Excitation and sensitization to heat of nociceptors by bradykinin (BK) were examined using an isolated rat skin‐saphenous nerve preparation. A total of 52 C‐fibres was tested: 42 were mechano‐heat sensitive (CMH) and 40 % of them were excited and sensitized to heat by BK superfusion (10−5m, 5 min) of their receptive fields; heat responses were augmented by more than five times and heat thresholds dropped to 36.4 °C, on average. Sixty per cent of the CMH did not respond to BK itself, but 3/4 of these units showed an increase in their heat responses by more than 100 % following BK exposure. Ten high‐threshold mechanosensitive C‐fibres did not discharge upon BK application but following this five of them responded to heat in a well‐graded manner. In all fibres, the sensitizing effect of BK was abolished within 9 min or less of wash‐out, and it could be reproduced several times at equal magnitude, whereas the excitatory effect of BK regularly showed profound tachyphylaxis. Sustained superfusion (20 min) of BK induced a desensitizing excitatory response while superimposed heat responses showed constant degrees of sensitization. The large extent and high prevalence of BK‐induced sensitization (almost 80 % of all fibres tested) and de novo recruitment of heat sensitivity suggest a prominent role of BK not only in hyperalgesia but also in sustained inflammatory pain which may be driven by body or even lower local temperatures acting on sensitized nociceptors. Based on the latter assumption, a hypothesis is put forward that excludes a direct excitatory effect of BK on nociceptors, but assumes a temperature‐controlled activation as a result of rapid and profound sensitization.

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Fifteen strains of mAb specific for HA of the A/Hong Kong/482/97 (H5N1) influenza virus were generated. The HA antigenic sites of the human A/Hong Kong/482/97 (H5N1) influenza virus were defined by using yeast cell surface-displaying system and anti-H5 HA mAb. Evolution analysis of H5 HA identified residues that exhibit diversifying selection in the antigenic sites and demonstrated surprising differences between residue variation of H5 HA and H3 HA. A conserved neutralizing epitope in the H5 HA protein recognized by mAb H5M9 was found using viruses isolated from [1997][1998][1999][2000][2001][2002][2003][2004][2005][2006]. Seven single amino acid substitutions were introduced into the HA antigenic sites, respectively, and the alteration of antigenicity was assessed. The structure obtained by homology-modeling and molecular dynamic methods showed that a subtle substitution at residue 124 propagates throughout its nearby loop (152)(153)(154)(155)(156)(157)(158)(159). We discuss how the structural changes caused by point mutation might explain the altered antigenicity of the HA protein. The results demonstrate the existence of immunodominant positions in the H5 HA protein, alteration of these residues might improve the immunogenicity of vaccine strains.Key words: Antigenic sites . Evolution analysis . HA . Immunodominant positions IntroductionIn 1997, an H5N1 influenza virus crossed the species barrier to directly transmit from birds to humans in Hong Kong and caused 18 cases of illness including six deaths [1]. New genotypes of the H5N1 virus continued to circulate and occasionally caused severe human disease in Asia [2][3][4]. A lack of protective immunity in the human population against the H5 influenza virus subtype presents a significant risk of an influenza pandemic.Antigenic drift of the influenza virus is caused by the accumulation of mutations in the antigenic regions of HA. Research on influenza virus antigenic drift has mainly been based on the analysis of the human H3N2 subtype [5,6]. The HA protein of all influenza viruses consists of two chains, HA1 and HA2. The HA1 3498mutates more frequently than HA2 and undergoes strong Darwinian selection for novel variants [7]. Using natural and laboratory-selected antigenic variants, five antigenic domains (sites A-E) were revealed in the H3 subtype HA molecule [8]. Of the 329 amino acids in HA1, 131 lie in or near to the five overlapping antigenic sites [5]. Codon usage at the H3 HA epitopic residues are biased toward diversification relative to the nonepitopic residues [7]. In other words, codon biases in the epitopic residues of H3 HA indicate that these residues are under strong selection for substitutional change to escape Ab pressure. Antigenic drift has also been shown to occur with avian influenza viruses, especially those circulating in poultry in the field and in antigenic variant viruses emerging from the H5N1 HPAI virus [9,10]. Therefore, the study of antigenic variation and codon usage at the H5 HA antigenic regions is necessary for the selection o...

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Background:The epidemic outbreak of severe acute respiratory syndrome (SARS) posed a worldwide threat to public health and economic stability. Although the pandemic has been contained, concerns over its recurrence remain. It is essential to identify specific diagnostic agents and antiviral vaccine candidates to fight this highly contagious disease. Methods: We generated 14 monoclonal antibodies (mAbs) specific to the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein and used these to thoroughly map the N protein antigenic determinants. We identified the immunodominant antigenic sites responsible for the antibodies in sera from SARS patients and antisera from small animals and differentiated the linear from the conformational antibody-combining sites comprising the natural epitopes by use of yeast surface display. Results: We identified 5 conformational and 3 linear epitopes within the entire N protein; 3 conformational and 3 linear epitopes were immunodominant. The antibody responses to the N protein fragments in mammalian sera revealed that 3 regions of the N protein are strong antigenic domains. We expanded the specificity of the N protein epitope and identified 4 novel confor-