[Immunization: adverse event reporting system].
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Adverse Drug Reaction Reporting Systems↗
Biomedical subjects
A Jacquet
Publications and source records attributed to A Jacquet.
Randomised double masked trial comparing the efficacy and tolerance of 0.05% mequitazine eye drops versus 0.05% levocabastine and placebo in allergic conjunctivitis induced by a conjunctival provocation test with Dermatophagoides pteronyssinus.
AIM: A double masked randomised trial comparing 0.05% mequitazine eye drops with 0.05% levocabastine and placebo was carried out in otherwise healthy volunteers allergic to house dust mites (Dermatophagoides pteronyssinus). METHOD: Double masked, randomised, single centre study, comparing three parallel treatment groups. 60 healthy adults with a confirmed history of allergic conjunctivitis to house dust mites for at least 2 years were included and completed the trial. Conjunctival provocation tests (CPT) were done at screening, at visit 2 (V2) (1 week later), and at visit 3 (V3) (2 weeks after V2). Treatment was instilled in the same eye, 5 minutes after the CPT at V2, and twice daily until V3. CPT were scored 5, 10, 15, and 60 minutes after instillation of the dose of Dermatophagoides pteronyssinus antigen determined at inclusion (V2, curative test) or resulting in positivity (V3, preventive test) RESULTS: In the V2 (curative) test the difference between the active treatments and placebo on the redness+itching scores was not significant. At the V3 (preventive) CPT there was a lower number of reactions at the threshold dose with mequitazine (20%) compared to placebo (60%, p = 0.01) or levocabastine (45%, p = 0.10). CONCLUSION: This trial failed to clearly demonstrate curative superiority of topical antihistamines with placebo, when a single dose of treatment was instilled following CPT. However mequitazine 0.05% eye drops were superior to placebo in preventing a reaction to CPT, after 2 weeks of treatment.
Adolescent↗
Absence of immunoglobulin E synthesis and airway eosinophilia by vaccination with plasmid DNA encoding ProDer p 1.
BACKGROUND: Various studies have shown that immunization with naked DNA encoding allergens induces T helper 1(Th1)-biased non-allergic responses. OBJECTIVE: To evaluate the polarization of the immune responses induced by vaccinations with plasmid DNA encoding the major mite allergen precursor ProDer p 1. METHODS: A DNA vaccine was constructed on the basis of a synthetic cDNA encoding ProDer p 1 with optimized codon usage. The immunogenicity of ProDer p 1 DNA in CBA/J mice was compared with that of purified natural Der p 1 or recombinant ProDer p 1 adjuvanted with alum. Vaccinated mice were subsequently exposed to aerosolized house dust mite extracts to provoke airway inflammation. The presence of inflammatory cells was examined in bronchoalveolar lavage (BAL) fluids and allergen-specific T cell reactivity was measured. RESULTS: Naive mice immunized with ProDer p 1 DNA developed Th1 immune responses characterized by high titres of specific IgG2a antibodies, low titres of specific IgG1 and, remarkably, the absence of anti-ProDer p 1 IgE. No specific responses were observed in animals vaccinated with the blank DNA vector. By contrast, natural Der p 1 or recombinant ProDer p 1 adsorbed to alum induced pronounced Th2 allergic responses with strong specific IgG1 and IgE titres. Spleen cells from DNA ProDer p 1-vaccinated mice secreted high levels of IFN-gamma and low production of IL-5. Conversely, both adjuvanted allergens stimulated typical Th2-type cytokine profile characterized by high and low levels of IL-5 and IFN-gamma, respectively. Whereas BAL eosinophilia was clearly observed in Der p 1-immunized animals, ProDer p 1 DNA as well as ProDer p 1 vaccinations prevented airway eosinophil infiltrations. CONCLUSIONS: These results suggest that vaccination with DNA encoding ProDer p 1 effectively fails to induce the allergen-induced IgE synthesis and airway cell infiltration. Plasmid DNA encoding ProDer p 1 may provide a novel approach for the treatment of house dust mite allergy.
