Impaired fear memories are correlated with subregion-specific deficits in hippocampal and amygdalar LTP

Inbred mouse strains have different genetic backgrounds that likely influence memory and long-term potentiation (LTP). LTP, a form of synaptic plasticity, is a candidate cellular mechanism for some forms of learning and memory. Strains with impaired fear memory may have selective LTP deficits in different hippocampal subregions or in the amygdala. The authors assessed fear memory in 4 inbred strains: C57BL/6NCrlBR (B6), 129S1/SvImJ (129), C3H/HeJ (C3H), and DBA/2J (D2). The authors also measured LTP in the hippocampal Schaeffer collateral (SC) and medial perforant pathways (MPP) and in the basolateral amygdala. Contextual and cued fear memory, and SC and amygdalar LTP, were intact in B6 and 129, but all were impaired in C3H and D2. MPP LTP was similar in all 4 strains. Thus, SC, but not MPP, LTP correlates with hippocampus-dependent contextual memory expression, and amygdalar LTP correlates with amygdala-dependent cued memory expression, in these inbred strains.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB₄). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB₄ using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB₄ gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB₄, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB₄ is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB₄ is not reduced in opposing gradients as compared to that in a single LTB₄ gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.This revised version was published online in May 2005 with corrected author affiliations     

Effect of dosage on immunogenicity of a Vi conjugate vaccine injected twice into 2- to 5-year-old Vietnamese children

In a double-blind, randomized, and placebo-controlled previous trial, the efficacy of Vi-rEPA for typhoid fever in 2- to 5-year-olds was 89.0% for 46 months. Vi-rEPA contained 25 microg of Vi and induced a greater-than-eightfold rise in immunoglobulin G (IgG) anti-Vi in all of the vaccinees tested. In this investigation, we conducted a dosage-immunogenicity study of 5, 12.5, and 25 microg of Vi-rEPA in this age group. Two doses of Vi-rEPA were injected 6 weeks apart. Blood samples were taken before and at 10 weeks (4 weeks after the second injection) and 1 year later. All postimmunization geometric mean (GM) levels were higher than the preimmune levels (P < 0.0001). At 10 weeks, the GM IgG anti-Vi level elicited by 25 microg (102 EU/ml) was higher than those elicited by 12.5 microg (74.7 EU/ml) and 5 microg (43 EU/ml) (P < 0.004): all of the children had > or = 3.52 EU/ml (estimated minimum protective level). One year later, the levels declined about sevenfold (13.3 and 11.3 versus 6.43 EU/ml, P < 0.0001) but remained significantly higher than the preimmune levels (P < 0.0001), and >96% of the children had a greater-than-eightfold rise. This study also confirmed the safety and consistent immunogenicity of the four lots of Vi-rEPA used in this and previous trials.     

Effect of loteprednol etabonate nasal spray suspension on seasonal allergic rhinitis assessed by allergen challenge in an environmental exposure unit

BACKGROUND: Loteprednol etabonate (LE) is a novel soft steroid that was designed to improve the benefit/risk ratio of topical corticosteroid therapy. This study assesses the clinical efficacy and safety of three different doses of LE nasal spray in seasonal allergic rhinitis (SAR). METHODS: In this single-center, double-blind, placebo-controlled, parallel-group trial 165 subjects with SAR to grass pollen received daily single doses of either 100, 200, 400 microg LE nasal spray, or placebo for 14 days. The patients underwent three 4-h allergen challenges with grass pollen in an environmental exposure unit at a screening visit (baseline) and on days 7 and 14 of treatment. Standardized nasal symptom scores were obtained every 20 min. Nasal flow, nasal secretions, and FEV(1) were measured every hour during allergen challenges. RESULTS: After 14 days of treatment, patients who received 400 microg LE had significantly lower total nasal symptom scores compared with those receiving placebo (P = 0.007). LE400 reduced rhinorrhea, nasal congestion, nasal itching, the amount of nasal secretions, and improved nasal flow as compared with placebo (P < 0.05). LE100 and LE200 were not significantly different from placebo. All treatments were well tolerated. CONCLUSIONS: Loteprednol 400 microg once daily is superior to placebo and the only effective dose tested in improving nasal symptoms and objective parameters in patients with SAR.     

Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities ( approximately 108 M(-1)) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications.     

Chronic diarrhea,hemorrhagic colitis,and hemolytic-uremic syndrome associated with HEp-2 adherent Escherichia coli in adults infected with human immunodeficiency virus in Bangui,Central African Republic

In human immunodeficiency virus (HIV)-infected adults from the Central African Republic, the occurrence of chronic diarrhea due to HEp-2 adherent Escherichia coli (EAEC) harboring virulence markers (eaeA, BFP, EAF, astA determinant of EAST/1, positive FAS test, enteropathogenic E. coli O serogroup) was shown to be associated with AIDS. We also show that EAEC that produce verotoxin (Stx2) but do not harbor the genetic markers for classical enterohemorrhagic E. coli are involved in hemorrhagic colitis and hemolytic-uremic syndrome in patients with HIV.     

Pulmonary granuloma caused by Pseudomonas andersonii sp nov

Pulmonary granuloma is a common lesion for which gram-negative bacteria are rarely implicated as a cause. Hence, most physicians are unaware of this etiology. We isolated a gram-negative bacterium from a surgically resected pulmonary granuloma in a 42-year-old, nonimmunocompromised woman. Within the necrotizing granuloma, numerous organisms also were demonstrated by Gram stain, suggesting a cause-disease relationship. Characterization of the bacterium by sequence analysis of the 16S ribosomal gene, cellular fatty acid profiling, and microbiologic studies revealed a novel bacterium with a close relationship to Pseudomonas. We propose a new species for the bacterium, Pseudomonas andersonii. These results suggest that the differential diagnosis of a lung granuloma also should include this gram-negative bacterium as a potential causative agent, in addition to the more common infections caused by acid-fast bacilli and fungi. This bacterium was shown to be susceptible to most antibiotics that are active against gram-negative bacteria.     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas δ-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

Plasmid-mediated quinolone resistance determinant qnrS in Enterobacter cloacae

PCR was used to investigate the occurrence of the plasmid-encoded quinolone resistance determinants qnrA and qnrS among diarrhoeagenic enterobacterial isolates recovered from Hanoi, Vietnam, during the period March 2001 to April 2002. In total, 162 Escherichia coli isolates, 28 Shigella isolates and three Enterobacter cloacae isolates were negative for qnrA, while a single Ent. cloacae isolate harboured a 50-kb qnrS-positive conjugative plasmid. Cloning and sequencing identified a qnrS gene bracketed by open reading frames identical to those surrounding the qnrS gene of a Shigella flexneri isolate from Japan, thereby suggesting a common mechanism of acquisition.