Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis

To date, there are no proven vaccines against any form of leishmaniasis. The development of live attenuated vectors shows promise in the field of Leishmania vaccination because these organisms mimic more effectively the course of real infections and can elicit potent activation of the immune system. In the present study, we investigated the potential of a parasitic protozoan that is nonpathogenic to humans, Leishmania tarentolae, as a live candidate vaccine that efficiently targets dendritic cells and lymphoid organs, thus enhancing antigen presentation and consequently influencing the magnitude and quality of T-cell immune responses. We demonstrated that L. tarentolae activates the dendritic cell maturation process and induces T-cell proliferation and the production of gamma interferon, thus skewing CD4(+) T cells toward a Th1 cell phenotype. More importantly, we found that a single intraperitoneal injection of L. tarentolae could elicit a protective immune response against infectious challenge with Leishmania donovani in susceptible BALB/c mice. These results suggest that the use of L. tarentolae as a live vaccine vector may represent a promising approach for improving the effectiveness and safety of candidate live vaccines against Leishmania infections and possibly other intracellular pathogens for which T-cell mediated responses are critical for the development of protective immunity.     

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induce IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine σ-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.     

Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.     

Quantification of HIV-1 p24 by a highly improved ELISA: an alternative to HIV-1 RNA based treatment monitoring in patients from Abidjan, C?te d'Ivoire.

BACKGROUND: Quantification of HIV-1 RNA remains difficult to implement in Africa. Simple and inexpensive tests for antiretroviral treatment (ART) monitoring are needed. OBJECTIVE: To evaluate an HIV-1 p24 ELISA, which combines efficient virus disruption, heat-denaturation and signal amplification, in a West African setting. STUDY DESIGN: Eighty-six HIV-1 infected patients from Abidjan, C?te d'Ivoire, were tested for p24, HIV-1 RNA, and CD4+ count at baseline, and twice within 8 months after ART initiation. RESULTS: All patients responded to ART with a minimal HIV-1 RNA drop of 0.5 log(10) at first follow-up. Forty-one (47.7%) then rebounded >0.5 log(10) or persisted above 1000 copies/mL by week 24. The predicted baseline concentration of p24 corresponding to 100,000 copies/mL of HIV-1 RNA, above which ART is recommended, was 4546 fg/mL (95% confidence interval 3148-6566). A prediction model of virologic failure, occurring after an initial response to ART, correctly classified 84% of patients using baseline p24, p24 change on therapy, and achievement of undetectable p24 as explanatory variables. The model and further bootstrap evaluation suggested a good ability to discriminate between sustained or failing virologic response to ART. CONCLUSION: HIV-1 p24 and RNA based-ART monitoring in a low-resource country dominated by HIV-1 CRF02 AG appeared comparable.     

Gene expression of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase in skeletal muscle from type 2 diabetic subjects

The gene of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase gives rise to several splice variants. We hypothesized that the expression of p85alpha splice variants may be altered in skeletal muscle from subjects with type 2 diabetes mellitus. Skeletal muscle biopsies were obtained from nine type 2 diabetic and eight healthy men, matched for age, body mass index (BMI) and physical fitness. PI 3-kinase activity in skeletal muscle following in vitro insulin stimulation was reduced in subjects with type 2 diabetes. p85alpha mRNA was elevated fourfold in type 2 diabetic as compared to healthy control subjects ( P<0.05). p85alpha mRNA abundance was positively correlated with plasma insulin concentration ( P<0.01) and serum glucose concentration ( P<0.01). Despite this, protein levels of p85alpha, p55alpha, and the novel human p50alpha were not altered in type 2 diabetic subjects. Thus, although gene expression of full-length p85alpha is increased in skeletal muscle from type 2 diabetics, this is not reflected by increased protein levels. Therefore, defects in PI 3-kinase activity are likely due to impaired activation of the enzyme rather than changes in protein expression of the isoforms of the regulatory subunit.     