Animals↗
High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.
BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1. METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.
Antigens, Dermatophagoides↗
Th2 polarization by Der p 1--pulsed monocyte-derived dendritic cells is due to the allergic status of the donors.
The polarization of the immune response toward a Th2 or a Th1 profile can be mediated by dendritic cells (DCs) following antigen presentation and interaction with T cells. Costimulatory molecules such as CD80 and CD86 expressed by DCs, the polarizing cytokine environment during DC--T-cell interaction, and also the nature of the antigen are critical in the orientation of the immune response. In this study, the effect of the cysteine protease Der p 1, one of the major allergens of the house dust mite Dermatophagoides pteronyssinus, on these different parameters was evaluated comparatively on monocyte-derived DCs obtained from healthy donors, from pollen-sensitive patients, or from patients sensitive to Dermatophagoides pteronyssinus. Results showed that Der p 1 induced an increase in CD86 expression only on DCs from house dust mite--sensitive patients. This was also associated with a higher capacity to induce T-cell proliferation, a rapid increase in the production of proinflammatory cytokines, tumor necrosis factor--alpha and interleukin (IL)-1 beta, and the type 2 cytokine IL-10. No changes in the release of IL-12 p70 were induced by Der p 1. Finally, purified T cells from house dust mite-sensitive patients stimulated by autologous Der p 1--pulsed DCs preferentially produced IL-4 rather than interferon-gamma. These effects were abolished in the presence of the inactive precursor of Der p 1 (ProDer p 1). Taken together, these data suggest that DCs from house dust mite--sensitive patients, in contrast to DCs from healthy donors and from pollen-sensitive patients, exposed to Der p 1 play a pivotal role in the enhancement of the Th2 response associated with the allergic reaction developed in response to house dust mite exposure. (Blood. 2001;98:1135-1141)
Animals↗
The surface antigen SAG3 mediates the attachment of Toxoplasma gondii to cell-surface proteoglycans.
The attachment of Toxoplasma gondii to target cells is mediated by recognition of cellular heparan sulfate proteoglycans (HSPGs). The present study was performed to determine whether SAG1 and SAG3, two of the parasite surface antigens anchored to the membrane via glycosylphosphatidylinositol groups (GPIs), are involved in the tachyzoite binding to proteoglycans. The use of recombinant soluble forms of these proteins allowed us to demonstrate that SAG3, but not SAG1, interacts specifically with cellular HSPGs. Indeed, soluble recombinant SAG3 protein (recSAG3) was found to bind to immobilized heparin, whereas recSAG1 did not interact with this glycoaminoglycan. The specific adherence of recSAG3 to CHO cells was inhibited by soluble glycoconjugates, of which heparin, fucoidan and dextran sulfate were the most effective. Moreover, binding of recSAG3 to two HSPGs-deficient cell mutants was reduced by up to 80%. Proteoglycan sulfation was critical for SAG3 adherence to HSPGs as incubation of cells in the presence of sodium chlorate drastically reduced the recSAG3 binding. Finally, preincubation of CHO cells with recSAG3 blocked the adsorption of radiolabelled Toxoplasma tachyzoites. Taken together, these results indicate that SAG3 is a first glycoaminoglycan-binding protein associated with Toxoplasma, and SAG3-HSPGs interactions are involved in the parasite attachment to target cells.
Animals↗
High-level expression in mammalian cells of recombinant house dust mite allergen ProDer p 1 with optimized codon usage.
BACKGROUND: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. METHODS: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. RESULTS: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5--10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. CONCLUSIONS: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.
Amino Acid Sequence↗
Cloning, expression and purification of recombinant cotton rat interleukin-5.
The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.
Amino Acid Sequence↗
Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cells.
