Comparative Studies on Naturally Occurring Antikeratin Antibodies in Human Sera

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

Distribution Pattern of Psoriatic Keratoblasts - Computer-assisted Image-analysis for Combined Evaluation of Dna-synthesis and Expression of 67 Kd Keratin Polypeptides in the Epidermis of Stable Plaques of Psoriasis

Psoriatic epidermis is characterized by increased DNA synthesis and disturbed differentiation. Even though these processes are closely associated, most investigations do not give insight into temporal/spatial relationships between both events. We previously developed a double labeling method for the simultaneous demonstration of the germinative and differentiated epidermal compartments in normal human skin by using tritium-labeled thymidine ([3H] Thd) incorporation and immunoperoxidase staining of 67kD keratin polypeptides. In this paper we report the results of combined evaluation of these compartments in stable plaques of psoriasis. Scanning of skin sections with an automatic image analyzer allows objective quantification of areas of total epidermis, 67kD- differentiated epidermis and numbers of [3H] Thdr+ nuclei. Our data indicate that the 67kD- undifferentiated psoriatic epidermis is expanded. Increased numbers of [3H] Thd+ basal and suprabasal psoriatic keratinocytes are present and most of them (97.9%) pertain to the 67kD- compartment. Keratin identification in scales taken from the same sites showed a variable but distinct decrease of 67kD keratin polypeptides. Hence, the hyperplastic epidermis of stable plaques of psoriasis is characterized by the presence of increased numbers of [3H] Thd+ cells, which primarily belong to the undifferentiated (67kD-) basal and suprabasal compartments, especially in the lowermost parts of the elongated interpapillary rete ridges. These changes are associated with a relative decrease of synthesis of 67kD polypeptides and the presence in the scales of keratins that confer a characteristic hyperproliferative epidermal keratin pattern to the psoriatic plaque

Microtubule-associated Protein 1b, a Neuronal Marker Involved in Odontoblast Differentiation

International audienceIntroduction: Map-1B belongs to the family of proteins that govern the dynamic state and organization of microtubules within cells. MAP-1B is a microtubuleassociated 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

Cytokine production by human odontoblast-like cells upon Toll-like receptor-2 engagement

International audienceRecent 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

Expression of NOD2 is increased in inflamed human dental pulps and lipoteichoic acid-stimulated odontoblast-like cells

International audienceHuman 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 Pam(2)CSK(4) 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

Lipopolysaccharide-binding protein inhibits toll-like receptor 2 activation by lipoteichoic acid in human odontoblast-like cells

International audienceIntroduction: 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