Synthesis and characterization of α-hydroxy, α-thiol and α-chloroformyl oligomers of poly(butylene terephthalate)

α-Hydroxy and α-chloroformyl oligomers of poly(butylene terephthalate) (PBT) were prepared in 1,1,2,2-tetrachloroethane by condensing terephthaloyl chloride and 1,4-butanediol and using benzoyl chloride and 4-hydroxybutyl benzoate as chain limitator. The average molecular weight was determined by ¹H NMR analysis, and thermal properties were assessed by differential scanning calorimetry (DSC). Preparation of α-thiol oligomers of PBT was also investigated by esterification of α-hydroxy oligomers with thioglycolic acid and using p-toluenesulfonic acid as catalyst. The DSC and TGA analyses pointed out that the introduction of a thiol group by esterification has no influence on the thermal properties of the PBT oligomers.     

Cortical auditory processing and communication in children with autism: electrophysiological/behavioral relations.

The purpose of the present study was to investigate the relations between late auditory evoked potentials (AEPs) recorded at temporal sites (the N1c wave or Tb) and verbal and non-verbal abilities in children with autism. The study was performed in 26 mentally retarded children with autism (AUT) aged 4-8 years (mean age +/- S.E.M. = 71 +/- 2 months; mean verbal and non-verbal developmental quotient +/- S.E.M. = 36 +/- 4 and 48 +/- 3). The stimuli used were 750 Hz tone bursts of 200 ms duration delivered binaurally at different intensity levels (50, 60, 70, 80 dB SPL) with 3-5 s interstimulus intervals. Temporal AEPs were first compared to those of a group of 16 normal children (NOR) in the same age range (mean age +/- S.E.M. = 69 +/- 3 months). We then focused on the AUT group and considered relations between temporal AEPs and the severity of disorders of verbal and non-verbal communication assessed using a behavior rating scale. AEPs recorded on left and right temporal sites were of smaller amplitude in the AUT group than in the NOR group. Increasing intensity-related amplitude was observed on both sides in NOR and only on the right side in AUT. The lack of intensity effect on the left side resulted in a particular pattern of asymmetry at the highest level of intensity (80 dB SPL) with greater N1c amplitude on the right than on the left side (the reverse was found in the NOR group). Electro-clinical correlations indicated that the greater the amplitude of the right temporal N1c responses, the higher the verbal and non-verbal communication abilities. This suggests a developmental reorganization of left-right hemisphere functions in autism, with preferential activation of the right hemisphere for functions usually allocated to the left hemisphere, particularly those involving the secondary auditory areas situated on the lateral surface of the superior temporal gyrus where the N1c/Tb wave is generated.     

Cholesterol crystal embolization diagnosed on bladder transurethral resection

Cholesterol crystal embolization (CCE) is a severe systemic disorder caused by vascular migration of cholesterol crystals originating from ulcerative atherosclerotic plaques located in large arteries. We report 2 cases of CCE diagnosed on bladder transurethral resection in 2 men aged 94 and 72 years. Both patients had atherosclerosis disease. One patient had been treated by heparin 1 month before for pulmonary embolism and the other had had a coronary angiography and bypass graft surgery 5 months before for silent myocardial infarction. One patient presented with hematuria and the other with acute renal failure. Cystoscopy showed multiple papillary tumors of the bladder wall. Bladder transurethral resections showed transitional cell carcinoma with cholesterol crystals occluding the lumen of small arterioles in the submucosa. Eight cases of CCE in the bladder wall have been reported in the literature in 3 women and 5 men aged 56 to 79 years. Cholesterol crystal embolization is often discovered in the bladder wall on necropsy specimens. Only 2 cases have been fortuitously discovered on bladder transurethral resection performed for transitional cell carcinoma. Cholesterol crystal embolization in the bladder wall is often a marker of severe disease although the evolution is quite favorable in our patients, still alive 1 and 2 years after diagnosis.