Secondary rearrangements and hypermutation generate sufficient B cell diversity to mount protective antiviral immunoglobulin responses.

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

[Glycopeptide-induced cutaneous adverse reaction: results of an immunoallergic investigation in eight patients]. / Toxidermies aux glycopeptides : résultats des explorations immunoallergologiques dans une série de huit cas.

BACKGROUND: Vancomycin (V) and teicoplanin (T) are glycopeptides used in severe infections and can induce different kinds of cutaneous adverse reactions (CAR). AIMS: To determine the value of immunoallergic investigations in CAR in which glycopeptides are suspected. METHODS: Retrospective study (2000-2007) in eight patients with CAR suspected of being caused by glycopeptides. Six weeks after abatement of the reaction, in accordance with ESCD's guideline for drug testing, immunoallergic skin tests investigations were carried out (drug patch-tests, prick-tests and intradermal tests) in succession for all the drugs taken during the CAR. If negative, a glycopeptide challenge was proposed. RESULTS: The study included eight patients (five women, three men; mean age=53); three patients presented a reaction to vancomycin, four reacted to teicoplanin and one reacted to both drugs. CARs consisted of six maculopapular rashes, one case of DRESS and one of urticaria. Skin tests confirmed involvement of glycopeptides in four of eight cases with cross-reactivity between V and T in two patients. Four patients exhibited good tolerance to rechallenge tests with glycopeptides. CONCLUSIONS: This study shows that skin tests may be useful in glycopeptide-induced CAR in determining the responsible drug and also in the event of rechallenge. Allergic cross-reactivity (V and T), observed in two of our patients, although already been reported in the literature, but does not occur systematically.

Lack of superinfection interference in retroviral vector producer cells.

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.

IgA responses in the intestinal mucosa against pathogenic and non-pathogenic microorganisms.

IgA is the most abundant immunoglobulin produced in mammals; most is secreted as a dimer across mucous membranes. This review discusses the different mechanisms of induction of IgA, and its role in protecting mucosal surfaces against pathogenic and non-pathogenic microorganisms.

Supplementation with carotenoids corrects increased lipid peroxidation in children with cystic fibrosis.

Evidence of lipid peroxidation previously documented in cystic fibrosis (CF) implies an imbalance between free radical generation and antioxidant defense mechanisms. The aim of the present study was to examine the relation between plasma concentrations of malondialdehyde, a marker of lipid peroxidation, and the exogenous antioxidant line of defense. Malondialdehyde concentrations (90.2 +/- 4.7 nmol/L) in 25 children with CF aged 9.6 +/- 0.8 y were higher (P < 0.001) than concentrations (69.1 +/- 2.6 nmol/L) in 17 children used as control subjects and were not correlated with any marker of disease severity. In contrast with their all-rac-alpha-tocopherol status, which was normal as a result of routine supplementation with a 200-mg dose of all-rac-alpha-tocopheryl acetate/d, beta-carotene was very low. A 2-mo open trial in which 12 children with CF aged 11.5 +/- 0.8 y were given 4.42 mg (8.23 mumol) beta-carotene three times per day led to normalization of the malondialdehyde concentration in all but 1 patient, in conjunction with an increase of plasma beta-carotene from 0.08 +/- 0.03 to 3.99 +/- 0.92 mumol/L. Their plasma concentrations were inversely correlated (r = -0.54, P = 0.006) [corrected] with malondialdehyde when the values measured pre- and posttreatment were pooled. We conclude that beta-carotene deficiency contributes to lipid peroxidation in CF and that supplementation may eventually prove to be a useful adjunct for the management of the disease.

Comparison of cardiovascular adjustments to exercise in adolescents 8 to 15 years of age after correction of tetralogy of fallot, ventricular septal defect or atrial septal defect.

