[Non-specific interstitial pneumonia : a rare clinical entity in adolescents]. / Pneumopathie interstitielle non spécifique : une entité rare chez l'adolescent.

Non-specific Interstitial Pneumonia (NSIP) is an anatomo-clinical entity within the group of Diffuse Infiltrative Pulmonary Diseases (DPID). It is very rarely found in pediatrics. Main symptoms are dry cough and dyspnea. Bronchoalveolar lavage and biology are non specific. The thoracic CT scan suspects the diagnosis, but histological examination of a lung biopsy remains the reference examination and makes the diagnosis highly probable according to the ATS / ERS criteria. An autoimmune assessment should be performed because NSIPs are often associated with connective tissue disease or may even be the first sign of connectivitive tissues diseases. The treatment of the acute phase is mainly based on the administration of corticosteroids and the prognosis is generally good. In this article, we describe the management of NSIP, based on a pediatric clinical case.

La Pneumopathie Interstitielle Non Spécifique (PINS) est une entité anatomo-clinique au sein du groupe des Pneumopathies Infiltrantes Diffuses (PID). Elle est très rarement retrouvée en pédiatrie. Elle se manifeste principalement par une toux sèche et une dyspnée. Le lavage broncho-alvéolaire et la biologie sont aspécifiques. Le scanner thoracique permet de suspecter le diagnostic, mais c'est l'examen histologique d'une biopsie pulmonaire qui reste l'examen de référence et qui permet, selon les critères de l'ATS/ERS, de poser un diagnostic avec grande probabilité. Un bilan auto-immun doit être réalisé car la PINS est très souvent associée à des connectivites ou peut même en être le premier signe. Le traitement de la phase aiguë repose, essentiellement, sur l'administration de corticoïdes, et le pronostic est, en général, bon. Dans cet article, nous décrivons la prise en charge d'une PINS, à partir d'un cas clinique rencontré en pédiatrie.

Higher doses of opioids in patients who need palliative sedation prior to death: cause or consequence?

BACKGROUND: Palliative sedation (PS) is necessary in a significant percentage of patients dying on an acute palliative care unit (PCU). Common indications are terminal restlessness, pain and dyspnoea. On our PCU, terminal restlessness was the main indication for PS but pain was the most prevalent symptom during admission. Because delirium is often drug induced in terminal cancer patients and opioids are amongst the most frequently implicated drugs, we hypothesised that the underlying pain problem and its treatment might have been related to the need for sedation. PATIENTS AND METHODS: To test this hypothesis, we did a retrospective analysis on the use of medication with potential cognitive side-effects, focusing on analgesics, in 68 patients who died on the PCU after PS and 89 patients who died without PS. RESULTS: Ultimately sedated patients used opioids in significantly higher doses; they were more often treated with a rotation to another opioid and with amitriptyline. The dose of opioids used at various time points between admission and death was strongly related to the probability of PS. CONCLUSIONS: Our findings support the hypothesis that, although pain was not the main indication for PS, pain and its treatment might have been primarily related to the need for palliative sedation in this patient cohort.

Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy.

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.

Phase II study of weekly gemcitabine in patients with metastatic breast cancer relapsing or failing both an anthracycline and a taxane.

A phase II study was performed to investigate the efficacy and tolerability of gemcitabine as third-line chemotherapy for patients with metastatic breast cancer, previously treated with both an anthracycline- and taxane-containing regimen. Twenty-three patients were treated with gemcitabine 1200 mg/m2 in a 30-min infusion on day 1, 8 and 15 of a 28 day cycle. Seventy-four percent of the patients had visceral metastases. No complete or partial responses were observed. Six patients (26%) had stable disease with a median duration of 4.0 months. The median time to progression was 1.9 months and the median survival time was 7.8 months. Neutropenia grade 3 and 4 was observed in four patients (18%). Non-hematological toxicity grade 3 included nausea and vomiting in 14%, skin toxicity in 9% and elevation of transaminases in 23% of the patients. Gemcitabine is ineffective as third-line single agent therapy in patients failing anthracycline and taxane treatment for metastatic breast cancer.

