Neonatal screening for sickle cell disease in France.

BACKGROUND: As a result of population growth in African-Caribbean regions of overseas France, and now immigration essentially from North and sub-Saharan Africa to mainland France, neonatal screening for sickle cell disease (SCD) has been performed in France since 1985 in Guadalupe and dependencies, as a universal test. After several pilot studies, screening was gradually extended to mainland France in 1996. Since 2000, the test has been performed at national level for all newborns defined as being "at risk" for SCD based on ethnic origin. METHODS: A dry blood sample is obtained by heel stick and analysed by isoelectric focusing as a first-line method, followed by either high-performance liquid chromatography or acid agar electrophoresis for confirmation, whenever a variant haemoglobin is observed on isoelectric focusing. RESULTS: In 2007, 28.45% of all newborns in mainland France were screened for SCD. Since 1996, a total of 3,890 newborns have been found to have SCD, and they have been followed up by reference paediatricians. CONCLUSION: Although screening for SCD at birth in France is not universal, it appears that missed babies are relatively infrequent. Despite obvious sociological problems inherent to the at-risk population, the follow-up of SCD babies is rather successful. Due to the birth prevalence of SCD in France, especially in comparison with other common genetic diseases, screening all newborns regardless of ethnic origin is an issue that is being addressed.

Influence of culture conditions on the alpha 1,2-fucosyltransferase and MUC gene expression of a transformed cell line MM-39 derived from human tracheal gland cells.

Human tracheal glands cells (HTGC) in culture are able to respond to adrenergic, cholinergic and purinergic agonists by increasing their serous and mucin secretions. These secretagogues are also able to maintain an optimal responsiveness of serous cells to stimulation when they are regularly and briefly delivered to the cells, making the HTGC a suitable model to study the serous secretion (Merten, in press). Our interest has been focused on the effects of cholinergic and purinergic secretagogues associated to histamine, on the mucous function of the transformed human tracheal gland cell line MM-39, which has a mixed, both serous and mucous, phenotype. When the cells were exposed to short stimulation every 2 days for 3 weeks with 10 or 100 microM carbachol, UTP and histamine, modifications of their mucous phenotype were observed. The expression of MUC genes appeared dependent on the culture conditions. Transcripts of MUC1, MUC4, and MUC5B genes were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 10 microM, in contrast to the unstimulated expression of MUC1 and MUC4 in control cells. MUC1, MUC4, MUC7, MUC6 and MUC11 transcripts were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 100 microM. These culture conditions were also able to induce an alpha 1,2-fucosyltransferase activity absent in the MM-39 cells cultivated with standard conditions. There was no marked effect on the alpha 2,3-sialyltransferase activity although the expression pattern of the sialyltransferase genes was reduced to the unique presence of ST3Gal III. In conclusion, MM-39 cells exposed to repeated stimulation by secretagogues at different concentrations express different sero-mucous phenotypes.

Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis.

Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity. Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7). The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines. Human airway mucins are highly glycosylated (70-80% per weight). They contain from one single to several hundred carbohydrate chains. The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules. Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars). The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases. The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin. There is growing evidence that the apomucin sequences influence the first glycosylation reactions. The elongation of the chains leads to various linear or branched extensions. Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants. The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases. Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa. Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease. However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected. Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection. These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNFalpha. In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells.

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNFalpha. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNFalpha; a 52% increase of alpha2,3-sialyltransferase activity was also observed in TNFalpha-stimulated MM-39 cells. After metabolic radio-labelling with [(3)H]glucosamine and [(3)H]fucose, the mucins released in the culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39-1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNFalpha was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the alpha2,3-sialyltransferase activity by TNFalpha argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNFalpha. In conclusion, the influence of TNFalpha on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Characterization of a sulfotransferase from human airways responsible for the 3-O-sulfation of terminal galactose in N-acetyllactosamine-containing mucin carbohydrate chains.

A galactose 3-O-sulfotransferase activity able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate to methyl galactosides or terminal N-acetyllactosamine-containing carbohydrate chains from human respiratory mucins was characterized in microsomal fractions prepared from human respiratory mucosa. The reaction products, methyl alpha- or beta-galactose 3-sulfate and three oligosaccharide alditols containing the sequence HSO3-3Gal beta 1-4GlcNAc beta 1-6GalNAc-itol were identified by high performance anion-exchange chromatography. Using methyl beta-galactoside as a substrate, the optimum activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, and 10 mM AMP in a 30 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1. The apparent Km for methyl beta-galactoside and for adenosine 3'-phosphate 5'-phosphosulfate were observed at 0.69 x 10(-3) M and at 4 x 10(-6) M respectively. This sulfotransferase is different from that responsible for sulfatide synthesis.

