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Biomedical subjects

B Delpech

Publications and source records attributed to B Delpech.

At least 55 records · Page 3Linked to original sources
PubMed1996

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.

Amino Acid Sequence↗
PubMed1996

CD44 and hyaluronan binding by human myeloid cells.

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

Antibodies, Monoclonal↗
PubMed1995

Enzyme-linked hyaluronectin: a unique reagent for hyaluronan assay and tissue location and for hyaluronidase activity detection.

Several techniques for assaying and localizing hyaluronan (HA), all based on the affinity to hyaluronan of proteins isolated from cartilage, chondrosarcoma, or brain, have been proposed. We show here that a unique reagent, alkaline phosphatase-linked hyaluronectin, can be used to assay hyaluronan in biological fluids or tissue extracts (enzyme-linked sorbent assay method) and to characterize it in cells or tissue sections in two steps: reagent incubation and staining. Results of assays in biological fluids or tissue extracts showed a good correlation with results of the previously described technique using antibodies to detect hyaluronectin bound to a plastic microtest plate (B. Delpech et al., 1985, Anal. Biochem. 149, 555-565) for both low concentrations (< 1 mg/liter, r = 0.973, P < 0.001) and high concentrations (> 1 mg/liter, r = 0.953, P < 0.001). The interassay variations were 8.5% when the assay was performed at 4 degrees C and 18.5% at 37 degrees C. The intraassay variations under those conditions were, respectively, 14.4 and 6.5%. Tissue HA could be detected easily with the reagent, as shown in fetal tissues and in tumors. Specificity of the reaction was controlled either by blocking the reagent with an excess of hyaluronan (which was not possible with other glycosaminoglycans) or by destroying tissue hyaluronan with streptomyces hyaluronidase. Alkaline phosphatase-linked hyaluronectin was also used to assay hyaluronidase activity in several biological fluids. One-hour incubation of hyaluronidase preparations on HA-coated plates made it possible to detect as low as 1 mU bovine testis hyaluronidase and 0.1 mTRU streptomyces hyaluronidase. Four-hour incubation made it possible to detect activity in a 1/12,500 dilution of human serum.

Alkaline Phosphatase↗
PubMed1995

CNS myelinogenesis in vitro: time course and pattern of rat oligodendrocyte development.

Oligodendrocyte precursor cells that develop into myelin-forming cells of the central nervous system (CNS) were cultured from newborn rat brain to study how they proliferate and differentiate in normal conditioning medium, and their cell development was characterized by scanning electron microscopy (SEM) observation and immunocytochemical studies. We have identified A2B5-negative pre-O2A progenitor cells (so-called "type-1" oligodendrocytes) in the secondary cultures on the astrocyte feeder layer. These cells are very small (diameter: 3.5 microns), round, and glossy, and develop into the process-bearing O2A progenitor cells (called "type-2" oligodendrocytes), which also express myelin basic protein (MBP) both in the cell body and in their cell processes. Finally, they develop into mature oligodendrocytes (called "type-3" oligodendrocytes). After MBP expression is elicited in these cells and MBP accumulates in the cell process in the area in contact with the axon, these cells are capable of forming the myelin sheath. Therefore, we examined the mechanism of myelin-sheath formation of "type-3" oligodendrocytes using video time-lapse movies, and demonstrated that these cells initially sent out processes to search for axons several times before the onset of myelination. Then thick filopodia extended towards the axon, and at the same time, the axonal part of neuron moved forward. Finally the ruffling lamellipodial parts wrapped up the axon similarly to a transverse wave with the secured thick filopodial process on the axon acting as scaffolding. These results suggest that our experimental systems are useful in studying normal oligodendrocyte development and their cellular biochemistry, as well as investigating the mechanism of myelin formation by oligodendrocytes.

Animals↗
PubMed1995

Neuroendocrine primary small cell carcinoma of the breast. Report of a case and review of the literature.

