A rare association between group A Streptococcus purpura fulminans and diffuse alveolar hemorrhage in a young girl: A case report.

We report the case of a 7-year-old girl with septic shock and coagulopathy associated with purpura fulminans (PF) and diffuse alveolar hemorrhage (DAH) due to group A Streptococcus (GAS) infection identified with 16S ribosomal RNA analysis performed on the skin biopsy. GAS infection with PF associated with DAH is rare in healthy young children but pediatricians should be aware of this condition because of the poor prognosis. The initial treatment for circulatory failure and severe disseminated intravascular coagulation as well as the prompt initiation of antibiotic treatment may be crucial for the outcomes of S. pyogenes PF.

Isolated neonatal MRI punctate white matter lesions in very preterm neonates and quality of life at school age.

OBJECTIVE: To study the quality of life at school age of very preterm infants presenting isolated punctate periventricular white matter lesions (IPWL) on late-preterm or term magnetic resonance imaging (MRI). METHODS: In 1996-2000, 16 of the 131 very preterm neonates explored by MRI were found to have IPWL. At the age of 9-14, 12 children from the IPWL group were compared with 54 children born preterm but with a normal MRI (no lesion). Quality of life (Health Status Classification System Pre School questionnaire), school performance, and motor outcome were investigated. RESULTS: Overall quality of life did not differ between the groups (classified as perfect in 2/12 of the IPWL vs 20/54 in the no-lesion). The sub-items mobility and dexterity differed significantly between the two groups, with impairment in the IPWL group (p < 0.001 and p < 0.05). This group also displayed higher levels of motor impairment: they began walking later [20(4) vs. 15(3) months), p < 0.01], had higher frequencies of cerebral palsy (6/12 vs. 2/54, p < 0.05), and dyspraxia (4/12 vs. 0/54, p < 0.001). The rate of grade retention did not differ between the groups (3/12 in the IPWL group vs. 17/54 in the no-lesions group) but, as expected, was higher than that of the French general population (17.4%) during the study period. CONCLUSION: This long-term follow-up study detected no increase in the risk of subsequent cognitive impairment in very preterm infants with IPWL, but suggests that these children may have a significantly higher risk of dyspraxia, and motor impairment.

Cell-cell and cell-matrix interactions during initial enamel organ histomorphogenesis in the mouse.

Relationships between cell-cell/cell-matrix interactions and enamel organ histomorphogenesis were examined by immunostaining and electron microscopy. During the cap-bell transition in the mouse molar, laminin-5 (LN5) disappeared from the basement membrane (BM) associated with the inner dental epithelium (IDE), and nondividing IDE cells from the enamel knot (EK) underwent a tooth-specific segregation in as many subpopulations as cusps develop. In the incisor, the basement membrane (BM) in contact with EK cells showed strong staining for LN5 and integrin alpha 6 beta 4. LN5 seems to provide stable adhesion, while its proteolytic processing might facilitate cell segregation. In both teeth, immunostaining for antigens associated with desmosomes or adherens junctions was similar for EK cells and neighboring IDE cells. Outside the EK, IDE cell-BM interactions changed locally during the initial molar cusp delimitation and on the labial part of the incisor cervical loop. Conversely, cell-cell junctions stabilized the anterior part of the incisor during completion of morphogenesis. Time and space regulation of cell-matrix and cell-cell interactions might thus play complementary roles in allowing plasticity during tooth morphogenesis and stabilization at later stages of epithelial histogenesis.

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts.

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MT1-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for alpha4 and alphav, with no difference measurable under stretching, while the mRNAs encoding for alpha6 and beta1 were increased and the one encoding for alpha5 was decreased. HGFs increased the mRNAs encoding for alpha2, alpha6, beta1, and beta3 and decreased the one encoding for alpha3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.

Effects of hepatocyte growth factor anti-sense oligodeoxynucleotides or met D/D genotype on mouse molar crown morphogenesis.

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelio-mesenchymal interactions during early organogenesis and to be also involved in the development of murine molars. In the developing tooth, HGF is expressed in the cells of the dental papillae, and c-Met, its receptor, in the cells of dental epithelia. In order to study the functional role played by HGF in tooth development, we tested the effects of HGF translation arrest by anti-sense phosphorothioate oligodeoxynucleotides on E-14 molars cultured in vitro. We also analyzed the histo-morphogenesis and crown cytodifferentiation of transgenic met E-14 molars cultured in vitro. 3D reconstructions revealed perturbations of the cusp pattern. However, histo-morphogenesis and crown cytodifferentiation were normal at the histological level.

Genetic comparison between different populations of Eulemur macaco flavifrons in northwest Madagascar using RAPD markers.

Eulemur macaco flavifrons, the Sclater's black lemur, is a critically endangered subspecies of northwest Madagascar, which is not yet protected by any reserve. In order to study the feasibility of creating such a reserve, an area of outstanding biological importance was selected in the region of Maromandia-Sahamalaza, which is probably the only remaining place which would permit the long-term survival of the Sclater's black lemur. To determine if genetic management is needed for the Sahamalaza black lemur population, its genetic variability was estimated with random amplified polymorphic DNA (RAPD) markers and compared with other populations. These comparisons demonstrate that the Sahamalaza black lemurs have a genetic variability equivalent to those in other areas. Thus, we conclude that no genetic management is required at the present time.

