In situ observation of the Yb2+ emission in the radiodarkening process of Yb-doped optical preform.

This Letter relates the clear evidence of Yb2+ formation under 2.5 MeV electron irradiation in optical fiber preforms showing a darkening of the core. We thus detected by in situ photoluminescence measurements the green emission of divalent Yb2+ under the 355 nm excitation. Moreover, we showed the existence of two types of Yb2+ ion species with different stabilities. We demonstrated that the radiodarkening mechanism is based on a pair association of Yb2+ with aluminum oxygen hole center point defects.

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry.

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.

MHC class II beta-chain and alphaIIbbeta3 integrin are expressed on T-cell progenitors in embryonic bone marrow.

The RR5 monoclonal antibody (mAb) was obtained after immunization of mice with hemopoietic cells from chicken embryos. The cDNA encoding the protein recognized by RR5 was cloned using COS-7 cells transfected with an embryonic bone marrow (BM) cDNA library. The epitope recognized by the RR5 mAb was located on the non-polymorphic MHC class II beta-chain molecule. In the embryonic BM, RR5 labeled 50% of the c-kit expressing cells. Previous experiments have shown that the T-cell progenitors are present in the MHC class II(+)/c-kit(+) BM population along with myeloid progenitors and that T-cell and myeloid progenitors also express the integrin alphaIIbbeta3. In this study, using intrathymic cell transfer experiments in chicks, we have tested the T-cell differentiation potential of MHC class II/alphaIIbbeta3 double positive cells. It proved to be similar to that of the c-kit/MHC class II positive cells. However, injection of triple positive cells resulted in a selection of cells with an increased T-cell potential. Most of the MHC class II positive cells which do not express c-kit are prone to apoptosis, indicating that these progenitors might need a survival signal via c-kit. Interestingly, the MHC class II positive progenitors lose this expression after intrathymic transfer. Taken together our data suggest that the presence of the MHC class II beta-chain molecule on the surface of BM progenitor cells could be implicated in differentiation toward myeloid and lymphoid lineages.

MHC class II and c-kit expression allows rapid enrichment of T-cell progenitors from total bone marrow cells.

T-cell progenitors in the embryonic bone marrow express the tyrosine kinase receptor c-kit. RR5, an anti-MHC class II beta chain monoclonal antibody, subdivides this c-kit positive population. Intrathymic transfer experiments showed that most of the T-cell progenitors belong to the MHC class II(+)/c-kit(+) bone marrow population in the embryo and young adult. On transplantation, these bone marrow progenitors lose this expression and differentiate into CD4 CD8 T lymphocytes. In contrast, erythroid progenitors are restricted to the MHC class II(-)/c-kit(+) population. The MHC class II(+)/c-kit(+) pro-T cells are metabolically active, because they stain brightly with rhodamin 123. Their cyclin A and B expression level suggests that they are in the mitotic phase of the cell cycle. Thus, we define an easy sorting protocol, which allows enrichment of T-cell progenitors from total bone marrow hemopoietic cells. (Blood. 2000;96:3988-3990)

Expression of CD44 during early development of the chick embryo.

CD44, the major cell-surface receptor for hyaluronate, is expressed on many cell types to mediate different functions including cell activation, homing and adhesion. The early pattern of CD44 expression was determined in the avian embryo by using a specific monoclonal antibody in whole-mount and tissue sections. CD44 was first expressed on cephalic neural fold cells and later on by subpopulations of pre-and migratory cranial neural crest cells. Trunk neural crest cells did not express CD44. At the 18-20 somite stage, CD44 expressing cells were also localized in the caudal region of the embryo, in the mesoderm of the remaining primitive streak and in the caudal ectoderm and above the secondary neural tube during the process of cavitation. In addition, some hemopoietic cells present in the blood stream were also CD44 positive.

[Receptors and development of endothelial and hematopoietic cells]. / Récepteurs et développement des cellules endothéliales et hématopoïétiques.

During vertebrate embryonic development, the endothelial and hemopoietic systems are the first system to be specified. In this review, we will summarize recent findings about the molecular mechanisms responsible for the successive steps of the development of these systems: the differentiation of mesodermal cells to endothelial and hemopoietic cells, their proliferation and their interactions to form the vascular system.

[Endothelial cell precursors in the avian embryo]. / Les précurseurs des cellules endothéliales chez l'embryon d'oiseau.

