAP-1B expression in in vitro and in vivo human odontoblasts during development and carious processes. The expression of MAP2 and tau was also studied., [Materials and Methods]: In cultured cells, MAP-1B expression was analyzed by real-time polymerase chain reaction, flow cytometry, and Western blot. Its distribution was visualized by in situ hybridization and immunochemistry both in vitro and in vivo. The expression of FMRP, MAP2, and tau was identified by real-time polymerase chain reaction and immunochemistry., [Results]: MAP-1B is specifically expressed in odontoblasts from adult third molars as well as incisor germs from human embryos. In adult carious teeth, it is also expressed in newly differentiated dentin-forming cells. In vitro, MAP-1B expression is related to the differentiation state of odontoblasts. MAP-1B clearly underlines the cellular architecture of cell bodies and processes of differentiated cells. FMRP, MAP2, and tau are also detected in vivo., [Conclusion]: On the basis of these data, MAP-1B could be considered as a new protein involved in the terminal differentiation of odontoblasts.

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.

Recent studies have demonstrated that human dental pulp cells sense pathogens and elicit innate and/or adaptive immunity. Particular attention has been paid to odontoblasts that are situated at the pulp-dentin interface and constitute the first line of defense to cariogenic bacteria entering dentin after enamel disruption. In this review, recent in vitro and in vivo data suggesting that odontoblasts initiate immune/inflammatory events within the dental pulp in response to cariogenic bacteria are discussed. These data include sensing of pathogens by Toll-like receptors (TLRs), production of chemokines upon cell stimulation with microbial by-products and induction of dendritic cell migration. Additional results presented here reveal that all TLR genes are expressed in the healthy human dental pulp that is thus well equipped to combat pathogens entering the tissue. Seventeen chemokine genes including CXCL12, CCL2, CXCL9, CX3CL1, CCL8, CXCL10, CCL16, CCL5, CXCL2, CCL4, CXCL11 and CCL3, and 9 chemokine receptor genes including CXCR4, CCR1, CCR5, CX3CR1, CCR10 and CXCR3, are also expressed in pulp. TLR2, CCL2 and CXCL1 are upregulated in odontoblasts both under caries lesions and upon stimulation with pathogen by-products. These molecules thus appear as preferential targets for the design of therapeutic agents able to reduce the immune/inflammatory response to cariogenic bacteria and favor pulp healing.

Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the caries process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2-, TLR3-, and TLR4-specific agonists (lipoteichoic acid [LTA], double-stranded RNA, and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26, and CXCL11 only in fibroblasts. Supernatants of stimulated cultures increased migration of immature dendritic cells compared with controls, odontoblast-like cells being more potent attractants than fibroblasts. Analysis of these data suggests that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.

Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.

Previously, we established a subtractive cDNA library enriched in odontoblast-specific genes and hypothesized that new, previously unidentified, markers would be present, associated with the odontoblast phenotype. In this paper, we report the first characterization of a new gene we have named HUGO, and its associated deduced protein sequence. This gene expression is under the control of two alternative promoters, resulting in the synthesis of two proteins, one of which, HUGO2, is included in the other, HUGO1. HUGO proteins are mainly composed of a proline-rich region at the N-terminus, 8 type III-fibronectin modules, and a transmembranous helix at the C-terminus. In odontoblasts, the proteins are located in Golgi vesicles. However, they display a broader expression pattern, since they are also expressed by nerve fibers in the dental pulp and other tissues ( e.g., trachea, brain, kidney), as demonstrated by immunohistochemistry and qPCR, respectively. Their location in odontoblasts suggests a role in collagen and glycosaminoglycan synthesis.

Premise of the study: Microsatellite markers were isolated in the rainforest tree Lophira alata (Ochnaceae), an important timber tree from Central Africa, and cross-amplified on its savannah counterpart, L. lanceolata. Methods and Results: From a microsatellite-enriched library sequenced on a 454 GS FLX platform, 13 primer combinations were identified. Amplification was optimized in two multiplex reactions. The primers amplified di- and trinucelotide repeats, with two to seven alleles per locus. Eleven primers also amplified in L. lanceolata. Conclusions: Microsatellite primers developed for the genus Lophira displayed sufficient variation to investigate hybridization between congeneric species in the rainforest-savannah transition., SCOPUS: ar.j, info:eu-repo/semantics/published

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharrnacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.

