Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Allergen measurements are used extensively in the formulation of allergy diagnostics and vaccines, yet no purified international allergen standards are available for calibration purposes. The aims of the European Union CREATE project were to develop international standards with verifiable allergen content. Purified natural and recombinant allergens were analyzed by means of SDS-PAGE, mass spectrometry, circular dichroism spectra, and small-angle x-ray scattering. IgE reactivity was assessed by means of direct RAST, RAST inhibition, immunoblotting, and basophil histamine release with sera from 961 allergic patients. Three recombinant allergens, rBet v 1, rPh1 p 5a, and rDer p 2, were structurally indistinguishable from their natural counterparts and showed excellent IgE reactivity suitable for use as certified reference materials. A second tier of allergens (rPhl p 5b, rOle e1, rDer p 1, rDer f 1, and rDer f 2) was identified that could provide suitable candidates for certified reference materials with minor improvements to the recombinant proteins. Only rPhl p I was considered unsuitable as a reference material. Quantitative ELISAs were identified that accurately measured each allergen, except for rPhl p 1. The CREATE project has provided a major step forward in allergen standardization and provides a model for the development of a comprehensive panel of international reference preparations that will harmonize allergen measurements worldwide. (J Allergy Clin Immunol 2008;122:882-9.)

The sentence beginning in line 8 of the abstract shouldread: ‘‘Expression of mRNA for other cytokines and inflam-matory mediators, IL-1a, IL-6, TGFb1, TNFa, MIP-1a,MIP-2, JE, KC, PDGFa and PDGFb although constitutivelyproduced was found to be up-regulated in injured adult murinesamples compared to normal control samples.’’

Despite binding to receptors distinct from those of type I interferons (IFNs), human interleukins-28A, -28B and -29 (IL-28A, IL-28B and IL-29; alternatively named IFN lambda-2 {IFN-λ2}, IFN-λ3 and IFN-λ1, respectively, or collectively, type III IFNs), a small family of three structurally-related cytokines, are, like IFNs, known to induce antiviral activity. To further biologically characterize IL-28A and IL-29, we compared their activities with those of IFNs in a range of human cell lines. We found that they induced antiviral activity in fewer cell lines and more weakly than IFNs; also IL-28A was less active than IL-29. Additionally, we showed IL-28A and IL-29 induced reporter genes – protein MxA promoter linked to luciferase, or interferon stimulated response element (ISRE) linked to secreted alkaline phosphatase (SEAP) – more weakly than IFN. Antiproliferative activity was induced by IFNs in most cell lines, but only in one human glioblastoma cell line, LN319, was dose-dependent IL-29-growth inhibition demonstrable. Polymerase chain reaction (PCR) quantification of messenger (m) RNA of IL-28/29 receptor subunits, IL-28Rα and IL-10Rβ, indicated variable expression levels; although their expression was highest in the responsive LN319 cell line, lower but significant expression of both mRNAs was found in relatively unresponsive cell lines. In conclusion, we found IL-28A and IL-29 act similarly to IFNs, but are less effective generally and have activity in a more limited range of cell lines.

Inflammatory mediators have been shown to play a major role in the complex series of co-ordinated events that occur in wound healing responses following injury. However, to date most of the studies carried out have addressed the expression, interactions and role of only one or two cytokines that are thought to be involved in wound repair. This study has evaluated, in murine skin samples taken at 0, 3, 12, 18, 24, 48, 72, 120 and 168 h post-wounding, the expression of a wide range of cytokines with potential for a role in wound repair. Various techniques (reverse transcription polymerase chain reaction (RT-PCR), bioassays and ELISA) were used to evaluate cytokine expression in these samples at both the mRNA and protein expressions level. Semi-quantitative analysis using RT-PCR revealed that IL-1beta, IP10, bFGF, and TGFbeta3 up-regulated in wounded samples, compared to non-injured control samples. Expression of mRNA for other cytokines and inflammatory mediators, IL-1alpha, IL-6, TGFbeta1, TNFalpha, MIP-1alpha, MIP-2, JE, KC, PDGFalpha and PDGFbeta, were found to be down-regulated in injured adult murine samples compared to normal control samples. Interestingly we failed to find evidence of mRNA expression for the cytokines IL-2, IL-4, IL-12, GM-CSF, IFNgamma and RANTES, in both non-injured and injured samples. These observations were also generally supported by the results obtained using bioassays for IL-1 and IL-6 and ELISA for IL-1alpha, IL-1beta, IL-6, TNFalpha, and IFNgamma.