Guidelines of the French Society of Otorhinolaryngology-Head and Neck Surgery (SFORL) and the French Society of Audiology (SFA) for Speech-in-Noise Testing in Adults.

OBJECTIVES: This document presents the fundamentals of speech audiometry in noise, general requirements for implementation and criteria for choice among the tests available in French according to the health-professional's needs. MATERIAL AND METHODS: The recommendations are based on a systematic analysis of the literature carried out by a multidisciplinary group of doctors, audiologists and audioprosthetists from all over France. They are graded A, B, C or expert opinion according to decreasing level of scientific evidence. RESULTS: Eight tests of speech audiometry in noise can be used in France. CONCLUSION: To be complete, evaluation of hearing status requires testing understanding of speech in noise. The examination must begin with a minimum of two measurements familiarizing the subject with the test procedure. For initial diagnosis, adaptive procedures establishing the 50% speech reception threshold (SRT50) in noise are to be preferred in order to obtain a rapid and standardized measurement of perception of speech in noise. When the aim is to measure real-life speech comprehension, tests based on sentences, cocktail-party noise and free-field stimulation are to be preferred. Prosthetic gain is evaluated exclusively in free field. This is the only way to evaluate the contribution of binaurality and to measure perception in noise in an environment as close as possible to real life. In order to avoid acoustic interference in free field, at least five loudspeakers should be used, in particular for evaluating the effectiveness of directional microphones, CROS devices enabling sounds picked up in the damaged ear to be rerouted to the functional ear, or bimodal fitting (i.e., when hearing is enabled by two modalities: for example, hearing aid for one ear, cochlear implant for the other).

The SK3/K(Ca)2.3 potassium channel is a new cellular target for edelfosine.

BACKGROUND AND PURPOSE: The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function. EXPERIMENTAL APPROACH: Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and ¹²5I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca²(+) -activated K(+) channel, SK3/K(Ca)2.3. KEY RESULTS: Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/K(Ca)2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit ¹²5I-Apamin binding to this SK(Ca) channel; rather, it reduced the calcium sensitivity of SK3/K(Ca)2.3 channel and dramatically decreased intracellular Ca²(+) concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SK(Ca) channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/K(Ca)2.3 protein in a SK3/K(Ca)2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine. CONCLUSIONS AND IMPLICATIONS: Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/K(Ca)2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.

Mucosal or systemic administration of rE2 glycoprotein antigen loaded PLGA microspheres.

We have evaluated the ability of recombinant E2 antigen, as a surfactant free formulation of poly (D,L-lactide-co-glycolide) (PLGA) microspheres, to elicit a systemic immune response after administration by mucosal routes (oral and nasal) in comparison to intramuscular route. The sequence encoding a truncated E2 glycoprotein of the classical swine fever virus (CSFV) was expressed in insect cells following infection with recombinant baculovirus, as a His-tagged recombinant antigen. The recombinant E2 glycoprotein (rE2) antigen was co-encapsulated with rabbit serum albumin (RSA) as a protein stabilizer. rE2/RSA loaded PLGA microspheres, with a mean diameter of 4 microm were obtained by a water in oil in water solvent extraction method (w/o/w). Rabbits were immunized with 10 microg of rE2 formulated in PLGA microspheres administrated by three different routes (oral, nasal and intramuscular). After 60 days, each rabbit in all three groups was challenge with 5 microg of rE2 glycoprotein solution by intradermal administration. Blood samples were collected weekly for 90 days and specific rE2 antigen antibodies measured. This work showed that rE2 antigen loaded microspheres was able to initiate an immune response. The intradermal challenge after nasal and oral administration had a clear boost effect on the systemic immune response. Moreover, the response after nasal administration was more intense and less variable than oral route. In conclusion, these data demonstrate a high potential of rE2 loaded PLGA microspheres for their use as a mucosal subunit vaccine.

Classical swine fever (CSF) marker vaccine. Trial I. Challenge studies in weaner pigs.

Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0-20%) than directly challenged pigs, probably mirroring the delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests.

Protein gamma-radiolysis in frozen solutions is a macromolecular surface phenomenon: fragmentation of lysozyme, citrate synthase and alpha-lactalbumin in native or denatured states.

PURPOSE: To test whether radiolysis-induced fragmentation in frozen aqueous protein solution is dependent on solvent access to the surface of the protein or to the molecular mass of the polypeptide chain. MATERIALS AND METHODS: 60Co gamma-irradiation of three proteins at -78 degrees C: lysozyme, citrate synthase and alpha-lactalbumin in their native state, with or without bound substrate, or denatured (random coil in urea/acid-denatured state). RESULTS: By SDS-polyacrylamide gel electrophoresis/analysis of the protein-fragmentation process, it was found that for a given protein D37 values (dose to decrease the measured amount of protein, with an unaltered polypeptidic chain, to 37% of the initial amount) varied according to the state of the protein. D37 for denatured proteins was always much smaller than for native states, indicating a greater susceptibility to fragmentation. In urea, contrary to the native state, no well-defined fragments were observed. Radiolysis decay constants (K= 1/D37) increased with solvent-accessible surface area of these proteins estimated from their radii of gyration in the various states. This is shown also in previous data on native or SDS-denatured proteins. Denatured proteins which have a large surface area exposed to the solvent compared with native ones are more fragmented at equal doses. CONCLUSIONS: It is concluded that D37 is directly related to the exposed surface area and not to the molecular mass of the polypeptide chain.