One case of breast neuroendocrine primary small cell carcinoma with light microscopic and immunohistochemical findings is reported. The patient died of unrelated disease 21 months after diagnosis and treatment by modified radical mastectomy, radiotherapy and subsequent chemotherapy. Immunohistochemical studies revealed cytokeratin and neuroendocrine markers (chromogranin, neuron-specific enolase) immunostaining on tumoral cells. Expression for neuropeptides (met-enkephalin, leu-enkephalin, beta-endorphin) and CALLA antigen was found. Based on this case report and six other previously reported cases, breast neuroendocrine primary small cell carcinoma appears to be a very aggressive tumor for which no firm conclusions regarding treatment can be drawn.

Aged↗
PubMed1994

Expression of opioid peptides in cells and stroma of human breast cancer and adenofibromas.

The expression of beta-endorphin, Met-enkephalin and Leu-enkephalin was studied in 63 malignant and benign human breast tumors using immunohistochemical methods. Among invasive ductal carcinomas, 93% were positive for beta-endorphin, 87% for Leu-enkephalin and 90% for Met-enkephalin, in both the tumor stroma and the cell bodies. Enkephalin was predominant in cells, whereas endorphin was predominant in stroma. Nearly the same distribution was found in adenofibromas. In pericancerous normal tissue, neuropeptides were predominantly expressed in the stroma. Although the neuropeptide expression is not cancer-specific, it could be cancer-related, since the results suggest that the neuropeptide expression could reflect the host response to cancer cells and not only the cancer cell activity. The possibility of a direct action of the nervous system on stroma reaction and then on cancer cells is discussed.

Adenofibroma↗
PubMed1994

Localization and solubilization of hyaluronan and of the hyaluronan-binding protein hyaluronectin in human normal and arteriosclerotic arterial walls.

Hyaluronectin (HN), a hyaluronan (hyaluronic acid, HA)-binding glycoprotein isolated from human brain, was studied in normal and atherosclerotic human arteries. It can be detected and assayed in tissue samples by immunohistochemistry. In addition, its high and specific affinity for HA makes it possible to develop specific histological localization of HA using HN as a probe. We tested the presence of HN and HA in human carotid artery samples from adults and newborns. In atheroma-free arterial samples HN was found in the intima, between smooth muscle cells and in the adventitial extracellular matrix. In atherosclerotic lesions, HN was strongly expressed in the diffuse thickened intima and surrounding extracellular microcrystalline calcium deposits, and very little in the lipid core. HA was found in the same locations. The similar localizations of HN and HA shown by immunohistology and demonstration of HN-HA complexes by high pressure liquid chromatography (HPLC) suggest that they are associated in vivo.

Adult↗
PubMed1994

Expression and effects of hyaluronan and of the hyaluronan-binding protein hyaluronectin in newborn rat brain glial cell cultures.

