Tatjana Heinrich

Antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children

Background Infants who develop house dust mite (HDM) allergy and HDM-sensitised children with severe persistent asthma have low antibody responses to the P6 antigen of Haemophilus influenzae. Objective To measure the development of antibody to two ubiquitous bacteria of the respiratory mucosa in a prospective birth cohort at high risk of allergic disease and to assess which responses are associated with asthma and atopy. Methods IgG1 and IgG4 antibody to H influenzae (P4 and P6) and Streptoccocus pneumoniae (PspA and PspC) surface antigens was measured in yearly blood samples of children aged 1–5 years. IgE to the P6 antigen was examined for the 5-year group. The children were stratified based on HDM sensitisation and asthma at 5 years of age. Results HDM-sensitised children had lower IgG1 antibody titres to the bacterial antigens, and early responses (<3 years and before the development of HDM sensitisation and asthma) corrected for multiple antigens were significantly reduced for P4, P6 and PspC (p=0.008, p=0.004 and p=0.028, respectively). Similar associations with asthma were also found (p=0.008, p=0.004 and p=0.032 for P4, P6 and PspC, respectively). The IgG4 antibody titre and prevalence were similar in both HDM-sensitised and non-sensitised groups, but sensitised children had a slower downregulation of the IgG4 response. Children with asthma (27/145 at 5 years) had lower anti-P6 IgE responses (p<0.05). Conclusions HDM-sensitised children have early defective antibody responses to bacteria that are associated with asthma. Surprisingly, antibacterial IgE was associated with a reduced risk for asthma.

The chitinase allergens Der p 15 and Der p 18 from Dermatophagoides pteronyssinus

Background House dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae cause allergic disease in humans as well as in dogs. In geographical regions where the two mite species coexist, they both elicit specific immunoglobulin (Ig E) responses in humans whereas dogs preferentially react to D. farinae extracts. In dogs the main IgE binding is directed to the D. farinae chitinase allergens Der f 15 and Der f 18 and not to the groups 1 and 2 allergens as found for humans. Although the IgE response of humans to Der f 18 has been investigated there is no report on Der f 15‐specific IgE in humans.

Pyroglyphid House Dust Mite Allergens

Mites of the family Pyroglyphidae are the most important source of house dust mite allergens. A small number of allergens, namely those of groups 1, 2, 4, 5 and 7 constitute the known major and mid-potency specificities, with possible important contributions of the groups 11, 14 and 15 requiring further definition. Most of the allergens can be identified by sequence homologies and the structures of the major allergens have been solved. There are however challenges in determining the nature of the group 5 and 7 allergens and in obtaining detailed structures of the significant allergens to be used for genetic engineering.

Maternal Antibodies to Pneumolysin but Not to Pneumococcal Surface Protein A Delay Early Pneumococcal Carriage in High-Risk Papua New Guinean Infants

ABSTRACT Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (ρ = 0.824, P < 0.001), PspA1 (ρ = 0.746, P < 0.001), and PspA2 (ρ = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.

GFP-complementation assay to detect functional CPP and protein delivery into living cells

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

Protein silencing with Phylomers: a new tool for target validation and generating lead biologicals targeting protein interactions

As the field of proteomics expands, the need for functional annotation of the proteome increases to allow the rational choice of particular targets from amongst a bewildering and increasing set of candidates. Similarly, the description of the interactome will continue to generate a plethora of candidate interactions that will need to be validated in functional assays. Although knockout mice and RNAi knockdowns have proven to be invaluable as primary tools in functional genomics, it should be remembered that these techniques apply at the gene and transcript level respectively and are, therefore, not always suitable for true functional proteomics investigations. Protein levels do not always correlate with RNA levels, particularly where active protein levels are regulated by protein stability or post-translational modifications. Moreover, off-target regulation of genes by RNAi/siRNA can complicate the validation of targets using this approach. In recent years, a myriad of potential protein targets including complexes has been emerging from high-throughput proteomics. Thus, the dilemma facing many investigators is having too many potential targets and too few means of validating them. Therefore, there is an unmet need for the development of technologies which allow the targeted disruption of particular proteins. Intracellular antibodies (intrabodies) and peptide aptamers are beginning to be applied in this area. A new class of peptide, known as Phylomers®, also has significant potential in the area of disruption of protein–protein interactions. Phylomers, which are derived from protein subdomains, are small enough to synthesise and are of a suitable size for delivery to tissues and even into cells, making them an ideal candidates for a next-generation tool for functional validation of the proteome.

Determinants of house dust mite allergenicity

Results: The studies summarized here show that quantitative measurements of allergen panels provide a better assessment of total anti-mite IgE than assays with mite extracts, and that major allergen Der p 1 induced correspondingly higher T helper cell type 2 (Th2) and Th1 cytokine responses than subordinate allergens Der p 5 and 7. The importance of the knowledge of variants of the major allergens is presented as well recent advances in the characterization of the molecules. Conclusions: Der p 1 and Der p 2 are major allergens of Dermatophagoides pteronyssinus. The minor allergens, however, constitute 50% of the IgE binding and the use of a panel of allergens provides a better measure of house dust mite allergy than IgE binding to extracts. The minor allergens will also be important for studying the immunoregulatory pathways that lead to high and low allergenicity.

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Sensitisation to Airborne Environmental Allergens: What Do We Know and What are the Problems?

The most widely distributed sources of allergens are the pyroglyphid Dermato phagoides pteronyssinus and Dermatophagoides farinae mites [1], temperate grass pollens [2] and cats [3]. Other important allergens with less global distributions are birch [4], olive [5], ragweed and mugwort pollens [6]. Cockroach allergy is important for inner-city dwellers in America [7]. Dog allergy has been more evident in regions with low exposure to other allergens but is also a frequent source of sensitisation elsewhere [8]. The glycyphagid mite Blomia tropicalis is important in highly populated tropical and subtropical regions [9]. The conifers Japanese cedar in Japan and mountain cedar in USA and to a lesser degree cypress are regionally important [10]. Allergens from Aspergillus, Alternaria, Cladosporium and Penicillium moulds sensitise 5–10% of most populations and are associated with severe asthma [11]. Emerging sources of sensitisation are domestic exposure to mice in inner city environments, and pollens from the weeds Salsola kali (Russian thistle or tumble weed) and Chenopodium album (lambs quarter or goosefoot) [5]. The pollens occur worldwide but have attracted interest in areas of desertification.