Phase I and pharmacological study of weekly administration of the polyamine synthesis inhibitor SAM 486A (CGP 48 664) in patients with solid tumors. European Organization for Research and Treatment of Cancer Early Clinical Studies Group.

A single-agent dose-escalating Phase I and pharmacological study of the polyamine synthesis inhibitor SAM 486A was performed. A dosing regimen of four weekly infusions followed by 2 weeks off therapy was studied. Fifty patients were entered into the study. Dose levels studied were 1.25, 2.5, 5, 8, 16, 32, 48, 70, 110, 170, 270, and 325 mg/m2/week. Pharmacokinetic sampling was done on day 1, and trough samples were taken weekly during the first treatment cycle. Pharmacodynamic sampling was done on days 1 and 22. At 325 mg/m2/week, dose-limiting toxicity was seen (one patient each with grade 4 febrile neutropenia, grade 3 neurotoxicity, and grade 3 hypotension with syncope and T-wave inversions on electrocardiogram). The recommended dose for further testing was set at 270 mg/m2/week. Infusion time was increased from 10 to 180 min due to facial paresthesias and flushing and somnolence. Drug exposure increased linearly with dose. Mean +/- SD t1,2 at 70-325 mg/m2 doses was 61.4+/-26.2 h, with a large volume of distribution at steady state. In peripheral blood leukocytes, a clear relationship between dose and inhibitory effect on S-adenosylmethionine decarboxylase or changes in intracellular polyamine pools was not recorded. SAM 486A can be administered safely using a dosing regimen of four weekly infusions followed by 2 weeks off therapy. The recommended dose for Phase II studies using this regimen is 270 mg/m2/week.

Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

Glycosylation beyond the Asn78-linked GlcNAc residue has a significant enhancing effect on the stability of the alpha subunit of human chorionic gonadotropin.

The effects of glycosylation beyond the Asn-linked GlcNAc residues on the stability of the alpha subunit of human chorionic gonadotropin are investigated, using enzymatic deglycosylation and NMR spectroscopy. Comparison of thermal denaturation profiles of both the intact alpha subunit and the alpha subunit carrying only GlcNAc monomers at both Asn52 and Asn78 established a small but significant decrease in thermal stability of the deglycosylated form. Since there is no secondary structure around Asn52 in the free subunit these results demonstrate that glycosylation beyond the Asn78-linked GlcNAc residue enhances the thermal stability of the alpha subunit of hCG. This feature has implications for understanding the effect of glycosylation on protein stabilization in general.

NMR studies of the free alpha subunit of human chorionic gonadotropin. Structural influences of N-glycosylation and the beta subunit on the conformation of the alpha subunit.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Asn52 of its alpha subunit (alpha hCG) is essential for signal transduction, whereas the N-glycan at Asn78 stabilizes the structure of the protein. In this study, an almost complete 1H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of alpha hCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of alpha hCG is solution, which was determined based on NOE data, is partially similar to that of the alpha subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33-58. In the crystal structure of hCG, residues 33-37 and 54-58 of the alpha subunit are part of an intersubunit seven-stranded beta-barrel and residues 41-47 constitute a 3(10)-helix. In contrast, in free alpha hCG in solution, amino acids 33-58 are part of a large disordered loop, indicating that in intact hCG interactions with the beta subunit of hCG stabilize the conformation of the alpha subunit. The NMR data of alpha hCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 1H-NOEs between the GlcNAc-1 residue at Asn78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the alpha subunit. 1H-NOEs across the glycosidic linkages of the glycan, resonance-line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.

Structure of the HNK-1 carbohydrate epitope on bovine peripheral myelin glycoprotein P0.

