Marie-Jeanne Staquet

Lipoteichoic Acid Increases TLR and Functional Chemokine Expression while Reducing Dentin Formation in In Vitro Differentiated Human Odontoblasts

Gram-positive bacteria entering the dentinal tissue during the carious process are suspected to influence the immune response in human dental pulp. Odontoblasts situated at the pulp/dentin interface are the first cells encountered by these bacteria and therefore could play a crucial role in this response. In the present study, we found that in vitro-differentiated odontoblasts constitutively expressed the pattern recognition receptor TLR1–6 and 9 genes but not TLR7, 8, and 10. Furthermore, lipoteichoic acid (LTA), a wall component of Gram-positive bacteria, triggered the activation of the odontoblasts. LTA up-regulated the expression of its own receptor TLR2, as well as the production of several chemokines. In particular, an increased amount of CCL2 and CXCL10 was detected in supernatants from LTA-stimulated odontoblasts, and those supernatants augmented the migration of immature dendritic cells in vitro compared with controls. Clinical relevance of these observations came from immunohistochemical analysis showing that CCL2 was expressed in vivo by odontoblasts and blood vessels present under active carious lesions but not in healthy dental pulps. In contrast with this inflammatory response, gene expression of major dentin matrix components (type I collagen, dentin sialophosphoprotein) and TGF-β1 was sharply down-regulated in odontoblasts by LTA. Taken together, these data suggest that odontoblasts activated through TLR2 by Gram-positive bacteria LTA are able to initiate an innate immune response by secreting chemokines that recruit immature dendritic cells while down-regulating their specialized functions of dentin matrix synthesis and mineralization.

Keratin polypeptides distribution in normal and diseased human epidermis and oral mucosa

Immune sera against total keratin and keratin polypeptide subunits were induced in guinea pigs, using the different bands of SDS polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum, derived from normal human epidermis. The distribution of the different polypeptides was studied in numerous human biopsies of normal epidermis, normal oral mucosa and epidermal and mucosal inflammatory, premalignant and malignant lesions using the indirect immunoperoxidase method. Antisera against total keratin (TK) and against the keratin polypeptide of M.W. 55,000 dalton (55K) labelled all keratinocytes in normal and pathological conditions. These antisera may be useful for the histodifferentiation in diagnostic pathology. Atisera against the keratin polypeptides of M.W. 67,000 (67K) and 62,000 dalton (62K) identified only keratin antigens in the spinous, granular and keratinized layer of normal epidermis and oral mucosa. No labelling of the basal layer was achieved with these immune sera. However, there were important differences in the distribution of these keratin antigens in altered epithelia which may be of value in the differential diagnosis of inflammatory, premalignant and malignant lesions of the skin and oral mucosa.

Experimental Production of Antibodies Against Stratum Corneum Keratin Polypeptides

SummaryAnti-keratin polypeptide sera (K.P.S.) were obtained by immunizing guinea pigs with fibrous proteins from stratum corneum, which were acquired from normal human epidermis by means of S.D.S. polyacrylamide gel electrophoresis. After absorption with red blood cells and liver powder the sera were tested by indirect immunofluorescence technique on different substrates. Antibodies against polypeptides P1 and P2 of M.W. 67,000 and 62,000 dalton, respectively, were directed toward cytoplasmic Ag of keratinocytes of spinous and granular layer of normal human and rabbit epidermis. No labeling could be detected in the basal cell layer. This finding is in favor of various differentiation stages of the keratinizing cells.P3 of M.W. 53,000 dalton induced low titre antibodies which labelled the whole epidermis, including the basal cell layer. The fourth polypeptide of M.W. 49,000 dalton seemed not to be immunogenic in such experiences.In tumors, such as basal cell carcinoma, squamous cell carcinoma, and warts, the expression of keratin antigens is markedly diminished.No analogy could be drawn between experimental keratin polypeptide antibodies and the human epidermal cytoplasmic antibodies which were detected in some patient sera.ZusammenfassungAntikeratin-Polypeptidseren (K.P.S.) wurden durch Immunisierung von Meerschweinchen mit fibrösen Proteinen des Stratum corneum erhalten, welches über die S.D.S. Polyacrylamid-Gel-Elektrophorese von normaler menschlicher Epidermis gewonnen wurde. Nach Absorption mit Erythrocyten und Leberpuder wurden diese Sera mit Hilfe der indirekten Immunfluorescenztechnik an verschiedenen Substraten getestet. Es wurden Antikörper gegen Polypeptide P1 und P2 mit einem Molekulargewicht von 67 000 und 62 000 Dalton nachgewiesen, die gegen cytoplasmatische Antikörper der Keratinocyten des Stratum spinosum und des Stratum granulosum der normalen menschlichen und Kaninchenepidermis gerichtet war. Ein Nachweis in den Basalzellagen konnte nicht erbracht werden. Dadurch wurden die verschiedenen Differenzierungsstadien der Zellen belegt, die sich zu Keratinocyten entwickeln und die oberhalb der Basalschicht lokalisiert sind.Polypeptide P3 des Molekulargewichts 53 000 Dalton induzieren niedrigere Antikörpertiter, die die gesamte Epidermis markieren einschließlich der Basalzellage. Polypeptide mit einem Molekulargewicht 49 000 Dalton schienen keine immunogenetische Wirkung in diesen Experimenten zu haben.Tumoren, wie Basalzellcarcinoma und Bindezellcarcinom, wie auch Warzen, wiesen eine deutlich verminderte Expression des Keratinantigens auf. Es wurde keine Analogie zwischen experimentell erzeugten Keratinpolypeptid-Antikörper und cytoplasmatischen Antikörpern der menschlichen Epidermis.

Immunocytochemical demonstration of filamentous structures in the parotid gland

SummaryThe 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.

Use of human reconstructed epidermis to analyze the regulation of β-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS

Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human β-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 thatin vitro reconstructed epidermis represents a useful model for studying regulation of expression of β-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to thein vivo situation.

Immunization onto shaved skin with a bacterial enterotoxin adjuvant protects mice against respiratory syncytial virus (RSV).

This study evaluated the potential of the skin as a non invasive route for RSV vaccination using two G protein-derived molecules, G2Na and G5 in mice. G2Na contains T and B-cell epitopes whether G5 is a pure B-cell epitope. In contrast to G5, G2Na coadministered with CT three times at 1 month interval onto 1cm of square area shaved skin, elicited a consistent serum anti-G2Na and anti-CT IgG response. The anti-G2Na IgG response was dominated by IgG1 isotype, an indirect marker of a Th2 type of response. Dramatic reduction and decrease of RSV titers in lung tissues and in the nasal tract, respectively, following intranasal virus challenge revealed biological relevance of the transcutaneous immunization in the context of RSV vaccine. These results suggest that the transcutaneous route may offer a promising potential for novel RSV vaccine strategy, simple, painless and economical.

Relationship between expression of matrix metalloproteinases and migration of epidermal and in vitro generated Langerhans cells

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 Bandrowskis 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.

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.

Microtubule-associated protein 1b, a neuronal marker involved in odontoblast differentiation.

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.

Withdrawal of TNF-alpha after the fifth day of differentiation of CD34+ cord blood progenitors generates a homogeneous population of Langerhans cells and delays their maturation.

Abstract: Human cord blood CD34+ progenitors cultured in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumor necrosis factor‐α (TNF‐α) and transforming growth factor‐β (TGF‐β) 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‐α 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‐α. The absence of TNF‐α was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogat the expression of CD83, CD86, HLA‐DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF‐α abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF‐α‐deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF‐α. These data indicate that the suppression of TNF‐α after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.