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Biomedical subjects

A Lamarre

Publications and source records attributed to A Lamarre.

At least 19 recordsLinked to original sources
PubMed2001

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.

Cells, Cultured↗
PubMed2001

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Adolescent↗
PubMed1995

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1992

The family of the asthmatic child.

The authors report clinical study of 34 asthmatic children between the ages of ten and 15, followed at a specialized outpatient clinic of a large pediatric hospital in Montreal. Evaluation of the children and their families involved the use of clinical scores with inter-rater agreement. The study found strong associations between certain personality traits and an excessive use of medication, and between personality traits and family structure. Regardless of the severity of their asthma, children with high levels of anxiety and dependence were more likely to live with highly cohesive families and to use greater quantities of cortisone than children with better adapted personality structures. Pathological family settings are known to cause more emotional and behaviour problems in children. We suggest there is a reciprocal influence, and we consider the effects on the family of an early childhood disease that is persistent, worrisome, unpredictable, and necessitates repeated hospitalization. Prospective studies of the high-risk subgroups identified in this study could facilitate early intervention for asthmatic children and their families.

Adolescent↗
PubMed1992

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.

Adolescent↗
PubMed1992

A protocol for aerosol therapy in acute hospitalized asthmatics.

The most important element in the treatment of acute hospitalized asthmatics is salbutamol administered via wet nebulization prescribed usually on a subjective evaluation of the patient's status. This study outlines a novel protocol that incorporates the FEV1 variable as the major objective parameter in a feedback loop. Data from patients admitted in the year prior to the initiation of the protocol (N = 348) were compared with similar data from patients hospitalized the year following institution of the protocol (N = 238). There were no withdrawals from the protocol due to adverse effects nor were there any deteriorations requiring ICU admission. A significant decrease in the length of hospitalization and the number of salbutamol prescriptions per hospitalization was observed after, relative to before, institution of the protocol (2.2 vs. 2.6 days; 2.3 vs. 3.8; p < .05, respectively). It is concluded that this protocol is safe and efficient, and may also be more efficacious than a nonstandardized approach for treating the hospitalized asthmatic child.

Acute Disease↗