Whereas the origin and migration of endothelial cells (ECs) have been studied primarily in the avian embryo, the molecular mechanisms governing these events in birds and mammals were unraveled following the identification of specific growth factors and of their receptors. In particular, analytic and experimental studies of vascular endothelial growth factor (VEGF) and its receptors have provided significant insights into the developmental biology of the vascular system. VEGFR2 is the earliest marker expressed by EC precursors in chickens and mice. Based on the localization of VEGFR2-positive cells in the avian embryo and on findings from clonal culture experiments, two types of EC precursors can be differentiated as early as the gastrulation stage, namely posterior mesoderm hemangioblasts capable of differentiating into both ECs and hematopoietic cells and anterior angioblasts capable of yielding only ECs.

[Prevalence of anti-Chlamydia pneumoniae antibodies in preadolescent children in Congo]. / Prévalence des anticorps anti-Chlamydia pneumoniae chez l'enfant préadolescent au Congo.

Chlamydia pneumoniae (CPn) is recognised as a common cause of both upper and lower respiratory tract infections. Seroepidemiological studies seem to indicate a world-wide distribution of this organism. In order to evaluate the prevalence of antibodies to CPn in a healthy pediatric population, we measured anti-C. pneumoniae antibodies in a group of 253 infants without respiratory tract infections, aged from 1 to 12 years. Sera were obtained from children seen at immunization clinics and schools in Lubumbashi (Congo). Antibodies to CPn were evaluated using micro-immunofluorescence assay. IgG antibody to CPn in a titre 16 was considered as positive. The antibody prevalence was found to be 25.7%. This prevalence was 6.2% in children aged from 1 to 6 years, and 37.8% in children aged from 7 to 12 years. It was less than 10% under five years, increasing to 50% at 12 years. The progressive increasing of seropositivity related to age suggests that reinfections may be frequent. This study shows an important spread of this bacteria in the preadolescent population of an African country.

Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells.

The fibrinogen receptor GPIIb-IIIa integrin is known to be expressed on cells of the megakaryocytic lineage, but its presence on hematopoietic progenitors has been a controversial issue. To resolve this ambiguity unequivocally, we performed clonogenic assays and intrathymic cell-transfer experiments in congenic animals. As the ontogeny of the avian hematopoietic system is well documented, we used this experimental model to trace GPIIb-IIIa expression during embryogenesis. Consequently, we now report that the GPIIb-IIIa integrin is expressed as early as embryonic day 3.5 (E3.5) to 4 in intraaortic hematopoietic clusters, the first site of intraembryonic hematopoietic progenitor emergence, and later in E6 paraaortic foci. Myeloid and erythroid progenitors were also detected within the GPIIb-IIIa+ CD45(+) population isolated from the E3.5 to 4 aortic area, while in embryonic and adult bone marrow, myeloid, erythroid, and T-cell progenitors were present in the GPIIb-IIIa+ c-kit+ population. Furthermore, we also provide the first evidence, that GPIIb-IIIa+ bone marrow cells can differentiate into T cells. Hence, GPIIb-IIIa can be used as a marker for multilineage hematopoietic progenitors, permitting identification of early intraembryonic sites of hematopoiesis, as well as the isolation of embryonic and adult hematopoietic progenitors.

Avian VEGF-C: cloning, embryonic expression pattern and stimulation of the differentiation of VEGFR2-expressing endothelial cell precursors.

VEGF-C is a recently discovered secreted polypeptide related to the angiogenic mitogen VEGF. We have isolated the quail VEGF-C cDNA and shown that its protein product is secreted from transfected cells and interacts with the avian VEGFR3 and VEGFR2. In situ hybridization shows that quail VEGF-C mRNA is strongly expressed in regions destined to be rich in lymphatic vessels, particularly the mesenteries, mesocardium and myotome, in the region surrounding the jugular veins, and in the kidney. These expression sites are similar to those observed in the mouse embryo (E. Kukk, A. Lymboussaki, S. Taira, A. Kaipainen, M. Jeltsch, V. Joukov and K. Alitalo, 1996, Development 122, 3829-3837). We have observed VEGFR3-positive endothelial cells in proximity to most of the VEGF-C-expressing sites, suggesting functional relationships between this receptor-ligand couple. The comparison of the VEGF and VEGFR2 knockout phenotypes had suggested the existence of another ligand for VEGFR2. We therefore investigated the effect of VEGF-C on VEGFR2-positive cells isolated from the posterior mesoderm of gastrulating embryos. We have recently shown that VEGF binding triggers endothelial differentiation of these cells, whereas hemopoietic differentiation appears to be mediated by binding of a so far unidentified VEGFR2 ligand. We show here that VEGF-C also triggers endothelial differentiation of these cells, presumably via VEGFR2. These results indicate that VEGF and VEGF-C can act in a redundant manner via VEGFR2. In conclusion, VEGF-C appears to act during two different developmental phases, one early in posterior mesodermal VEGFR2-positive endothelial cell precursors which are negative for VEGFR3 and one later in regions rich in lymphatic vessels at a time when endothelial cells express both VEGFR2 and VEGFR3.

