Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method.

AIMS: To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. METHODS AND RESULTS: Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. CONCLUSIONS: The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries.

Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines.

Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n=145) and mothers living in an urban area (n=147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45-48%), whereas the majority of the remaining samples had low antibody titers (2-16). Despite these low titers, the breast milk still reduced vaccine strain titers (2×10(6) plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P<0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants.

Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam.

Noroviruses (NoV) and sapoviruses (SaV) are recognized as important causes of acute gastroenteritis in children worldwide. In this study, the prevalence and genetic variability of NoV and SaV were determined in hospitalized children <5 years of age with acute gastroenteritis in Hanoi, Vietnam. A total of 501 fecal specimens collected between November-2007 and October-2008, that previously had been tested for rotavirus (RV), were tested for NoV and SaV by realtime RT-PCR. Positive samples were genotyped by conventional RT-PCR followed by sequencing. GII NoV was detected in 180 (36%) and SaV in 7 (1.4%) of the samples. NoV was detected year-round ranging from 9.5% in April to 81.5% in September among RV negative samples. NoV GII.4 Minerva (2006b) was the dominant genotype (93%) with a few other genotypes detected including GII.3 (4.4%), GII.13 (1.7%), and GII.2 (0.6%) but no GI strains. Only GI and GII SaV strains were detected in this study. No difference in NoV prevalence between age groups was noted. Frequency of vomiting or fever was similar between children with NoV and RV infection, yet, NoV caused diarrhea with longer duration. In conclusion, NoV is the second most frequent cause of diarrhea in hospitalized children in North Vietnam.

Protein kinase C in rat brain cortex and hippocampus: effect of repeated administration of fluoxetine and desipramine.

1. Recent evidence indicates that changes in the activity of cyclic AMP-dependent protein kinase may be involved in neuroadaptive mechanisms after chronic treatment with antidepressants. The aim of this study was to investigate the effect of repeated administration of fluoxetine (FL) and desipramine (DMI) on the distribution and activity of protein kinase C (PKC) in subcellular fractions of rat cortex (Cx) and hippocampus (Hc) under basal conditions and in response to a single in vivo administration of 5-HT2A/2C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). 2. Rats were treated for 21 days with FL (5 mg kg-1 day-1, i.p.) or DMI (10 mg kg-1 day-1, i.p.). DOI was injected to groups of rats receiving repeated doses of antidepressants or to control rats 1 h before ex vivo PKC assay. Distribution of PKC was determined by [3H]-phorbol-12,13-dibutyrate ([3H]-PDBu) binding and PKC activity by the Amersham enzyme assay system. 3. Autoradiography of tissue sections revealed decreased [3H]-PDBu binding in CA1 region of hippocampus (by 18%) and paraventricular thalamic nucleus (by 28%) of rats after repeated administration of FL. 4. In vitro exposure of brain sections to 50 microM FL resulted in significant decreases (by 23-32%) of [3H]-PDBu binding in six out of seven regions examined; exposure to 100 microM FL reduced [3H]-PDBu binding (by 36-52%) in all regions. In contrast, exposure of brain sections to 100 microM DMI failed to alter specific [3H]-PDBu binding in brain sections. 5. The activity of PKC in subcellular fractions of Cx and Hc was significantly (by 40-50%) decreased in rats given repeated doses of FL or DMI. A single administration of either drug was without effect.6. A single in vivo administration of DOI to control rats resulted in reduced PKC activity (by 30-40%)in the particulate fraction of both Cx and Hc. This response to DOI was similar in DMI-treated rats but was not seen in rats given repeated doses of FL. A single administration of DOI to animals given repeated doses of FL resulted in PKC activities higher than those seen in rats treated with FL alone.7. The results indicate that repeated administration of FL and DMI produced similar changes in basal PKC activity but differentially affected the PKC response to the 5-HT2A/2c receptor agonist, DOI. The effect on basal PKC activity may result from a post-receptor action of antidepressants; the alteration of PKC response to DOI after fluoxetine could be due to receptor-mediated desensitization of the signalling system.

Chronic fluoxetine treatment upregulates 5-HT uptake sites and 5-HT2 receptors in rat brain: an autoradiographic study.

This study was undertaken to investigate the effect of chronic treatment with fluoxetine, a selective serotonin uptake inhibitor used widely in the treatment of depression, on the distribution and density of 5-HT uptake sites, 5-HT2 receptors, and vesicular amine uptake sites in rat brain. Fluoxetine (10 mg/kg i.p.) was administered daily for 21 days. The density of 5-HT uptake sites labelled by [3H]paroxetine, 5-HT2 receptors labelled by [3H]ketanserin in presence of tetrabenazine and vesicular amine uptake sites labelled by [3H]ketanserin in the presence of mianserin were measured by quantitative autoradiography in 22 areas of rat brain, using coronal tissue sections. Chronic administration of fluoxetine produced significant increases in the density of 5-HT uptake sites in layers of frontoparietal cortex (by 32-43%), of striate cortex (by 55%), in CA1 field of hippocampus (by 111%) and in superior colliculus (by 20%). Fluoxetine treatment also resulted in upregulation of 5-HT2 receptors in layers of frontoparietal cortex (31-38%) and in CA2-3 fields of hippocampus (by 39%). The density of tetrabenazine-sensitive vesicular amine uptake sites in the caudate-putamen was also significantly increased (by 66%). The observed alterations in 5-HT uptake site and 5-HT2 receptor densities are likely a part of adaptive neuronal changes that occur after chronic administration of fluoxetine and may be related to the antidepressant effect of the drug.

5-HT uptake sites and 5-HT2 receptors in brain of antidepressant-free suicide victims/depressives: increase in 5-HT2 sites in cortex and amygdala.

The density (Bmax) of 5-HT2 receptors labelled with [3H]ketanserin was significantly increased in prefrontal cortex (by 67%) and amygdala (by 97%) from suicide/depressives in comparison with controls. There were no differences in Kd of [3H]ketanserin binding between the two groups. The density (Bmax) and affinity (Kd) of [3H]paroxetine sites were not significantly different in the suicide/depressives and controls. The ratio between the density of presynaptic 5-HT uptake sites and postsynaptic 5-HT2 receptors in amygdala was significantly lower in suicide/depressives than in controls. The data confirm and extend some of the previous findings of increases in 5-HT2 receptors in post-mortem brains of suicide victims and depressives who died of natural causes and lend support to the view that an abnormality in brain serotonergic system is associated with depression and suicidal behaviour.