Renal synthesis of leukaemia inhibitory factor (LIF), under normal and inflammatory conditions.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine that is particularly involved in nephrogenesis and repair of the extracellular matrix. Transgenic mice overexpressing LIF have mesangial proliferative glomerulonephritis. Also, during local inflammatory reactions, such as kidney graft rejection or urinary tract infections, urinary LIF excretion is enhanced. The aim of the study therefore was to study LIF production by normal and inflammatory diseased kidneys (glomerulonephritis or graft rejection), maintained in short cultures. To determine the responsibility of the kidney itself in LIF synthesis, we measured LIF secretion into the culture supernatants of human mesangial or renal tubular epithelial cells. Fragments from diseased kidneys, whether grafts or not, released more LIF than normal human kidney fragments, mesangial or renal tubular epithelial cells. However, LIF production was delayed in renal transplants compared to glomerulonephritic samples taken from untreated patients. In every case, LIF production was enhanced by interleukin 1beta (IL-1beta) and inhibited by IL-4 or dexamethasone, except in two severe rejection episodes. So, LIF appeared to respond to pro- and anti-inflammatory stimuli, in vitro and in vivo. Considering its biological effects, LIF could play a role in inflammatory renal diseases.

Cadmium nephrotoxicity assessed in isolated rat glomeruli and cultured mesangial cells: evidence for contraction of glomerular cells.

Cadmium (Cd), an important pollutant, causes severe damage at the renal tubular level. Numerous previous studies have focused upon Cd tubular nephrotoxicity. The present study of Cd-induced glomerular damage examined the vasoactive effect of Cd in freshly isolated glomeruli and mesangial cells. Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by culture for outgrowth of cells. Quantitative evaluation of glomerular and cellular contractions was performed by morphometric measurement of the area with an automatized image analyzer following different incubation times with Hanks' balanced salt solution or Cd2+. Each glomerulus or mesangial cell served as its own control. Cd lethality was measured with microassay methods (neutral red, MTT uptake, and lactate dehydrogenase release), allowing the determination of an IC50. This ranged from 35 to 60 microM. CdCl2 induced a time-dependent contractile effect on isolated glomeruli; planar surface area decreases were 6.9% (1 microM), 7.5% (0.1 microM), and 7% (0.01 microM). The decrease started as soon as Cd was in contact with glomeruli and ended 40 min later: T5 (2%), T10 (3.5%), T20 (4. 2%), T30 (6.3%), T40 (7%). Cell size reduction was 19% (1 microM), 14% (0.1 microM), and 18% (0.01 microM) and was also time-dependent. To confirm that contractile events occurred during the cell shape changes, examination of the mesangial alpha-actin network was performed concurrently. These results indicate that Cd contracts glomerular structures. This may, in part, explain the reduction in glomerular filtration seen in Cd nephrotoxicity.

Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-alpha-glutamate carboxypeptidase/prostate-specific membrane antigen.

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.

Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D.

A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.

Bacterial contig map of the 21q11 region associated with Alzheimer's disease and abnormal myelopoiesis in Down syndrome.

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.

Effective priming of neonates born to immune dams against the immunogenic pseudorabies virus glycoprotein gD by replication-incompetent adenovirus-mediated gene transfer at birth.

One of the main limitations of the vaccination of neonates from vaccinated or infected mothers is the interference by inherited maternal antibodies, which are known to inhibit the immune response against both live and inactivated vaccines. The efficiency of bypassing this inhibition by the transfer of an immunogenic glycoprotein gene, the gD gene of pseudorabies virus (PRV), into neonates was explored. The experiments were conducted in 1-day-old piglets, which are immunocompetent at birth. The same transcription unit (gD of PRV under the control of the adenovirus major late promoter) was delivered intramuscularly at birth either in the form of naked DNA or cloned in the genome of a replication-defective adenovirus. A booster injection of a conventional live PRV vaccine strain was given at 10 weeks of age, the replication of which was greatly restricted by the residual amounts of colostral antibodies in control animals. Piglets were challenged at the age of 16 weeks with a virulent PRV strain. The replication-defective adenovirus was able to efficiently prime piglets born to immune dams against gD in such a way that inoculation with the Bartha strain protected them against a subsequent challenge with the same level of efficacy in piglets born to naive or immune dams. In contrast, piglets born to immune dams into which the gD gene was not transferred, or transferred as naked DNA at birth, were not protected. These results open the way for early immunization of neonates born to vaccinated or infected mothers.

Characterization of the chicken telokin heterogeneity by time-of-flight mass spectrometry.