The selection of efficacious anti-tumor monoclonal antibodies (MAbs) for biological applications is a lengthy and labor-intensive process. In vitro characterization of one hybridoma fusion may reveal large numbers of tumor antigen-specific hybridomas. Very often, many of these tumor-specific antibodies need to be assessed in vivo using several different murine xenograft tumor prevention models to determine biological efficacy. The production and purification of sufficient quantities of many antigen-specific hybridomas is time-consuming, and several months can pass between initial determination of MAb specificity and bioactivity. Moreover, many tumor-specific MAbs selected using in vitro binding studies have no in vivo anti-tumor efficacy. These studies describe an in vivo screening method either to eliminate non-efficacious MAbs or to rank-order several tumor-specific MAbs in an expeditious manner. Proof-of-concept studies were conducted using two hybridomas secreting fully characterized neutralizing human anti-tumor MAbs (CNTO MAbs). Nu-/nu- mice were injected with CNTO MAb-secreting hybridoma cells in Matrigel cell matrix, followed by injection of target human tumor cells 4 days later (when circulating CNTO MAbs were detected in serum). Both the tumor take-rate and the mean tumor volumes were reduced significantly in mice treated with CNTO MAbsecreting hybridomas compared with mice treated with non-antibody-secreting cells. A panel of human antitumor antigen-specific MAbs with unknown biological efficacy was then evaluated by this method. The hybridomas exhibited a varied pattern of anti-tumor protection, indicating that some hybrids were secreting neutralizing anti-tumor MAbs, while others appeared to be less efficacious. These studies demonstrate a rapid, biologically relevant "yes/no" in vivo screening method for the evaluation of anti-tumor antigen MAbs.

Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164–44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6–receptor interaction.

International audience; Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.

Langerhans cells (LC) are dendritic cells that capture foreign antigens and migrate with them to the regional lymph nodes where they are presented to naive T cells. The possible role of matrix metalloproteinase-9 (MMP-9) in migration was suggested following experiments in a mouse model and in human skin explants. Using in vitro generated LC (iLC) derived from CD34+ cord blood cells and epidermal LC (eLC), we investigated the correlation between MMP-9 and other MMPs production and cell migration. Cells were activated by Bandrowski's base (BB), a chemical allergen, or by recombinant birch pollen allergen 1 (rBetv 1). Contact with allergens triggered migration of these cells, with a maximum rate being reached after 24 h. Migration was preceded by production of MMP-2 and MMP-9; part of the molecules were recovered as pro-MMPs in cell culture supernatant and part were associated with cell membrane proteins. At the cellular level, membrane-type 1 (MT1) and MT3-MMP were also identified. Addition of tumor necrosis factor-alpha (TNF-alpha) initiated pro-MMP-2 and pro-MMP-9 production followed by cell migration in a dose-dependent manner. These data imply that TNF-alpha is a key molecule for MMP production and cell migration. Furthermore, activation of iLC with BB or rBet v 1 induced synthesis of TNF-a and expression of TNF RII on the cell membrane, suggesting an autocrine loop. In conclusion, membrane-associated MMP-2 and-9 rather than soluble MMPs appear to be involved in cell migration.