Mucous and serous secretions of human bronchial epithelial cells in secondary culture.

Human bronchial surface epithelial cells were maintained in secondary culture on a collagen gel substrate in a defined, serum-free medium. These conditions have previously been reported to promote mucous cell differentiation. After 3 wk in culture, approximately 40% of the cells were stained by an antibody directed against human respiratory mucin. Analysis of media from cells cultured in the presence of the radioactive precursors [3H]glucosamine and [35S]sulfate revealed that the cells secreted high molecular weight glycoproteins with properties of typical respiratory mucins. In addition, hyaluronic acid and proteoglycans containing chondroitin sulfate and/or heparan sulfate glycosaminoglycans were identified in cell conditioned media. Finally, Western blot analyses showed that the cells secreted lysozyme and mucous proteinase inhibitor, proteins that are generally considered to be markers for submucosal gland serous cells. These results show that human bronchial cells from the surface epithelium in secondary culture secreted a range of glycoconjugates and proteins that were typical secretory products of both mucous and serous cells.

Differential apomucin expression in normal and neoplastic human gastrointestinal tissues.

BACKGROUND/AIMS: The cloning of genes encoding human mucins is the basis for the study of their normal tissue distribution and the alterations associated with cancer. The aim of this study was to determine the normal and tumor tissue expression of MUC1, MUC2, MUC5B, and MUC5C. METHODS: The reactivity of apomucin-specific antibodies with fresh normal and tumor tissues was analyzed using immunohistochemical techniques. RESULTS: Anti-MUC1 antibodies reacted with most glandular epithelia. Anti-MUC2 antibody was mainly reactive with intestinal goblet cells and cervical mucous cells. Anti-MUC5B was reactive with a wide range of epithelial tissues whereas anti-MUC5C was reactive with stomach, trachea, and endocervix. Double-labeling experiments showed coexpression of MUC1/MUC2 and MUC2/MUC5C in colonic tissue. Multiple apomucins were detected in colon cancers, but no relationship to histochemical mucus stains was observed. CONCLUSIONS: It is concluded that (1) each apomucin shows a distinct tissue expression pattern; (2) multiple apomucins are present in a single tissue and at the single cell level; and (3) altered apomucin expression takes place in pathological colonic tissue.

Human respiratory mucins.

Human respiratory mucins are secreted by goblet cells and mucous glands of the respiratory mucosa. They consist of a broad family of complex glycoproteins with different peptides, or apomucins, corresponding to several genes located on at least three different chromosomes. Glycosylation, the major posttranslational phenomenon, is responsible for about 70-80% of the weight of mucins: it produces an extraordinary diversity of O-glycosidically linked carbohydrate chains which are expressed as several hundreds of different chains in the mucins of a single individual. The variety of mucin peptides and the diversity of carbohydrate chains probably allows many interactions, especially with microorganisms: this may be an essential factor in the defence of the underlying respiratory mucosa.

Structural features of the core proteins of human airway mucins ascertained by cDNA cloning.

Tracheobronchial secretions are one of the most important elements of the mucociliary system that protects the respiratory mucosa. They contain bronchial mucus, which is composed of a group of macromolecules secreted by the goblet cells of the epithelium and the submucosal glands. Bronchial mucins are the most characteristic molecules of this mucus. They form a group of complex, polydispersed O-linked glycoproteins containing sugars, which make up 80% of their weight. The protein core of human airway mucin has been difficult to sequence by traditional technologies because of its high content of serine and threonine residues linked to numerous oligosaccharide chains. We therefore prepared a lambda gt11 cDNA library from one sample of human tracheobronchial mucosa and screened this library with a polyclonal antibody directed against the apopeptides of human bronchial mucins. We obtained 20 positive clones that were sequenced. These sequences were classified into three different types. The use of the nucleotide probes from these clones in Northern blot analysis showed that the RNA messages were extremely polydispersed. At the current time, four of these probes allow us to map human tracheobronchial mucins genes to at least three different chromosomes. These results suggest that the peptide moiety of the human airway mucin is very heterogeneous.

