Aberrant antibody responses to allergens and bacterial antigens in children during asthma exacerbations and convalescence
B. J. Hales, L. Pearce, L. A. Hazell, W. Smith, W. R. Thomas with Dr A. C. Martin Princess Margaret Hospital and Professor P. N. LeSouef, Dr I. A. Liang and Dr C. M. Hayden UWA School of Paediatrics and Child Health.

The IgE antibody responses to house dust mite allergens of children admitted to an emergency department for the treatment of asthma attacks were indistinguishable from the responses of children recruited from a community cohort by the presence of skin test reactivity. They had the same titres and predominance of antibody to the major Der p 1 and 2 allergens. Children recruited with exacerbation however had an almost complete absence of IgG1 and IgG4 antibody and this was further reduced in the children with persistent asthma or frequent episodes. There was no restoration of IgG antibody after convalescence although the IgE antibody to the allergens decreased. The most consistent change in recovery was an increase in the IgE antibody titre to the P6 antigen of Haemophilus influenzae possibly induced by a recrudescence of a low level infection. Like the allergens the IgG anti-P6 titres were low in the children with exacerbations and lowest in those with persistent asthma or frequent episodes.  Exacerbations associated with rhinovirus infection only had quantitative differences in the antibody responses to children without infection although some of these such as more rapid reductions of IgE titres could be fruitful avenues of investigation. The results show susceptibility of allergic children to asthma exacerbation is associated with defects in anti-microbial and anti-allergen responses.

Development defects in antimicrobial immunity in asthma
B. J. Hales, L. J. Pearce, L. A. Hazell, W. R. Thomas with M.M.H. Kusel, P. D. Sly and P. G. Holt from Cell Biology and Clinical Sciences

Antibody titres to the protective P6 antigen of the mucosal colonising bacteria Haemophilus influenzae were found to develop slowly in pre-school aged children destined to become atopic. Importantly the developmental defect was evident at 2 years of age and thus preceded the development of allergy. This combined with the low IgG antibody titres to P6 of children with frequent and persistent asthma provides a measure of generally altered immune responses and a prognostic marker of children at risk of developing allergy and asthma.  Detailed investigation is required to determine the causal relationships of the association. It has now been shown that reduced IgG antibody titres are also found for virulence-associated protein antigens of Pneumococcus pneumoniae as well as another protective antigen of H. influenzae, P4.

Interaction of allergic sensitisation and Pasteurella pneumotropica infection in mice.
 S. B.  See and W. R. Thomas

Pasteurella pneumotropica naturally infects the mucosa of mice in a similar fashion to the infection of humans with Haemophilus influenzae.  The characterisation of its outer membrane protein antigens P4, P6, P26 and D15 has previously been reported as well as immune responses to these proteins and the vaccine potential of the P4 and P6 antigens.  Allergic responses induced in mice sensitised by the inhalation of the papain allergen have now been shown to have at least two different effects on this infection. Firstly allergic reactions in mice that had recovered from infection induced a transient recurrence of the infection in the lungs, an event consistent with notion that allergy may increase susceptibility to infection. In contrast, sensitised mice infected with P. pneumotropica shortly after a challenge with allergen had increased resistance to the infection and made higher IgG antibody responses including the Th1 dependent subclass. The bacterial infection also prolonged the eosinophilia found in the lungs.

Antibodies to rhinovirus antigens
B. J. Hales, S. C. Lee, L. A. Hazell, W. Smith, T. K. Heinrich with Dr I. A. Liang and Professor P. N. LeSouef, and Dr C. M. Hayden UWA School of Paediatrics and Child Health.

Rhinovirus infection frequently exacerbates asthma and may have a role in the development of sensitisation.  Immunological studies have however been limited partially because of the lack of suitable antigens and antibody assays have been limited to measuring the largely isolate-specific neutralising antibodies. While neutralising antibodies are important for the development of resistance to reinfection with the same serotype it is likely that the bulk of the immune response and its accompanying immunopathological effects is due to the many antigenic determinants that do not react with neutralising antibodies and are shared by similar isolates. To explore this and test the feasibility of performing antibody-isotype assays the VP1 capsid protein was made as a recombinant polypeptide expressed in E. coli. A representative from the type A and type B families of the rhinovirus was made in the expectation that the 80% sequence identity within each group will allow sufficient cross reactivity to detect responses to the isolates that infect children.  Antibody assays with the antigens showed high titre reactions for IgG1 antibody from children and the reactivity to the more ubiquitous type A protein was more frequent than the type B protein.  Sera were frequently specific for either the type A or the type B showing a low degree of cross reactivity and the unlikely potential of cross reactivity with even less similar members of the picornoviridae family.

