Predominance of a rare type of HIV-1 in Estonia

An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in Estonia. The objective of this study was to further investigate the molecular epidemiology and genetic structure of HIV-1 in Estonia. Most of the investigated individuals became infected after August 2000 when HIV-1 started to spread rapidly among Estonian intravenous drug users (IDUs). Two viral DNA regions, gag/pol and gp41, were sequenced and subtyped from peripheral blood mononuclear cells or plasma from 141 individuals. Phylogenetic analysis in the gp41 region revealed that the most frequent type of the virus among IDUs was a circulating recombinant form, CRF06_cpx, whereas a few samples showed highest sequence similarity to a subtype A strain circulating in Ukraine and Russia. Likewise, in the gag/pol region, most of the samples were classified as CRF06_cpx, with a few classified as subtype A. In this region, however, 16% of the sequences turned out to be mosaic unique recombinant forms consisting of CRF06_cpx and subtype A. At least 9 mosaic forms were identified, each with distinct patterns of multiple crossover. To characterize Estonian CRF06_cpx as well as recombinant isolates in more detail, 4 near-full-length HIV-1 genomes were sequenced.     

Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.     

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2

BACKGROUND: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. OBJECTIVE: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. METHODS: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. CONCLUSION: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.     

Revisiting serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma

This study analyzed serum thyroglobulin (Tg) during hypothyroidism in 207 patients with differentiated thyroid carcinoma treated with total thyroidectomy and radioiodine ablation and undetectable anti-Tg antibodies. Disease staging was defined by clinical examination, stimulated Tg, pre- and post-ablative radioiodine scanning, and other imaging methods (X-Ray, US, CT and MIBI-scan). The average interval from initial therapy was 2.3 years. 153 patients (74%) had no evident disease, 34 (16.4%) presented neck/mediastinal disease, and 20 (9.6%) had distant metastases (Mt). The best cut-off for Tg was 1 ng/ml, showing 100% sensitivity for distant Mt and 88.2% for local recurrence or lymph node Mt, and 88.8% specificity for any Mt and 74.8% for distant Mt. In patients with Tg <1 ng/ml, 2.8% showed cervical lymph nodes Mt. Cervical or mediastinal disease were 26% of cases with Tg between 1 and 5 ng/ml. Tg from 5 to 10 ng/ml was associated to distant Mt in 14.2% of the cases and others showed lymph nodes Mt. In patients with Tg >10 ng/ml, 51.3% presented distant Mt. We suggest the need for neck US even in cases with Tg <1 ng/ml. In addition, patients with Tg levels <5 ng/ml should be investigated by neck US and mediastinal CT only, and empirical therapy should be limited to patients with a minimum Tg level >5 ng/ml.