Hyaluronan (HA) is a polymerized nonsulfated extracellular matrix glycosaminoglycan that may be involved in brain development. We have tested the expression of HA and the HA-binding protein hyaluronectin (HN) in glial cell cultures from newborn rat brain. HA was secreted into the culture medium by type 1 astrocytes in the first stages of the primary cultures. The secretion was high during cell proliferation, reached a maximum when they were confluent, and then decreased. HA was not secreted at a detectable level by total O-2A lineage cell-enriched cultures. HA labeled small O-2A progenitor cells (GFA-, A2B5+, HA+), small O-2A progenitorlike (GFA-, A2B5-, HA+) cells, and type 2 astrocytes (GFA+, A2B5+, HA+), but not mature oligodendrocytes (Galc+, HA-). In contrast to HA, hyaluronectin labeled oligodendrocyte membranes (i.e., more mature cells) from day 8. A2B5+ GFA- cells were found to be either HA+ or HN+ at days 7-9, suggesting intermediary stages. The addition of HA to primary cultures and to O-2A progenitor-enriched cultures decreased significantly the increase in the number of O-2A progenitors, of mature (Galc+) oligodendrocytes proportionally to the decrease of the O-2A progenitor number, and of BrdU+ cells, suggesting that HA acts (directly or indirectly) on O-2A cell proliferation. This effect, which was seen for concentrations as low as 0.1 micrograms/ml, was HA specific and was not observed with other glycosaminoglycans. When primary cultures were performed in the presence of hyaluronidase-digested or HA-depleted (by passage on a HN column) fetal calf serum, the total number of O-2A lineage cells was dramatically increased (100%, p < 10(-4)) in comparison with control cultures in standard fetal calf serum. Platelet-derived growth factor increased the total number of O-2A lineage cells and of (Galc+) oligodendrocytes. This effect was opposed by HA dose dependently. The effect of HA was significantly inhibited by HN (30%, p < 10(-4)). HN had, however, no effect when it was added to culture in the presence of hyaluronidase in fetal calf serum, suggesting its effect was only due to its binding to HA. During cell maturation, HA disappears as HN appears. This and the fact that HA and PDGF have opposite effects suggest an effect of these factors, or of their balance, on myelination.

Animals↗
PubMed1993

Molecular cloning of a human beta 3-adrenergic receptor cDNA.

We report the molecular cloning of a beta 3-adrenergic receptor [beta 3-AR] cDNA from human brown adipose tissue. The cDNA-encoded protein is identical to the previously cloned beta 3-AR but with 6 additional amino acids at the C-terminus. The C-terminus is shared by the beta 3 receptors expressed in human neuroblastoma cells [SK-N-MC] [Mol. Pharmacol. 42 (1992) 964-970]. Furthermore, using a polymerase chain reaction strategy we have cloned and sequenced the beta 3-AR introns. Sequence analysis demonstrates that the human beta 3-AR gene comprises at least 3 exons and 2 introns and that the most abundant beta 3-AR transcripts encode a protein with an exon 3-derived C-terminus. Interestingly, although a similar organization has been found in rodent genes, the rat beta 3-AR transcripts encode a receptor with an exon 2-derived C-terminus.

Adipose Tissue, Brown↗
PubMed1993

Cannabinoid-receptor expression in human leukocytes.

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS), probably through the cannabinoid receptor, which has recently been cloned in rat and human. While numerous reports have also described effects of cannabinoids on the immune system, the observation of both mRNA and cannabinoid receptor has hitherto been exclusively confined to the brain, a reported detection in the testis being the sole example of its presence at the periphery. Here we report the expression of the cannabinoid receptor on human immune tissues using a highly sensitive polymerase-chain-reaction-based method for mRNA quantification. We show that, although present in a much lower abundance than in brain, cannabinoid receptor transcripts are found in human spleen, tonsils and peripheral blood leukocytes. The distribution pattern displays important variations of the mRNA level for the cannabinoid receptor among the main human blood cell subpopulations. The rank order of mRNA levels in these cells is B cells > natural killer cells > or = polymorphonuclear neutrophils > or = T8 cells > monocytes > T4 cells. Cannabinoid-receptor mRNA, which is also found in monocytic, as well as T and B leukemia cell lines but not in Jurkat cells, presents a great diversity of expression on these cells as well, B-cell lines expressing a much higher level than T-cell lines. The cannabinoid receptor PCR products from leukocytes and brain are identical both in size and sequence suggesting a strong similarity between central and peripheral cannabinoid receptors. The expression of this receptor was demonstrated on membranes of the myelomonocytic U937 cells using the synthetic cannabinoid [3H]CP-55940 as ligand. The Kd determined from Scatchard analysis was 0.1 nM and the Bmax for membranes was 525 fmol/mg protein. The demonstration of cannabinoid-receptor expression at both mRNA and protein levels on human leukocytes provides a molecular basis for cannabinoid action on these cells.

Animals↗
PubMed1993

Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain.