Multiple apomucin translation products from human respiratory mucosa mRNA.

Poly(A)-rich RNA was purified from a pool of five human tracheobronchial mucosa. After in vitro translation in a reticulocyte lysate and immunoprecipitation of the translated products, using either a polyclonal antiserum or a monoclonal antibody to deglycosylated respiratory mucin peptides, the products were characterized by SDS/PAGE. The respiratory mucin precursors migrated as a very large smear from almost the top of the resolving polyacrylamide gel to an area corresponding to a molecular mass of about 100 kDa. After hybridization with mucin cDNA probe TH 29 described by Crepin et al. [Crepin, M., Porchet, N., Aubert, J. P. & Degand, P. (1990) Biorheology 27, 471-484] respiratory mucin mRNAs also appeared polydisperse. Although degradation or incomplete translation of high-molecular-mass mRNA cannot be entirely ruled out, these results suggest that human respiratory apomucins consist of a family of peptides which share some common epitopes. This possibility is in agreement with (a) the diversity of mucin precursors observed previously with pulse/chase experiments performed with explants of human respiratory mucosa and (b) the polydispersity of secreted respiratory mucins observed by electron microscopy.

Evidence for the in vivo degradation of human respiratory mucins during Pseudomonas aeruginosa infection.

The comparison of distribution of glycopeptides of sputa from patients suffering from various chronic hypersecretions has already shown an increased acidity with a decreased proportion of neutral glycopeptides in the respiratory secretions of patients suffering from cystic fibrosis, as compared to those of patients with chronic bronchitis. In order to find out whether this decrease is specific to cystic fibrosis mucins or whether it is due to a degradation of mucus by Pseudomonas aeruginosa, which infects most of the sputa from patients with this disease, mucus glycopeptides from patients with different chronic bronchial disorders, infected by Pseudomonas or not, were prepared and fractionated by ion-exchange chromatography. The neutral fraction, which has never been studied in detail, was gel-filtered, and provided two fractions, one containing true mucin glycopeptides and the other containing a mixture of peptides and glycopeptides with a lower molecular mass. In the Pseudomonas-infected samples, the true mucin glycopeptide fraction was greatly diminished as compared to this same fraction in non-Pseudomonas-infected samples; this was not specific to cystic fibrosis secretions. In contrast, the glycopeptide fraction with a lower molecular mass was greatly increased in all the Pseudomonas-infected samples. Polyacrylamide gel electrophoresis of this second fraction showed unique glycopeptide bands between 40-50 kDa in the Pseudomonas-infected samples, regardless of the origin of the samples. These bands were revealed by an antibody directed against whole cystic fibrosis mucin. Infected chronic bronchitis sputa and cystic fibrosis samples without P. aeruginosa did not show these bands. These studies therefore suggest that there are P. aeruginosa-associated changes in mucins which may result from degradation of mucins.

Use of an antiserum against deglycosylated human mucins for cellular localization of their peptide precursors: antigenic similarities between bronchial and intestinal mucins.

Highly glycosylated regions of mucins, or glycopeptides, were obtained by proteolysis of human bronchial mucins. They were deglycosylated by treatment with a trifluoromethane sulfonic acid/anisole mixture and subsequent solvolysis with anhydrous liquid hydrogen fluoride. The resulting peptides were then used to raise an immune serum in rabbit. This immune serum was used to localize the peptide precursors of human respiratory mucins within bronchial cells, using an immunohistochemical method. Two main patterns of labeling were observed in the goblet cells: the entire cytoplasm of some goblet cells was immunoreactive, whereas in other cells the labeling was concentrated around the nucleus. In the respiratory mucous glands, the labeling was localized around or below the nucleus. The serous cells were not stained. Similar labeling was observed in human colon goblet cells. This immune serum seems to be specific for mucin-secreting cells and has a strong affinity for the perinuclear region of these cells.

Identification of human tracheo-bronchial mucin precursors.

