[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.

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.

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.

IgA production without mu or delta chain expression in developing B cells.

Surface, membrane-bound, immunoglobulin M (IgM) or IgD expression early in B cell ontogeny is considered essential for the differentiation of antibody-producing cells in mammals; only in IgM+ B cells is the heavy chain locus rearranged to express antibodies of other classes. We show here that IgA is selectively expressed in muMT mice, which lack IgM or IgD expression and have a pro-B cell developmental block. muMT IgA binds proteins of commensal intestinal bacteria and is weakly induced by Salmonella infection, although not through conventional immunization. This muMT IgA pathway requires extrasplenic peripheral lymphoid tissues and may be an evolutionarily primitive system in which immature B cells switch to IgA production at peripheral sites.

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.

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.

Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies.

Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

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.

Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E.

Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice to evaluate whether myelin-viral protein cross-reactive antibody responses could be generated. Each immunogen induced specific but not cross-reactive antibodies. We conclude that ns4b is expressed in infected cells and is immunogenic, although this does not involve amino acids shared with a self protein, at least in the experimental conditions used.

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.

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice.

The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag. Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv-treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.

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.

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.

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.

Patterns of prescribing isotretinoin: focus on women of childbearing potential.

OBJECTIVE: The primary objectives of this study were to determine how dermatologists currently prescribe isotretinoin and to determine if the potential for adverse effects, especially those affecting the fetus, has influenced dermatologists' prescribing patterns. DESIGN: A survey was mailed to 1618 dermatologists practicing in the US. The survey comprised 22 multiple-choice and fill-in-the-blank questions about the use of isotretinoin. Eight weeks were allowed for completion and return of the survey. SETTING: The setting of the study included dermatologists in private practice, those with academic appointments, those in administration, and a few dermatologists in other pursuits. PARTICIPANTS: The membership roster of the American Academy of Dermatology served as the sampling frame from which survey recipients were drawn. After arranging the list by zip code, a sample of dermatologists was taken by systematically choosing every fifth name on the list, giving the researchers the total sample of 1618 physicians. MAIN OUTCOME MEASURES: Questions were organized into the following sections: (1) practice status of respondent, (2) prescriber demographics, (3) influence of adverse effects on prescribing, (4) prescribing practices, (5) discontinuation of therapy, and (6) restriction of isotretinoin to dermatologists. The survey concluded by providing participants the opportunity to make further observations or comments. RESULTS: Of the 1618 surveys mailed, 670 usable responses were received (41.4 percent). Most respondents were in private practice. Data show that dermatologists were prescribing isotretinoin for indications other than those contained in the official labeling. Most physicians reported that they do perform a pregnancy test before prescribing the drug, and many require written informed consent before prescribing. Physicians report that, in general, their patients tolerate isotretinoin well. When therapy is discontinued, it is most often secondary to hypertriglyceridemia. Dermatologists believe that they should have sole authority for prescribing isotretinoin. CONCLUSIONS: Our results show that there is still a need for emphasizing the limited indications for isotretinoin and a need for effective patient education for women of childbearing potential who may be prescribed this drug.