Surgical correction of tetralogy of Fallot (TF) has generally been associated with a reduced maximal exercise tolerance, possibly related to the ventriculotomy inherent to the intracardiac repair procedure. This study documents the exercise hemodynamics of a group of patients operated on for TF who showed similar clinical and functional characteristics, and compares these responses to those of age-matched patients operated on for an isolated ventricular septal defect (VSD) or atrial septal defect (ASD) in an attempt to better understand the role of the ventriculotomy in the exercise limitation. Thirty patients, ages 12 to 19 years, operated on before 5 years of age for complete repair of TF (n = 13), VSD (n = 7) or ASD (n = 10) and 10 age-matched control subjects underwent a progressive maximal cycling test to determine the maximal oxygen uptake (VO2 max), and completed submaximal cycling at intensities of 33 and 66% VO2 max, respectively, to determine the cardiac output (CO2-rebreathing). No significant differences in VO2 max were observed (TF = 37.6 +/- 10; VDS = 34.0 +/- 9.2; ASD = 36.5 +/- 7; controls = 41.3 +/- 6.0 ml/kg/min). The maximal heart rate, however, remained lower in all patient groups in comparison with control subjects (p less than or equal to 0.05) (TF = 178 +/- 14; VSD = 172 +/- 17; ASD = 179 +/- 16; controls = 191 +/- 12 beats/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of phage-displayed recombinant anti-idiotypic antibody fragments against coronavirus-neutralizing monoclonal antibodies.

Murine coronaviruses provide useful animal models for human neurological disorders such as multiple sclerosis. In an effort to better understand the mechanisms involved in protection from coronavirus infection, we are studying the role of the idiotypic network in the modulation of viral infectivity. We have explored the feasibility of using single-chain antibodies displayed on phage surfaces for the isolation of recombinant anti-idiotypic antibodies (anti-Ids) with antigen-mimicking properties, which has proven to be difficult with conventional hybridoma approaches. A phage-display library containing more than 10(8) different antibody specificities was screened for the presence of anti-Ids by successive rounds of panning with three different in vitro neutralizing and in vivo protective antiviral monoclonal antibodies. After five rounds of panning, between 32% and 84% of all individual clones tested showed antibody-binding in an enzyme-linked immunosorbent assay (ELISA). Although several clones showed identical antibody sequences, a number of different clones were identified and further characterized. None of the selected clones induced the production of antiviral or neutralizing antibodies or conferred reproducible protection from viral challenge in BALB/c and C57BL/6 mice. These results demonstrate that anti-Ids can be isolated from a phage-display library, although high-affinity antigen-mimicking phages with antiviral protective capacities were apparently not represented in this library. This argues for the development of more diverse phage-display libraries.

Diffusion capacity and oxygen desaturation effects on exercise in patients with cystic fibrosis.

Although a fall in arterial oxygen saturation (SaO2) during exercise has been reported in patients with advanced lung disease due to cystic fibrosis (CF), not every patient with advanced disease desaturates, and pulmonary function tests have not been considered predictive as to which patient will desaturate. This study evaluated oxygen desaturation by ear oximetry during a progressive exercise test in 21 patients with CF and compared it to forced expiratory volume in one second (FEV1), the forced vital capacity (FVC) and the single breath diffusing capacity for carbon monoxide (DCO), all expressed as percent predicted. During exercise, the SaO2 fell less than 0.25 percent per ml of the maximal O2 consumption per kilogram of body weight to values never less than 90 percent in 15 patients (group A), whereas it fell more than this and always to values at the end of exercise of less than 90 percent in six others (group B). The FEV1 ranged from 103 percent predicted to 37 percent for group A compared to 28 to 17 percent in group B, while the range of FEV1/FVC was 87 to 52 percent for group A and 54 to 40 percent for group B. The range of DCO for group A was 129 to 84 percent compared to 64 to 54 percent. In conclusion, this study found that both the FEV1 and the DCO could separate those that had significant desaturation from those that did not and that no patient with a DCO of 80 percent or greater had significant desaturation during exercise.