Cat allergens
W. Smith , S. E. O'Neil, L. A. Hazell, B. J. Hales, W. R. Thomas, T. K. Heinrich and S. Piboonpocanun, Mahidol University, Bangkok

Although it probable that the major cat allergen Fel d 1 is the target of most of the IgE binding activity of cat dander there evidence for frequent IgE binding by other unidentified allergens especially from salivary proteins. The contribution of these allergens to allergic response to the cat is unknown and may be particularly relevant for people with rhinoconjunctivitis who have been shown in three independent studies to have low IgE titres to the major allergen Fel d 1. Previous analysis of cDNA libraries from different cat tissues has revealed the salivary lipocalin Fel d 4, which has frequent but generally low IgE binding.  In addition to these allergens a number of other potential cat allergens have now been tested for IgE and IgG antibody binding. These include cat albumin aka Fel d 2, the cystatin Fel d 3 allergen, the breast and salivary expressed (BASE) protein, haptoglobin and S100A12. The new IgE binding proteins were identified from IgE screening assays or in the case of BASE its homology with a major horse allergen. The folding of recombinant allergens was authenticated by circular dichroism. The most important allergens were Fel d 1, Fel d 4 and Fel d 2. For most of the sample representing predominantly adults with rhinoconjunctivitis the titres to Fel d 1 and Fel d 4 were similar although a minority (8%) had high titres to Fel d 1.  Very high titres of IgG1 antibody were consistently detected which on average were higher in the non-allergic group. No IgG1 antibodies to the other IgE-binding proteins were detected whereas for the bulk of the subjects, the IgG4 antibodies to Fel d 1 and Fel d 4 were similar and higher in allergic subjects.  The less potent allergens were not reactive. These results not only show the significance of Fel d 4 in addition to Fel d 1 but contrast markedly to mite allergy where IgG antibody is restricted to allergic subjects and the relative presence of the different IgG subclasses is the same for all allergens.

Bystander allergy and sublingual desensitisation of allergic responses to cysteine protease antigens
P. T. Cunningham, C. E. Elliot and W. R. Thomas with P.G. Holt Cell Biology

A model of inhalation allergy to the cysteine protease papain has been used to develop new desensitisation strategies relevant to the biochemically homologous Der p 1 allergen. The intranasal administration of activated papain induces persistent and boostable IgE antibody and sensitises for Th2 type lung inflammatory responses.  Sublingual desensitisation of primed mice has been shown to inhibit the ability to induce further IgE antibody and the release of IL-5 into the serum after an inhalation challenge but this was largely dependent on the use of activated papain.
Similar desensitisation with greatly diminished lung pathology could also be achieved by sublingual administration of non-activated papain provided that it was absorbed to chitosan nanoparticles.  The administration of the activated papain with Der p 2 was found to produce lasting allergic responses to Der p 2 instead of the usual transient reactions.

Anti-microbial peptides
T. K. Heinrich, T. L. Chai, with P. M. Watt, Drug Development and G. R. Pocock, Microbial Dynamics.

Acinetobacter baumannii is an important cause of infection of intensive care patients such as burns victims. It is of major concern because it has an intrinsic ability to develop antibiotic resistance and some isolates are resistant to all of the antibiotics available today. This bacterium is being used as a target to produce new types of peptide-based antibiotics. Most existing work on peptide antibiotics for other microorganism has examined naturally occurring peptides or peptides based on their structural motifs.  Here we have used a phage display strategy intended to produce a new range of peptides. A PCR based technique was used to produce 50-200 base pair random fragments from the genomes of a diverse range of bacteria and archea and these were used to construct a phage display library in the lytic T7 bacteriophage. Phage enriched from the library by cycles of absorption and elution from the bacteria were screened for binding activity to the bacteria by ELISA and synthetic peptides were made to represent the peptide encoded by the positive phage. A number of structurally distinct peptides with anti-microbial activity in the low micromolar range have been identified. Peptides were active when they were synthesised as proteolytic resistant retroinversopeptides and many had low toxicity for human cells. The peptides were shown to have varying mechanisms of action as demonstrated by their bacteriocidal and membrane depolarision activities. Clinical isolates resistant to the standard antibiotics were susceptible.