A structural basis for the selection of dominant alpha beta T cell receptors in antiviral immunity

We have examined the basis for immunodominant or public TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1alpha and CDR2alpha loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRalpha constant domain thought to form part of the docking site for CD3epsilon. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.

The effectiveness of personalized smoking cessation strategies for callers to a Quitline service

Aim To assess the effectiveness of a program of computer-generated tailored advice for callers to a telephone helpline, and to assess whether it enhanced a series of callback telephone counselling sessions in aiding smoking cessation. Design Randomized controlled trial comparing: (1) untailored self-help materials; (2) computer-generated tailored advice only, and (3) computer-generated tailored advice plus callback telephone counselling. Assessment surveys were conducted at baseline, 3, 6 and 12 months. Setting Victoria, Australia. Participants A total of 1578 smokers who called the Quitline service and agreed to participate. Measurements Smoking status at follow-up; duration of cessation, if quit; use of nicotine replacement therapy; and extent of participation in the callback service. Findings At the 3-month follow-up, significantly more (chi(2)(2) = 16.9; P < 0.001) participants in the computer-generated tailored advice plus telephone counselling condition were not smoking (21%) than in either the computer-generated advice only (12%) or the control condition (12%). Proportions reporting not smoking at the 12-month follow-up were 26%, 23% and 22%, respectively (NS) for point prevalence, and for 9 months sustained abstinence; 8.2, 6.0, and 5.0 (NS). In the telephone counselling group, those receiving callbacks were more likely than those who did not to have sustained abstinence at 12 months (10.2 compared with 4.0, P < 0.05). Logistic regression on 3-month data showed significant independent effects on cessation of telephone counselling and use of NRT, but not of computer-generated tailored advice. Conclusion Computer-generated tailored advice did not enhance telephone counselling, nor have any independent effect on cessation. This may be due to poor timing of the computer-generated tailored advice and poor integration of the two modes of advice.