The nature and function of previously described perineuronal nets are still obscure. In the present study their polyanionic components were demonstrated in the rat brain using colloidal iron hydroxide (CIH) staining. In subcortical regions, such as the red nucleus, cerebellar, and vestibular nuclei, most neurons were ensheathed by CIH-binding material. In the cerebral cortex perineuronal nets were seen around numerous nonpyramidal neurons. Biotinylated hyaluronectin revealed that hyaluronan occurs in perineuronal nets. Two plant lectins [Wisteria floribunda agglutinin (WFA) and Vicia villosa agglutinin (VVA)] with affinity for N-acetylgalactosamine visualized perineuronal nets similar to those rich in anionic components. Glutamic acid decarboxylase (GAD)-immunoreactive synaptic boutons were shown to occupy numerous meshes of perineuronal VVA-positive nets. Electron microscopically, VVA binding sites were scattered throughout perisynaptic profiles, but accumulated at membranes and in the extracellular space except not in synaptic clefts. To investigate the spatial relationship between glial cell processes and perineuronal nets, two astrocytic markers (S100-protein and glutamine synthetase) were visualized at the light and electron microscopic level. Two methods to detect microglia by the use of Griffonia simplicifolia agglutinin (GSA I-B4) and the monoclonal antibody, OX-42, were also applied. Labelled structures forming perineuronal nets were observed with both astrocytic, but not with microglial, markers. It is concluded that perineuronal nets are composed of a specialized type of glia-associated extracellular matrix rich in polyanionic groups and N-acetylgalactosamine. The net-like appearance is due to perisynaptic arrangement of the astrocytic processes and these extracellular components. Similar to the ensheathment of nodes of Ranvier, perineuronal nets may provide a special ion buffering capacity required around various, perhaps highly active, types of neurons.

Acetylgalactosamine↗
PubMed1993

Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.

Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P < 0.001). Besides their location in the intercellular part of gliomas, HA and HN displayed a perivascular location in 1/3 astrocytomas, 17/24 glioblastomas, and 3/7 meningiomas, suggesting they could be produced also by the vascular stroma of tumours and that they would characterise the neoangiogenesis. All cultivated glioma cells tested produced HA in vitro, whereas only 1/11 cell lines produced HN, at a low level. The results obtained suggest that glioma HA and HN are produced by both cancer cells and vascular stroma cells, which contribute to the edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

Adolescent↗
PubMed1993

Caffeine decreases glial cell number and increases hyaluronan secretion in newborn rat brain cultures.

Newborn rat brain astrocytes (type 1 astrocytes, O-2A progenitor cells, and O-2A progenitor-derived cells, i.e. oligodendrocytes and type 2 astrocytes) were cultivated to investigate the effect of addition of caffeine to the culture medium on glial cell development and secretion of hyaluronan (hyaluronic acid, HA). HA is a glycosaminoglycan, secreted by type 1 astrocytes especially, which is a major component of the extracellular matrix of immature brain involved in morphogenesis and differentiation. Caffeine was added to the culture medium of primary glial cell cultures at concentrations of 102 microM (20 mg/L) or 255 microM (50 mg/L), considered therapeutic and toxic levels, respectively, in human newborns. HA was measured in the culture medium by immunoenzyme assay using sheep brain hyaluronectin, a glycoprotein that exhibits a strong affinity for HA, as probe. In primary glial cell cultures, 102 microM (20 mg/L) caffeine had no visible effect on cell number or on HA secretion. At 255 microM (50 mg/L), there was a significant reduction of cell number (i.e. type 1 astrocytes, O-2A progenitor cells, and progenitor-derived cells) and a significant increase of HA secretion per cell. These results suggest that caffeine at a high concentration in brain could have a prejudicial effect on the number of proliferating glial cells (astrocytes and oligodendrocytes) and on the composition of the extracellular matrix, which could affect myelination onset.