Histamine challenge test in children using forced oscillation to measure total respiratory resistance.

This study analyzed the relationship between total respiratory resistance (Rrs) measured by forced oscillation technique and FEV1 during histamine provocation test in 31 children between seven and 17 years of age. Rrs was measured at frequencies between 6 (R6) and 26 Hz (R26). (R6-R26)/R26 was used as an index of frequency dependency of Rrs. A positive histamine test was defined as PC20 less than 8 mg/ml. Seventeen subjects had a positive test, and all of these had increases from baseline of R6 greater than 50 percent and (R6-R26)/R26 greater than 0.45. Of the 14 subjects whose PC20 was greater than 8 mg/ml, only two had changes in R6 and (R6-R26)/R26 of this magnitude. These two subjects had changes in FEV1 of 16 and 18 percent. There was a strong linear relationship between the changes in FEV1 and both R6 and (R6-R26)/R26 from baseline to the final value at the end of the test (r = 0.87 and 0.91 respectively). In conclusion, this study demonstrated that the evaluation of airway reactivity by histamine challenge may be done by forced oscillation technique. It is easy to administer and may allow testing of children unable to perform spirometry.

Comparison of cardiac output determinants in response to upright and supine exercise in patients with cystic fibrosis.

This study characterizes cardiac output response to progressive submaximal upright cycling in CF patients. Thirty-one CF patients as well as 11 aged-matched CF control subjects completed cardiac output determinations (CO2-rebreathing) at rest, and at submaximal exercise corresponding to 30, 50 and 75 percent VO2max, in both upright and supine positions. The VO2max was similar in three of four groups, but lower in those with severe CF. The cardiac output generally increased with exercise intensity in both positions, except in severe CF. The change from upright to supine posture resulted in a significant increase in SI at rest and for every submaximal exercise in control subjects, but not CF patients. These observations may suggest that the abnormal cardiac output response observed in severe CF could be related to a potential limitation in ventricular diastolic reserve found in all CF patients independent of disease severity which becomes more apparent under increased ventricular preload.

Melatonin protects against the effects of chronic stress on sexual behaviour in male rats.

The effects of chronic mild stress (CMS) on both sexual behaviour and wet dog shakes (WDS), a serotonergic type 2A (5-HT2A) receptor-mediated behaviour, were explored in the male rat. In addition, the possible attenuation of these effects by chronic treatment with melatonin, a putative 5-HT2A antagonist, was examined. The CMS procedure resulted in a significant increase in WDS and an overall decrease in all aspects of sexual behaviour. Concurrent melatonin administration attenuated the CMS-induced effects on sexual behaviour, but not the effects on either spontaneous WDS or WDS in response to the 5-HT2A agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, suggesting a mechanism of action other than exclusive 5-HT2A antagonism. These results are the first to demonstrate that melatonin significantly protects against the detrimental effects of a chronic stressor on sexual behaviour.

Increased viral titers and enhanced reactivity of antibodies to the spike glycoprotein of murine coronavirus produced by infection at pH 6.

Infection of cell monolayers by murine coronavirus A59 at pH 6 rather than 7 yielded a ten-fold increase in the infectious titer and a remarkable enhancement of the reactivities of monoclonal and polyclonal antibodies against the spike glycoprotein in immunoblotting, immunoprecipitation and enzyme-linked immunosorbent assays. These observations are very useful for detecting antibodies against the S glycoprotein of coronaviruses and enhancing infectious titers.

Vascular ring: clinical and physiological assessment of pulmonary function following surgical correction.

Twelve children were evaluated 7.1 years (mean) after surgical repair of a vascular ring causing tracheal compression. Nine patients complained of persistent respiratory symptoms, mostly cough, dyspnea, and/or wheezing. Functional evaluation revealed abnormal spirometry and/or lung volumes in seven subjects. Analysis of maximal expiratory-inspiratory flow-volume loops suggested the presence of residual upper airway obstruction in three patients and peripheral airway obstruction in three others. Eleven patients demonstrated bronchial hyperreactivity to histamine.