Tumour-derived factors suppress differentiation and function of in vitro generated DC. Here, we investigate the effect of two melanoma clones differing in their invasive and metastatic properties on the generation and/or functional maturation of human epidermal LC. LC were generated from CD34+ cord blood progenitors under GM-CSF/TNF-α/TGF-β1. CD34+ cells were co-cultured with or without melanoma cells using Transwell dishes. After 11 days of co-culture, CD34+-derived cells display a non-adherent undifferentiated morphology, a high level of monocytic CD14 marker, a down-regulated expression of LC markers (CD1a, E-cadherin) and DC markers (CD40, CD80, CD54, CD58, CD83, CD86, HLA-DR, HLA-class I). These cells were less potent than control LC in inducing allogeneic T cell proliferation. The generation of the CD14+ population was correlated with a decrease in the CD1a+ population, without any statistical differences between the two clones. Melanoma cells diverted the differentiation of CD34+ cells towards a dominant CD14+ population only if the progenitors were in an early growth phase. IL-10, TGF-β1 and VEGF were not responsible for these effects, as assessed by using blocking antibodies. By contrast, co-culture of fresh epidermal LC with melanoma cells did not affect their phenotype and function. Our data demonstrate that melanoma cells inhibit the earliest steps of LC differentiation, but failed to affect the functional maturation of epidermal LC. This suggests that melanoma cells participate in their own escape from immunosurveillance by preventing LC generation in the local cutaneous microenvironment. © 2001 Cancer Research Campaign http://www.bjcancer.com

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.

Evidence deriving from studies in the mouse showed that Langerhans cells (LC) capture antigens encountered in the epidermis and migrate subsequently from the epidermis to the skin-draining lymph nodes where they efficiently present antigens to T cells1,2,3. To migrate from epidermis to regional lymph nodes, antigen-bearing epidermal Langerhans cells must move through extracellular matrix (ECM) of various composition. The basement membrane (BM) of human skin presents a barrier rich in laminin (LM), type IV collagen, nidogen and heparan sulfate proteoglycans. The ECM of the superficial dermis contains interstitial collagens I and III essentially. Fibronectin (FN) is encountered mainly in afferent lymphatics. The effects of these ECM molecules on the LC behavior remain poorly understood.

Epidermal Langerhans cells (LC) reside in the suprabasal portion of squamous epithelia, and function as potent antigen presenting cells1. Previous studies2–4 showed that epicutaneous hapten application induces a significant accumulation of antigen-bearing dendritic cells in the regional lymph nodes. This suggests that LC are capable of migrating, and probably carrying foreign antigens from the epidermis to the lymph nodes in order to present these antigens to resting T cell. However, the precise mechanism of this migration is far from being clear.

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 β1 integrins. β1 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.

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.

Receptors for the Fc portion of IgG subclasses and IgA are present on the cell surface of alveolar macrophages (AM) from mice, rat, and humans (1–5). Fc-receptors for IgG (Fcγ-R) are known to induce a series of phagocyte functions including the respiratory burst, degranulation and phagocytosis (6). By contrast, there is little information on the function of Fc-R for IgA (Fcα-R). In the present in vitro study, we evaluate the Fc-αR mediated phagocytosis in normal human AM and compare it to the Fcγ-R mediated phagocytosis. Furthermore, the effect of in vivo macrophage activation on the Fc-R is investigated by studying AM from patients with pulmonary sarcoidosis.

The aim of this study was to analyze the filament distribution in the parotid gland and their tumors. A correlation to the histogenetic implications and histological properties was attempted. Normal rat and human parotid glands as well as pleomorphic adenomas and squamous cell carcinomas of this gland were examined by the indirect immunoperoxidase technique using antibodies to the keratin polypeptide of 67,000 dalton, and 55,000 dalton and anti-actin auto-antibodies. Both keratin and actin antigens were demonstrated in the duct system and in the myoepithelial cells of the normal salivary glands. The acinar cells remained negative. In pleomorphic adenomas, there were numerous keratin-positive spindle-shaped cells which represented the so-called myoepithelial cells. These cells were demonstrated to contain actin, too. The tubular duct-like structures were labeled by keratin antiserum and by anti-actin auto-antibodies. In squamous cell carcinomas, the majority of the tumor cells were strongly labeled by keratin antibodies. Actin was detected in these malignant cells, too. Our results show important differences in the cellular elements of the normal salivary glands with regard to their filament distribution. In normal and tumoral conditions, our findings support the hypothesis of the epithelial nature of the myoepithelial cells. Our preliminary results encourage the research of filamentous structures for scientific and diagnostic purposes.