Segregation of the embryonic vascular and hemopoietic systems.

The origin of endothelial cells and their subsequent assembly into the primary vascular system have been mostly analyzed in the avian embryo. Following the discovery of specific growth factors and their cognate receptors, the molecular mechanisms underlying these processes have been unraveled in both birds and mammals. In particular, experimental studies of the angiogenic vascular endothelial growth factor (VEGF) and its receptors, carried out in both vertebrate classes, have provided significant insight into the developmental biology of endothelial cells. The VEGF receptor VEGFR2 is the earliest marker known to be expressed by endothelial precursor cells of avian and mouse embryos. Based on the localization of VEGFR2+ cells in the avian embryo and on clonal culture experiments, two types of endothelial precursor cells can be distinguished from gastrulation stages onward: posterior mesodermal VEGFR2+ hemangioblasts, which have the capacity to differentiate into endothelial and hemopoietic cells, and anterior VEGFR2+ angioblasts, which can only give rise to endothelial cells.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2.

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.

Capture-ELISA: a new assay for the detection of immunoglobulin M isotype antibodies using Chlamydia trachomatis antigen.

We present here a new method of IgM antibody-capture enzyme-linked immunosorbent assay (IgM-Capture-ELISA) for the diagnosis of recently acquired infections with Chlamydia trachomatis. For this analysis, plates were coated with goat IgG anti-human Fc mu. The capture of serum IgM antibodies was revealed indirectly by the sequential addition of biotinylated chlamydial proteins and peroxidase-conjugated streptavidin. In chlamydial extracts, cysteine-rich proteins are preferential antigenic targets for the humoral response. 3-(N-maleimidopropionyl)-biocytin (MPB), which binds biotinylated moieties to sulfhydryl groups, was used for the labeling procedure. The preservation of the antigenic specificity of labeled proteins was controlled by a blotting of these proteins, which were, respectively, probed either with specific IgM antibodies or with streptavidin. This analysis revealed that, after labeling, recognized epitopes are more particularly present on the major outer membrane protein (MOMP) of Chlamydia trachomatis. The validation of IgM-Capture-ELISA was assessed by using 170 selected sera from patients suspected of being infected by Chlamydia. Results were respectively compared to conventional indirect immunofluorescence assays (MIF-IgM assays) and to Western blotting. Sixteen sera were found to possess IgM antibodies against Chlamydia trachomatis with IgM-Capture-ELISA. Among these 16 sera, 14 and 15 were, respectively, positive with MIF-IgM assays and in Western blotting. Data obtained with IgM-Capture-ELISA reveal the absence of false-positive results in sera containing rheumatoid factor, which has been shown to interfere in the two other methods. IgM-Capture-ELISA value was then confirmed using sera from patients consulting for genital or pulmonary diseases, from patients with confirmed chlamydial infections, and from patients with other pathologies. IgM-Capture-ELISA appears as an alternative simple semi-quantitative assay for the detection of early chlamydial infection.

BEN/SC1/DM-GRASP, a homophilic adhesion molecule, is required for in vitro myeloid colony formation by avian hemopoietic progenitors.

BEN/SC1/DM-GRASP is a membrane glycoprotein of the immunoglobulin superfamily isolated in the chick by several groups, including ours. Its expression is strictly developmentally regulated in several cell types of the nervous and hemopoietic systems and in certain epithelia. Each of these cell types expresses isoforms of BEN which differ by their level of N-glycosylation and by the presence or absence of the HNK-1 carbohydrate epitope. In the present work, the influence of glycosylation on BEN homophilic binding properties was investigated by two in vitro assays. First, each BEN isoform was covalently coupled to microspheres carrying different fluorescent dyes and an aggregation test was performed. We found that homophilic aggregates form indifferently between the same or different BEN isoforms, showing that glycosylation does not affect BEN homophilic binding properties. This was confirmed in the second test, where the BEN-coated microspheres bound to the neurites of BEN- expressing neurons, irrespective of the isoform considered. The transient expression of the BEN antigen on hemopoietic progenitors prompted us to see whether it might play a role in their proliferation and differentiation. When added to hemopoietic progenitor cells in an in vitro colony formation assay anti-BEN immunoglobulin strongly inhibited myeloid, but not erythroid, colony formation although both types of precursors express the molecule.