BACKGROUND: The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy. OBJECTIVE: This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells. METHODS: The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity. RESULTS: Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive. CONCLUSIONS: The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy.
Allergens↗
Plasma, urine and skin pharmacokinetics of cefepime in burns patients.
We studied the pharmacokinetics of cefepime (2 g bd) in six burns patients. Blood, urine and skin samples were collected to measure cefepime concentrations. A two-compartment model was fitted to the data. At day 1, t(1/2beta) was 2.45 +/- 0.56 h, V(ss) 0.36 +/- 0.1 L/kg, total clearance 152 +/- 25.2 mL/min, and AUC 217 +/- 34 mg*h/L. There was no statistical difference between day 1 and day 3 for any of the pharmacokinetic parameters. We demonstrated good penetration of cefepime in skin. These results show that it is not necessary to change the standard dosage of cefepime in burns patients.
Adult↗
Protective immunity against congenital toxoplasmosis with recombinant SAG1 protein in a guinea pig model.
Primary infection with Toxoplasma gondii during pregnancy can induce fetal pathology and abortion in both humans and animals. The present study describes the development of an experimental model of congenital toxoplasmosis in the guinea pig. In this animal model, we evaluated the protective effect of vaccination with a recombinant form of SAG1 against maternofetal transmission of tachyzoites. The presence of parasites in fetuses was determined by nested PCRs and by an in vivo readout after fetal brain homogenate injections in mice. The absence of parasites was demonstrated in 66 to 86% of fetuses derived from adult guinea pigs immunized with SAG1 and challenged with the mildly virulent T. gondii strain C56. In contrast, more than 80% of fetuses from mock-immunized guinea pigs were infected. The protection was not correlated with titers of antibody to SAG1. Our results indicated that this experimental model constitutes a relevant model for evaluation of vaccine candidates against congenital toxoplasmosis and that SAG1 elicits significant protection against maternofetal transmission.
Animals↗
Cloning, expression and purification of recombinant cotton rat interferon-gamma.
The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.
Amino Acid Sequence↗
Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation.
A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2(t) production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.
ATP-Binding Cassette Transporters↗
Pharmacokinetic-pharmacodynamic analysis of mnesic effects of lorazepam in healthy volunteers.
AIMS: To describe the pharmacokinetic-pharmacodynamic modelling of the psychomotor and mnesic effects of a single 2 mg oral dose of lorazepam in healthy volunteers. METHODS: This was a randomized double-blind, placebo-controlled two-way cross-over study. The effect of lorazepam was examined with the following tasks: choice reaction time, immediate and delayed cued recall of paired words and immediate and delayed free recall and recognition of pictures. RESULTS: The mean calculated EC50 values derived from the PK/PD modelling of the different tests ranged from 12.2 to 15.3 ng ml-1. On the basis of the statistical comparison of the EC50 values, the delayed recall trials seemed to be more impaired than the immediate recall trials; similar observations were made concerning the recognition vs recall tasks. CONCLUSIONS: The parameter values derived from PK/PD modelling, and especially the EC50 values, may provide sensitive indices that can be used, rather than the raw data derived from pharmacodynamic measurements, to compare CNS effects of benzodiazepines.
Amnesia, Anterograde↗
Differential neutralizing antibody responses to varicella-zoster virus glycoproteins B and E following naked DNA immunization.
The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.
Animals↗
Susceptibility to levofloxacin of clinical isolates of bacteria from intensive care and haematology/oncology patients in Switzerland: a multicentre study.