Effect of smaller droplet size of dornase alfa on lung function in mild cystic fibrosis. Dornase Alfa Nebulizer Group.

Aerosolized recombinant human DNase (dornase alfa) reduces mucus viscoelasticity in vitro and improves pulmonary function in patients with cystic fibrosis (CF). We postulated that if dornase alfa could be delivered more peripherally to small airways in the lung in the form of smaller aerosol droplets in patients with early airway obstruction, the increase in pulmonary function from baseline might be improved. CF patients (n = 749) with mild lung disease (baseline forced vital capacity > or = 70% predicted) were randomly assigned to receive dornase alfa 2.5 mg daily for 2 weeks by one of two nebulizer systems: 1) the Medic-Aid Durable SideStream nebulizer powered by the MobilAire Compressor (SS/MA) producing a droplet size with a mass median aerodynamic diameter (MMAD) of 2.1 microm; or 2) the Hudson T Up-draft nebulizer with a DeVilbiss Pulmo-Aide compressor (HT/PA) with an MMAD of 4.9 microm. Spirometry was performed at baseline and following 14 days of treatment. Dornase alfa delivered by both nebulizer systems produced small but statistically significant improvements in pulmonary function compared with baseline. There was a trend (P = 0.06) toward greater improvement in forced expiratory flow in 1 s in the SS/MA group (4.3%) compared with the HT/PA group (2.5%). These results indicate that the short-term spirometric response to dornase alfa is influenced in part by the physical characteristics of the aerosol in patients with mild lung disease. We speculate that this may be true for other therapeutic aerosols, and it appears that localization of disease in the lung plays a role in the response to inhaled agents.

Protection from lethal coronavirus infection by immunoglobulin fragments.

Molecular mechanisms of in vitro and in vivo virus neutralization by specific Ab remain largely undefined. Murine coronaviruses provide an excellent animal model for such studies. To determine the role of Ab bivalency and the contribution of its Fc portion in the neutralization of viral infectivity and passive protection of mice by an in vitro neutralizing and in vivo protective mAb (7-10A), F(ab')2 and Fab fragments were generated and their biologic properties were examined. The two fragments reacted in ELISA like the whole Ab against viral Ag or specific anti-idiotypic Abs. The affinity constants of the different Ab preparations were determined by surface plasmon resonance using immobilized anti-idiotypic Abs. The apparent affinity constant of the whole Ab molecule was 7.0 x 10(9) M-1 and was reduced 2-fold for F(ab')2 fragments and 14-fold for Fab molecules. Like whole Ab, both F(ab')2 and Fab fragments could neutralize virus in vitro and passively protect mice in vivo. However, the efficiency of in vivo neutralization by Fab fragments was reduced compared with the bivalent molecules, despite almost identical half-lives of both types of Ab fragments. These results demonstrate that in vitro and in vivo virus neutralization mechanisms by this Ab are independent of Fc-mediated functions and bivalency, but are probably influenced by Ab avidity. Also, this is the first report of in vivo protection against a viral infection by Fab fragments of antiviral Ab.

Effect of pH and temperature on the infectivity of human coronavirus 229E.

The stability of human coronavirus 229E infectivity was maximum at pH 6.0 when incubated at either 4 or 33 degrees C. However, the influence of pH was more pronounced at 33 degrees C. Viral infectivity was completely lost after a 14-day incubation period at 22, 33, or 37 degrees C but remained relatively constant at 4 degrees C for the same length of time. Finally, the infectious titer did not show any significant reduction when subjected to 25 cycles of thawing and freezing. These studies will contribute to optimize virus growth and storage conditions, which will facilitate the molecular characterization of this important pathogen.