Bronchial mucin peptide chains were obtained by performing a two-step chemical deglycosylation of the highly glycosylated regions (or glycopeptides) which are the most characteristic part of bronchial mucins. The deglycosylated preparation was used to prepare an antiserum directed against mucin peptide epitopes. This antiserum reacted with the area containing rough endoplasmic reticulum of goblet cells and of mucous gland of human bronchial mucosa but not with secretory vesicles. The antiserum was used for immunoprecipitation of radiolabelled mucin precursors in pulse-chase experiments with explants of human bronchial mucosa. SDS-polyacrylamide gel electrophoresis followed by fluorography revealed precursors with a molecular mass in the range of 200 to 400 kDa as early as after 10 min pulse labeling with [3H]threonine. These results suggest that the mucin polydispersity previously visualized by electron microscopy may be explained by the synthesis of several respiratory mucin peptide precursors with different molecular sizes and/or that precursors with different amounts of carbohydrate are rapidly formed.

The complexity of mucins.

Mucins represent the main components of gel-like secretions, or mucus, secreted by mucosae or some exocrine glands. These high-molecular-weight glycoproteins are characterized by the large number of carbohydrate chains O-glycosidically linked to the peptide. The determination of mucin molecular weight and conformation has been controversial for several reasons: 1) the methods used to solubilize mucus and to purify mucins are different and 2) the molecules have a strong tendency to aggregate or to bind to other molecules (peptides or lipids). Recently, electron microscopy has shown the filamentous shape of most mucins and their polydisperse character which, in some secretions, might correspond to a polymorphism of the peptide part of these molecules. The recent development of high pressure liquid chromatography and high-resolution proton NMR spectroscopy has allowed major progress in the structural study of mucin carbohydrate chains. These chains may have from 1 to about 20 sugars and bear different antigenic determinants, such as A, B, H, I, i, X, Y or Cad antigens. In some mucins, such as human respiratory mucins, the carbohydrate chain diversity is remarkable, which raises many questions. Mucins are molecules located at the interface between mucosae and the external environment. The carbohydrate chain diversity might allow many interactions between mucins and microorganisms and play a major role in the colonization or the defense of mucosae.

Evaluation by immunoblotting of the criteria for tubular lesions diagnostic with urinary SDS-PAGE.

Immunoblotting methods have allowed the identification of 14 urinary proteins. From this study, it appears that some low-relative-molecular mass proteins are derived from disrupted high-relative-molecular mass proteins. The only reliable bands for the diagnosis of tubular lesions, on the polyacrylamide gel stained with Coomassie Blue, are retinol-binding-protein and beta 2-microglobulin. alpha 1-microglobulin can overlap with breakdown products of albumin. The visualization of Ig light chains and lysozyme is rather poor after Coomassie Blue stainings and the accurate identification of these bands may be only improved by the use of immunoblotting.

Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments.

Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.

Action of trifluoromethanesulfonic acid on highly glycosylated regions of human bronchial mucins.

Highly glycosylated glycopeptides were prepared from human bronchial mucus. They were heterogeneous and contained an average of 45 residues of glycosylated hydroxyamino acid per 100 amino acid residues. The kinetics of deglycosylation of these glycopeptides by trifluoromethanesulfonic acid-anisole mixtures at 25 degrees was monitored by chemical analysis and by polyacrylamide gel electrophoresis. The peripheral sugars were almost completely cleaved in 45 min with 3:2 and 2:1 CF3SO3H-anisole. A maximum of 75% of the O-linked N-acetylgalactosamine residues were released and mixtures of glycopeptides and peptides were obtained. Increasing the reaction time caused peptide bond cleavage. Rather mild conditions (1.2:1 CF3SO3H-anisole at 25 degrees for 90 min) gave limited deglycosylation of highly glycosylated bronchial glycopeptides, allowing the uncovering of GalNAc-peptide linkages and peptide regions able to induce the formation of specific antibodies in the rabbit.

Reducing the artifacts produced by impure antisera in immunoblots of low-molecular-mass proteins in urine.

Immunoblots of several urinary low-molecular-mass proteins can be very useful in investigations of pathological proteinuria. However, use of certain commercial antisera in such procedures leads to artifacts corresponding to nonspecific bands; e.g., immunoglobulins from nonimmunized rabbit serum may bind to human urinary proteins, and this binding is not inhibited by Triton X-100. We have developed a procedure to improve the specificity of detection of urinary low-Mr proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by immunoblotting with commercial antisera: we treat the protein blot with a mixture of mercaptoethanol and sodium dodecyl sulfate before incubation with the first antiserum. This allows direct use of commercial antisera without prior absorption of contaminating antibodies.