The objective of this study was to examine the susceptibility of clinical isolates to levofloxacin, a fluoroquinolone with extended activity against Gram-positive bacteria, and other antibiotics in 12 Swiss clinical microbiology laboratories using the NCCLS disc diffusion technique. Isolates were prospectively collected from intensive care units (ICUs (59%), oncology wards (7%) and other units with haematology/oncology patients (34%) from June 1995 to March 1996. The levofloxacin breakpoints used were as recommended by the manufacturer. A total of 310 Gram-positive and 580 Gram-negative isolates from the respiratory tract (36%), skin/wounds (12%), blood (16%), urine (17%) and other sources (19%) were tested. The percentage of isolates susceptible to levofloxacin was 100% for Enterococcus spp. (38 strains), Streptococcus agalactiae (13), Streptococcus pneumoniae (65), Acinetobacter spp. (11), Citrobacter diversus (6), Citrobacter freundii (17), Klebsiella oxytoca (39), Morganella morganii (16), Proteus mirabilis (20), Proteus vulgaris (23), Serratia spp. (19), Stenotrophomonas maltophilia (10) and Haemophilus influenzae (41). The percentage of isolates susceptible to levofloxacin for Staphylococcus aureus (95 strains, including 2% MRSA) was 94%, coagulase-negative staphylococci (85) 65%, Enterobacter spp. (75) 99%, Escherichia coli (111) 97%, Klebsiella pneumoniae (45) 98% and Pseudomonas aeruginosa (124) 87%. In conclusion, levofloxacin is a new fluoroquinolone to which the most common clinical isolates in Switzerland are susceptible. The susceptibility of Enterococcus spp. and S. pneumoniae to levofloxacin was particularly remarkable. This compound appears to be a promising therapeutic alternative for the treatment of Gram-positive infections.
Anti-Infective Agents↗
The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation.
A truncated form of SAG1, the immunodominant surface antigen of Toxoplasma gondii, has been produced in the methylotrophic yeast, Pichia pastoris. By construction, the recombinant protein lacks C-terminal residues 308-336 which, in native SAG1, encompass the glycosylphosphatidylinositol anchorage site. Secretion of anchor-less SAG1 proceeded via the yeast prepro alpha-mating factor signal peptide and yielded two immunoreactive protein species having apparent molecular masses of 31.5 and 34.5 kDa, respectively, and differing only by N-glycosylation of the single Asn-X-Ser site present in the molecule. Purification of the anchor-less SAG1 was achieved by a combination of ion-exchange and size-exclusion chromatographies. N-terminal amino acid sequencing of the products indicated the presence of additional residues glutamic acid--alanine at the N-terminal end of the products. Despite incomplete processing and unnatural glycosylation, anchor-less SAG1 proteins apparently adopted a suitable conformation recognized by monoclonal and human serum-derived antibodies, specific for the native SAG1. In addition, the recombinant anchor-less SAG1 proved competent for inducing proliferation, in vitro, of mononuclear cells from seropositive individuals. Finally, properly adjuvanted anchor-less SAG1 was able to induce protection of mice against a lethal challenge with T. gondii tachyzoites.
Amino Acid Sequence↗
The varicella zoster virus glycoprotein B (gB) plays a role in virus binding to cell surface heparan sulfate proteoglycans.
Varicella-zoster virus (VZV) interacts with cell surface heparan sulfate proteoglycans during virus attachment. In the present study, we investigated the potential involvement of two VZV glycoproteins, gB and gE, in the virus adsorption process. We showed that gB, but not gE, binds specifically to cellular heparan sulfate proteoglycans (HSPGs). Indeed, soluble recombinant gB protein (recgB) was found to bind to immobilized heparin and to MRC5 and L cells, a binding which was inhibited by heparin. Furthermore, recgB binding to two heparan sulfate-minus mutant L cell lines, gro2C and sog9 cells, was markedly reduced as compared to the parental L strain. Under the same experimental conditions, soluble recombinant VZV gE protein did not interact with heparin or with cell surfaces. We also demonstrated that the gB-HSPGs interactions were relevant to the VZV attachment to cells. Indeed, although polyclonal antibodies directed to gB did not impair the VZV binding, recgB could delay the virus adsorption. Our results thus strongly suggest that the interactions between gB and heparan sulfate proteoglycans take part in the initial VZV attachment to cell surfaces.
Animals↗