The HNK-1 carbohydrate epitope, expressed by many neural recognition molecules, is involved in cell interactions that control cell type-specific neurite outgrowth and regeneration. It is also the target for autoimmune IgM antibodies in demyelinating neuropathies of the peripheral nervous system in humans. Despite its acknowledged importance in cell interactions, the HNK-1 carbohydrate structure, when expressed on glycoproteins, is still unknown. Here, we describe the structure of one of the predominant HNK-1-bearing glycans of bovine P0. The epitope consists of the sulfated trisaccharide SO4-3GlcAbeta1-3Galbeta1-4GlcNAc, attached to the alpha1-6 arm of a diantennary core with a bisecting N-acetylglucosamine. It is the first example of a terminal 3-sulfated glucuronic acid on an asparagine-linked carbohydrate. Because the similarity between the glycoprotein-derived structure and the glycosphingolipids carrying HNK-1 is restricted to the terminal sulfated trisaccharide, we conclude that this element is sufficient for HNK-1 immunoreactivity. Knowledge of the HNK-1 structure on proteins is an important prerequisite for the elucidation of its functional role in development and disease.

Site-specific and complete enzymic deglycosylation of the native human chorionic gonadotropin alpha-subunit.

Numerous studies have shown that glycosylation of the alpha-subunit of human chorionic gonadotropin (alpha hCG) is essential for the biological activity of this hormone. To obtain detailed insight into the function of N-glycosylation, the availability of site-specifically and fully deglycosylated alpha-subunits obtained under non-denaturing conditions is a prerequisite. NMR spectroscopy in combination with FAB-mapping demonstrates that only Asn52 of the alpha-subunit is accessible to digestion by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F under native conditions. Treatment of native alpha hCG with endo-beta-N-acetylglucosaminidase B results in full deglycosylation yielding alpha hCG with one GlcNAc residue at both Asn52 and Asn78.

Rapid and simple approach for the NMR resonance assignment of the carbohydrate chains of an intact glycoprotein. Application of gradient-enhanced natural abundance 1H-13C HSQC and HSQC-TOCSY to the alpha-subunit of human chorionic gonadotropin.

The structure assessment of an intact glycoprotein in solution requires an extensive assignment of the carbohydrate NMR resonances. However, assignment of homonuclear spectra is very complicated because of the severe overlap of protein and carbohydrate signals. Application of pulsed field gradients allowed high quality natural abundance 1H-13C HSQC and HSQC-TOCSY spectra to be recorded of the alpha-subunit of human chorionic gonadotropin. Most carbohydrate 1H-13C correlations appear in a distinct region between the aromatic region and the protein C alpha-H alpha region. The enormous reduction in overlap led to fast and unambiguous assignment of the anomeric 1H-13C correlations. Subsequently, correlations of the monosaccharide skeleton atoms were readily assigned in the HSQC-TOCSY spectrum.

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis.

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3-80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site lead to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4 bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.

(1----5)-linked beta-D-galactofuranosides are immunodominant in extracellular polysaccharides of Penicillium and Aspergillus species.

Aspergillus and Penicillium species produce extracellular polysaccharides which are immunologically active. Methyl beta-D-galactofuranoside interferes with the reaction between the polysaccharide antigens and the antibodies raised in rabbits. Of the different interlinked dimers of beta-D-galactofuranosides (1----2; 1----3; 1----5; 1----6) the (1----5) interlinked beta-D-galactofuranoside gave the highest inhibition. An increasing inhibitory effect of di-, tri-, tetra-, penta-, hexa-, and heptamer of (1----5) interlinked beta-D-galactofuranosides was observed. It was noticed that the penta-, hexa- and heptamer of (1----5) interlinked beta-D-galactofuranosides were able to link antibodies raised against the extracellular polysaccharides produced by Penicillium species. The tetramer molecule was able to neutralize the binding of antibodies, which are naturally present in human sera, to the polysaccharides produced by Penicillium and Aspergillus species.