Chicken gizzard telokin was purified to apparent homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This preparation yielded upon mass spectrometry analysis seven mass peaks spanning from 15 858 to 17 100 Da. Anion exchange-high performance liquid chromatography of the purified telokin revealed a high diversity of telokin molecules. By combining protein chemistry to chromatography and mass spectrometry, the telokin heterogeneity was analyzed. Three acetylated N-termini were found, AMI, MIS, and SGR. Cyanogen bromide cleavage of telokin yielded six different C-terminal peptides corresponding to the removal of one to six C-terminal glutamyl residues from the protein sequence deduced from the cDNA. Phosphorylation of telokin was detected, thus increasing the heterogeneity of the telokin preparation. In addition, peptide sequencing has shown that telokin contained either an aspartyl or a glutamyl residue at position 27, probably resulting from chicken polymorphism.

Endogenous basic fibroblast growth factor isoforms involved in different intracellular protein complexes.

Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins.

Radiation-inactivation analysis of the oligomeric structure of the renal sodium/D-glucose symporter.

The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/D-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294 +/- 40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84 +/- 25 kDa in the presence of a glucose gradient, and 92 +/- 20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225 +/- 49 kDa. The 294 kDa result supports earlier suggestions that the Na+ gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.

Study of the delivery of the gD gene of pseudorabies virus to one-day-old piglets by adenovirus or plasmid DNA as ways to by-pass the inhibition of immune response by colostral antibodies.

In the present study, it was shown that piglets with maternal antibodies, which had been primed with a replication-defective adenovirus that expresses the pseudorabies virus (PRV) glycoprotein gD and boosted with the Bartha vaccine strain at 10 weeks of age are equally protected clinically upon a challenge as piglets without maternal antibodies vaccinated with the same approach or with the Bartha vaccine strain alone. Priming with a plasmid that expresses gD was less efficient.

Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis.

Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Echogenic dilated bowel loops before 21 weeks' gestation: a new entity.

OBJECTIVE: The purpose of this study was to determine the outcome at birth for fetuses in which echogenic dilated bowel loops (EDBL) are detected before 21 weeks of gestation. MATERIALS AND METHODS: A retrospective study was undertaken of 45 patients with EDBL. Sonographic criteria included dilated bowel with an echogenic wall. EDBL was divided into two categories: the isolated and the complex (associated with other abnormalities). When the EDBL affected only one quadrant of the fetal abdomen, it was referred to as the local form, and when it was observed in more than one quadrant, it was termed the diffuse form. RESULTS: Intestinal dilatation of 2-8 mm was revealed on sonography in 21 fetuses with the isolated form of EDBL. Follow-up sonography showed resolution of EDBL in 20 cases. The outcome at birth was normal in 19 (90%) cases. The 20th fetus was born with jejunal atresia, and the remaining fetus died after cordocentesis. Twenty-four fetuses with the complex form of EDBL also had intestinal dilatation of 2-8 mm shown on sonography. Nine (38%) of these fetuses were born alive: six had gastroschisis; two had meconium peritonitis; and one had vertebral defects, imperforate anus, tracheoesophageal fistula, and radial and renal dysplasia (VATER). In these cases, resolution of EDBL was noticed at sonographic follow-up. Fourteen (58%) of the 24 fetuses were aborted. The remaining fetus died after amniocentesis. The incidence of normalcy is comparable in both local and diffuse dilatation. Amniocentesis was performed in 15 cases. Disaccharidase activity was measured in 13 patients. Low disaccharidase activity was detected in 10 (77%) of 13 cases. We saw cystic fibrosis in none of the 45 fetuses. Infection was detected before and at birth in five (11%) cases. CONCLUSION: EDBL is a new entity that is most likely related to temporary obstruction. A fetus with the isolated form has a good prognosis, whereas a fetus with the complex form has an outcome directly related to the severity of associated abnormalities.

Nitric oxide (NO) donor 3-morpholinosydnonimine antagonizes cyclosporin A-induced contraction in two in vitro glomerular models.

Cyclosporin A induces in vivo a severe nephrotoxicity characterized by a large decrease in renal hemodynamics. The aim of this study is to establish the ability of the known NO donor 3-morpholinosydnonimine (SIN-1) to prevent the cyclosporin A-induced contraction by using rat isolated glomeruli and cultured glomerular mesangial cells. Isolated rat glomeruli are obtained from the renal superficial cortex by a sieving method. Mesangial cells are cultured in RPMI 1640 with 15% fetal calf serum. The planar surface area (PSA) of either isolated glomeruli or mesangial cells is assessed using an image analyzer. Each glomerulus or mesangial cell serves as its own control through calculation of the area before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or in control solution with cyclosporin A alone or cyclosporin A and SIN-1. Cyclosporin A (10(-6) mol/L) induces an important time-dependent contraction of either glomerulus or mesangial cell. When pretreated with different concentrations of SIN-1 (10(-4) to 10(-9) mol/L), only a slight size decrease is noted. In conclusion, a direct constrictive effect of cyclosporin A in isolated glomeruli and mesangial cells can be prevented by the NO donor SIN-1, suggesting an important involvement of the nitric oxide pathway in the cyclosporin A-induced nephrotoxicity.

Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of beta-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate.

N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.