Genome-wide association study of IgG1 responses to the choline-binding protein PspC of Streptococcus pneumoniae.

Streptococcus pneumoniae causes invasive pneumococcal disease. Delayed development of antibodies to S. pneumoniae in infancy is associated with the development of atopy and asthma. Pneumococcal surface protein C (PspC) is a vaccine candidate and variation in its choline-binding region is associated with invasive disease. This study examined 523 060 single-nucleotide polymorphisms in The Western Australian Pregnancy Cohort (Raine) Study to find loci influencing immunoglobulin G1 (IgG1) responses to PspC measured at age 14 years (n=1152). Genome-wide significance (top SNP rs9275596; P=3.1 × 10(-14)) was only observed at human leucocyte antigen (HLA). Imputed HLA amino-acid polymorphisms showed the strongest associations at positions DRB1 47 (P=3.2 × 10(-11)), 13SRG (P=9.8 × 10(-10)) and 11SP (P=9.8 × 10(-10)), and at DQA1 34 (P=6.4 × 10(-10)), DQB1 167R (P=9.3 × 10(-6)) and HLA-B 95 W (P=1.2 × 10(-9)). Conditional analyses showed independent contributions from DRB1 47 and DQB1 167R to the signal at rs9275596, supported by an omnibus test showing a strong signal for the haplotype DRB1_47_DQB1_167 (P=9.02 × 10(-15)). In silico analysis showed that DRB1 four-digit allele groups defined by DRB1 47F bind to a greater complexity of core 9-mer epitopes compared with DRB1 47Y, especially across repeats in the C-term choline-binding region. Consequent differences in CD4 T-cell help for IgG1 to PspC could have implications for vaccine design. Further analysis in other cohorts is merited.

Developments in the field of allergy in 2014 through the eyes of Clinical and Experimental Allergy.

The pathogenesis of asthma continues to be a major topic of interest to our authors with reviews and original papers on the role of viruses, mechanisms of inflammation, biomarkers, and phenotypes of asthma being major topics. A number of papers described new treatments for asthma focusing on blocking the Th2 response reflecting the fact that two decades of work in this area is finally bearing fruit. The pathogenesis of chronic rhinosinusitis is a growing area of interest, but there has been less on the genetics of airways disease than in previous years possibly reflecting the degree of rigour (and therefore a smaller body of work), with which these sorts of studies are now being undertaken. There continues to be a wide range of papers dealing with mechanisms of allergic disease ranging from clinical-based studies to basic research and the use of in vivo animal models especially mice. As before, mechanisms and new approaches to immunotherapy are common themes. Several were published in the allergens section investigating modification of allergens to increase their effectiveness and reduce the risk of adverse events. Risk factors for allergic disease was a common theme in the epidemiology section and food allergy a common theme in clinical allergy with papers on the development of protocols to induce tolerance and attempts to find biomarkers to distinguish sensitization from allergic disease. This was another exciting year for the editors, and we hope the readers of the journal.

Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations.

BACKGROUND: Rhinoviruses from the Enterovirus genus cause frequent infections and induce remarkably high titres of anticapsid antigen antibodies in asthmatics, while the prevalence of neutralising antibodies to the gut-trophic echoviruses from the same genus is diminished. OBJECTIVE: To assess the absolute and specific antibody titres to VP1 antigens of the gut-trophic enteroviruses, echovirus 30 and Sabin 1 poliovirus, in asthmatic and non-asthmatic children. METHODS: Recombinant polypeptides representing the VP1 capsid antigens of echovirus 30 and Sabin poliovirus 1 were produced. Their ability to bind IgG1 antibodies from the plasma of asthmatic (n = 45) and non-asthmatic (n = 29) children were quantitated by immunoassays that incorporated immunoabsorptions to remove cross-reactivity. RESULTS: The IgG1 antibody titres and prevalence of antibody binding to echovirus 30 were significantly lower for asthmatic children compared to controls (P < 0.05) and inversely correlated with total IgE levels for the whole study population (r = -0.262; P < 0.05). There was no difference in the prevalence and titre between groups to the VP1 antigen of Sabin poliovirus. Anti-tetanus toxoid titres measured for comparison did not correlate with anti-echovirus or poliovirus, but correlated with anti-rhinovirus titres in controls but not asthmatics, where the titres were higher for the asthmatic group. CONCLUSIONS AND CLINICAL RELEVANCE: The associations of lower antibody titres of asthmatic children to echovirus reported here and those of our previous findings of a heightened response to rhinovirus suggest a dichotomy where respiratory enterovirus infection/immunity increases the probability of developing asthma and enteric infections lower the risk. This provides further support for the concept of intestinal infection playing a key role in the development of allergic respiratory disease.

Two newly identified cat allergens: the von Ebner gland protein Fel d 7 and the latherin-like protein Fel d 8.

INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Th2-associated immunity to bacteria in teenagers and susceptibility to asthma.

Bacterial colonisation of the airways is associated with increased risk of childhood asthma. Immunoglobulin (Ig)E against bacterial antigens has been reported in some asthmatics, suggesting a role for bacterial-specific type-2 immunity in disease pathogenesis. We aimed to investigate relationships between bacterial-specific IgE amongst teenagers and asthma susceptibility. We measured titres of IgE against Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus in 1,380 teenagers, and related these to asthma symptomatology and immunophenotypes. IgE titres against S. aureus-derived enterotoxins were highest amongst atopics and were associated with asthma risk. Surprisingly, IgE titres against H. influenzae and S. pneumoniae surface antigens were higher, not stratified by atopy and independently associated with decreased asthma risk. The positive association between type-2 immunity to S. aureus and asthma phenotypes probably reflects IgE-mediated effector cell activation via enterotoxin super antigens which are secreted in soluble form. The contrasting benign nature of type-2 immunity to H. influenzae and S. pneumoniae antigens may reflect their lower availability in soluble forms that can crosslink IgE receptors. We theorise that instead they may be processed by antigen presenting cells and presented to type-2 memory cells leading to mucosal secretion of interleukin (IL)-4/IL-13, a mechanism widely recognised in other tissues to attenuate T-helper-1 associated bacterial-induced inflammation.

Anti-bacterial IgE in the antibody responses of house dust mite allergic children convalescent from asthma exacerbation.

BACKGROUND: Atopic sensitization to the house dust mite (HDM) is associated with altered antibody responses to the nasopharyngeal colonizing bacterium Haemophilus influenzae and children admitted to the emergency department for asthma exacerbation have reduced IgG responses to HDM allergens. OBJECTIVE: To investigate anti-bacterial and anti-allergen antibody responses during convalescence from asthma exacerbation and differences found in exacerbations associated with and without viral infection. RESULTS: IgE antibodies to the P6 bacterial antigen increased in 60% of sera during convalescence and for many children achieved titres as high as IgE titres to allergens. In contrast IgE anti-HDM titres declined during convalescence. The anti-bacterial IgE titres were the same in subjects with and without virus infection while the anti-HDM IgE declined more rapidly in virus-infected subjects. IgG titres to the major HDM allergens showed no consistent increase and the overall IgG anti-HDM titres even declined in subjects without a virus infection. Anti-bacterial IgG antibodies in contrast to IgE did not change. Patients with frequent episodic or persistent asthma had similar IgE anti-bacterial titres to patients with infrequent asthma during the acute phase, although they had reduced IgG titres to both the bacteria and the HDM. CONCLUSIONS: During the period following an acute exacerbation of asthma there was a marked and specific increase in anti-bacterial IgE compared with a reduced IgE response to HDM. This provides further support for the concept of T-helper type 2 responses to bacterial antigens playing a role in asthma pathogenesis.

The radicals in cigarette tar: their nature and suggested physiological implications.

The paramagnetism of cigarette tar is found to be associated with at least four different types of species. One of the types is responsible for over 80 percent of the total paramagnetism and has a signal intensity that is independent of temperature from 60 to 250 K. This non-Curie-Weiss temperature dependence indicates that the principal paramagnetic species in tar is not an organic monoradical (doublet) species but instead is a donor-acceptor excimer with a paramagnetic excited state and a diamagnetic ground state. Modeling experiments suggest that the excimer consists of quinone (Q) and hydroquinone (QH2) molecules held in a tar matrix. Since such Q-QH2 species are catalysts for the oxidation of hydrocarbons and are very active redox systems, this paramagnetic species may be implicated in the cocarcinogenic properties of tar. Alternatively, since semiquinone radicals are known to bind to DNA, the tar paramagnetic species may be directly involved in the carcinogenic properties of tar.

Differences in the antibody response to a mucosal bacterial antigen between allergic and non-allergic subjects.

BACKGROUND: The immune response to bacterial antigens on mucosal surfaces may be modified in individuals allergic to aeroallergens due to a maturational or genetic difference or from the interaction between inhaled allergens and bacteria at the mucosa. METHODS: Plasma from children and adults allergic (n = 97) and non-allergic (n = 54) to aeroallergens were initially tested for IgG1 (Th1) and IgG4 (Th2) reactivity to P6, a conserved outer membrane protein of Haemophilus influenzae. IgE binding was measured for some allergic donors. The development of the antibody responses to P6 was subsequently examined in the plasma from 35 children aged 1, 2 and 5 years taken from a prospective birth cohort. RESULTS: IgG4 antibodies to P6 were more readily detected in allergic subjects than in non-allergic subjects (p<0.001), with a strong bias to the male gender. Some allergic subjects (35%) also had IgE antibody (1-10 ng/ml) that was not associated with IgG4 or gender. In the cohort study of infants, subjects who developed skin prick test positivity to mite allergens by 5 years of age had an 85% reduction in the IgG1 anti-P6 antibody at year 2 (p<0.05) and, unlike skin test negative infants, this group had IgG4 anti-P6 antibodies at 5 years of age. CONCLUSIONS: The antibodies of subjects allergic to a bacterial antigen included IgE and IgG4 (particularly for males) compared with the almost exclusive IgG1 response of non-allergic subjects. The IgG1 responses of 2-year-old children who became skin test positive was markedly reduced and P6-specific IgG4 became detectable at 5 years of age.