Animals↗
PubMed1993

Expression of the hyaluronan-binding glycoprotein hyaluronectin in leukemias.

Hyaluronectin (HN), a hyaluronan (hyaluronic acid, HA)-binding glycoprotein is normally expressed in the nervous system, found in the desmoplasia of tumours, and is also produced in vitro by peripheral blood mononuclear cells. We have therefore investigated the expression and the production of HN by leukemic cells, with the hypothesis that HN would be expressed in leukemias of the myeloid lineage. Fresh and frozen leukemic cells were studied from 70 patients of whom 53 had acute myeloblastic leukemia (AML). HN was strongly expressed (> 80% blood cells) in two out of 13 M4 AMLs and four out of four M5B AMLs. One further M4 AML displayed 25% positive cells and two 20% cell positivity cases were seen, in one case of M4 AML and in one case of chronic myelomonocytic leukemia (CMML). The rest of the cases of AML as well as all cases of acute lymphoblastic leukemia (ALL) showed almost no positivity (< 1%). The residual positive cells appeared to be normal blood promonocytes. Taken together > or = 20% positive cells was seen in eight out of 56 (14%) examined myeloid leukemias. The HN production was significantly higher (p < 0.0001) in cell culture media of M4 and M5 AML cells than in other AML or ALL cell culture media. A significant correlation was found (p < 0.0001) between the number of HN-positive leukemic cells and the number of cells with a monocytic morphology, suggesting that HN is a marker for the promonocyte.

Acute Disease↗
PubMed1993

111In-hyaluronectin, a new probe for radiodetection of tumours: biodistribution and imaging in grafted mice.

Hyaluronectin (HN), a glycoprotein which shows a strong specific affinity for Hyaluronic Acid (HA), a very high molecular weight glycosaminoglycan expressed in the stroma reaction of all tumours, was used to radiolocalize grafted autologous carcinomas (CB33 and CB03) in mice. After conjugation to DTPA (0.5-3.7 DTPA/HN) and radiolabelling with 111In (5 microCi/micrograms HN) HN retained 93% of its affinity for HA. Different preparations of HN purified from lamb brain were assayed. The best results were obtained when the HN molecules reactive with HA were selected by gel permeation to discard unreactive molecules which were unable to complex with HA. At 48 hours the tumour to blood ratio was 7.7 with a localization index of 2.2 in CB33; there was 2.8% uptake of the injected dose (ID) into CB33 and 1.6% into CB03. 111In-HN uptake per gram of tumour was tumour-size dependent: the smaller masses had the higher uptake. Tumours were visualized and either early images of 111In-HN distribution or simultaneous distribution images of 99mTc-phytate or 201Tl-DTPA were used for the subtraction treatments.

Animals↗
PubMed1992

Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.

Base Sequence↗
PubMed1992

Hyaluronan (hyaluronic acid) and hyaluronectin in the extracellular matrix of human breast carcinomas: comparison between invasive and non-invasive areas.

We performed quantitative determination of the distribution of hyaluronan (hyaluronic acid, HA) and the HA-binding protein, hyaluronectin (HN), 2 components of the extracellular matrix of tumor desmoplasia, within 71 human breast carcinomas. Results showed that HA and HN were more elevated in tumoral than in non-tumoral adjacent tissue, and that the peripheral invasive area of tumors contained increased levels of HA and HN as compared with the central non-invasive area (p less than 10(-3) and p less than 10(-5) respectively). HN and HA levels of 61 ductal carcinomas were related to the histological grade of tumors; no significant difference was found between grades for HA; HN was found to be significantly lower in grade III than in grade II tumors (p less than 0.01). HA and HN rates were correlated in grade I and grade II tumors and were not correlated in grade III. Mean percentage of HA saturation level by HN for whole tumors was found to be less than 4%, indicating that HA is essentially free of proteins and could be used as a target for cancer diagnosis or therapy.

Breast Neoplasms↗