Localization of antigens associated with adherens junctions, desmosomes, and hemidesmosomes during murine molar morphogenesis.

Epitheliomesenchymal interactions are known to play a crucial role during odontogenesis. Since epithelial cell-cell and cell-matrix interactions may also be involved in enamel organ histomorphogenesis, we investigated the localization of proteins associated with junctional complexes in mouse and rat first lower molars by indirect immunofluorescence. Adherens junctions were detected using antibodies directed against E-cadherin, beta-catenin, and plakoglobin (gamma-catenin). Desmosomes were localized with antibodies against desmoglein, and hemidesmosomes using antibodies against BP-230 and HD-1 proteins. When the inner dental epithelium differentiates, a decrease of E-cadherin, plakoglobin, and BP-230 is seen. An asymmetric distribution of plakoglobin, desmoglein, and BP-230 between the lateral and medial side of the tooth exists; desmoglein, which was first restricted to the gubernaculum dentis, progressively accumulated in the stellate reticulum, the stratum intermedium, and the basal pole of ameloblasts. The specific temporospatial distributions patterns of these antigens suggests a direct involvement of adherens junctions, desmosomes, and hemidesmosomes in the development of the murine first lower molar.

Expression of p21(WAF1/CIP1) during mouse odontogenesis.

p21(WAF1/CIP1) is a cyclin-dependent kinase (Cdk) inhibitor. This protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation. The expression pattern of p21 during mouse embryogenesis was correlated with terminal differentiation of multiple cell lineages including skeletal muscles, cartilage, skin and nasal epithelium. p21 expression was analyzed by in situ hybridization during odontogenesis as well as during in vitro tooth development in chemically defined medium with or without retinoic acid. p21 transcripts were detected in the restricted area of the inner dental epithelium during late cap and initial bell stages and then confined to the post-mitotic odontoblasts and ameloblasts. The replicating cells were devoid of any signal. The distribution of p21 mRNA in vitro, whatever the culture conditions, was similar to the in vivo pattern. p21 protein immunolocalization was superimposed on the transcripts distribution but more restricted in ameloblasts. TGFbeta1 is known to induce p21 expression. During dental cytodifferentiations, TGFbeta1 and p21 expressions overlap. Growth inhibition by TGFbeta1 may be associated with p21 induction.

Interaction of vinculin with the clathrin heavy chain.

To further document the interaction of vinculin with the clathrin heavy chain (CHC) which was observed by using gel overlay, co-sedimentation experiments were performed and attempts were made to localize the domains involved on both molecules. The binding properties of proteolytic fragments of vinculin were investigated after cleavage with V8 protease. Neither the isolated globular domain, nor the C-terminal rod domain were able to interact with the CHC. Either the interaction involved the portion of vinculin which links these two domains, or the region of vinculin mediating the interaction was present on one of the two major fragments, but the cleavage itself resulted in conformational changes which abolished the binding. The first hypothesis could be ruled out using alpha-chymotrypsin generated fragments of vinculin, suggesting that the native conformation of vinculin might play an important role. Proteolytic cleavage of CHC with trypsin demonstrated that the interaction with vinculin is mediated by the proximal or distal segment of the CHC. Presence of clathrin light chain (CLC) associated with the CHC did not affect its interaction with vinculin. Vinculin did not interact with the CLC.

Absence of interaction between the 165-kDa fibronectin-binding protein involved in mouse odontoblast differentiation and vinculin.

Previous data suggested that matrix could control the organization of microfilaments in differentiating odontoblasts and that this process involved a complex of fibronectin-165-kDa membrane protein-vinculin. The use of two different gel systems and microsequence analysis demonstrated that two distinct 165-kDa proteins interact, one with fibronectin and the other with vinculin.

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.

[Cell-matrix interactions and odontoblast differentiation]. / Interactions cellules-matrice et différenciation de l'odontoblaste.

The terminal differentiation of odontoblasts requires the integrity of the cytoskeleton and is controlled by cell-matrix interactions. These interactions implicate both matrix molecules and matrix-associated growth factors. On the one hand, predentin-dentin constituents were found to initiate odontoblast differentiation and to allow the maintenance of this state; TGF-beta or related molecules are implicated. Fibronectin on the other hand can induce the differentiation of second generation odontoblasts and interacts with three high molecular weight proteins present in membrane prepared from dental mesenchymal cells. One of these proteins (165 kDa) was localized on the surface of odontoblasts and is involved in the organization of microfilaments. Two main axes of research will have to be developed in the future in order to understand how matrix molecules and growth factors interactions can be modulated in time and space by epithelial and mesenchymal cells, and how such modulations can affect the phenotype of these cells.

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation.

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.

Characterization of a mouse monoclonal antibody to vimentin by indirect immunofluorescence microscopy and immunoblotting.

A murine IgM monoclonal antibody, termed MC15A13G was produced after immunization of Balb/c mouse with Swiss mouse dental papillae. This antibody, characterized by immunoblotting analysis and indirect immunofluorescence microscopy, recognized a 57 Kda protein identified as vimentin in different cell types with no cross reaction with other cytoskeletal proteins.