Orientation of the bacteriochlorophyll triplet and the primary ubiquinone acceptor of Rhodospirillum rubrum in membrane multilayers determined by ESR spectroscopy (I).

Chromatophores from Rhodospirillum rubrum were oriented as multilayers on quartz slides under reducing conditions. Irradiation of these multilayers in the resonance cavity of an ESR spectrometer at 6 K yielded the spectrum of the bacteriochlorophyll dimer triplet. The relative intesities of the main six lines of the triplet were dependent on the angle subtended by the direction of the external magnetic field with plane of the multilayers. The angular dependence of the intensities of these transitions can best be interpreted in terms of one of the principal axes of the triplet lying along the plane of the membrane while the other two axes are titled 10--20 degrees away from the parallel to and normal to the membrane directions. If we assume the porphyrin planes of the dimer to be parallel and the largest splitting of the triplet transitions to correspond to those transitions in a direction normal to this plane, then these data imply that the dimer planes are nearly perpendicular to the membrane plane. Purified iron-depleted phototrap complexes were similarly oriented in reconstituted phosphatidylcholine multilayers and the angular dependence of the light-induced spectrum recorded at room temperature. A computer analysis of this angular dependence suggests that the plane of the primary ubiquinone acceptor molecule is parallel to the plane of the membrane and therefore perpendicular to the donor.

In vivo interaction between nitrogenase molybdenum-iron protein and membrane in Azotobacter vinelandii and Rhodospirillum rubrum.

Oriented whole cell multilayers of Azotobacter vinelandii and Rhodospirillum rubrum were analyzed by electron spin resonance (ESR) spectroscopy to detect possible structural associations between nitrogenase molybdenum-iron (MoFe) protein and cytoplasmic or intracytoplasmic membrane. Initially, protocols were designed to obtain strong molybdenum-iron protein ESR signals in whole cell samples of each organism. Then, two-dimensional orientation of whole cell membranes was demonstrated in whole cell multilayers using doxyl stearate spin label in A. vinelandii and the bacteriochlorophyll a dimer triplet signal, (BCHl a)T2, from the intracytoplasmic membrane-bound photosynthetic apparatus of R. rubrum. Subsequent analysis of the low-field signals, g = 4.3 and g = 3.6, of molybdenum-iron protein in whole cell multilayers of each organism showed orientation-dependent characteristics, although the properties of each were different. Specifically, as the normal to the membrane plane was rotated from perpendicular to parallel with the ESR magnetic field, the amplitude of the g = 3.6 signal decreased from maximum to about 37% of maximum in A. vinelandii and from maximum to about 88% of maximum in R. rubrum. The angular dependence of the g = 4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to about 63% of maximum in R. rubrum. These data suggest that the molybdenum-iron protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association may exist between this nitrogenase component and membrane in these organisms.

Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters.

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S]+ clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-. The results indicate similar [4Fe-4S]+ clusters with analogous S = 1/2 and S = 3/2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1/2 and S = 3/2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1/2 and S = 3/2 forms of the [4Fe-4S]+ have been determined. Together with the VTMCD studies of [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-, which are shown to be predominantly S = 1/2 and 3/2, respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1/2 and S = 3/2 [4Fe-4S]+ clusters. Ground state zero-field splitting parameters for the S = 3/2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1/2 resonance. A rapidly relaxing axial resonance, g perpendicular = 1.94 and g parallel = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S]+ clusters in frozen solution is discussed in light of these results.

A role for CCL28-CCR3 in T-cell homing to the human upper airway mucosa.

Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

Strategies for controlling antibiotic use in a tertiary-care paediatric hospital.

The need to control antibiotic use has grown out of concern for costs and emerging resistance. The paediatric population poses some different opportunities and barriers for controlling antibiotic use. Several strategies to promote rational antibiotic use that have been introduced at The Hospital for Sick Children, Toronto, Ontario are presented. These include a formulary system, restricted drug program, therapeutic interchange program, sequential antibiotic therapy, automatic stop orders, antibiotic order forms, drug use evaluation program, guidelines for antibiotic use, standardized dosing, educational efforts, appropriate use of culture and sensitivity reporting, rational use of therapeutic drug monitoring and monitoring by pharmacists of antibiotic use.