Allergenic Can f 1 and its human homologue Lcn‐1 direct dendritic cells to induce divergent immune responses

Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. With two homologous lipocalins, the major respiratory dog allergen Can f 1 and the human endogenous, non‐allergenic Lipocalin‐1, we investigated their impact on human monocyte‐derived dendritic cells (DC). The two lipocalins had differential effects on DC according to their allergenic potential. Compared to Lipocalin‐1, Can f 1 persistently induced lower levels of the Th1 skewing maturation marker expression, tryptophan breakdown and interleukin (IL)‐12 production in DC. As a consequence, T cells stimulated by DC treated with Can f 1 produced more of the Th2 signature cytokine IL‐13 and lower levels of the Th1 signature cytokine interferon‐γ than T cells stimulated by Lipocalin‐1 treated DC. These data were partially verified by a second pair of homologous lipocalins, the cat allergen Fel d 4 and its putative human homologue major urinary protein. Our data indicate that the crosstalk of DC with lipocalins alone has the potential to direct the type of immune response to these particular antigens. A global gene expression analysis further supported these results and indicated significant differences in intracellular trafficking, sorting and antigen presentation pathways when comparing Can f 1 and Lipocalin‐1 stimulated DC. With this study we contribute to a better understanding of the induction phase of a Th2 immune response.     

Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen

In the last few years Ostrya carpinifolia pollen is consideredas an important cause of respiratoryallergy in Mediterranean areas. The concentration ofthe pollen was measured over a period of fifteen yearsfrom 1981 to 1996 in an area around Genoa; the resultsof this study have clearly indicated an increasingtrend that correlate with persons sensitization.In this study we sought to define the immunochemical andbiochemical properties of hop-hornbean pollen. Soluble proteins extracted from Ostryacarpinifolia pollen and from the taxonomicallyrelated species Corylus Avellana, were analyzedby polyacrylamide gel electrophoresis (SDS-PAGE), byhorizontal isoelectrofocusing (IEF) and by twodimensions electrophoresis (2D-PAGE). Allergenicproteins were identified with sera of sensibilizedpatients and cross-reactivity was evaluated byimmunoblotting techniques. The electrophoreticanalysis showed a partial identity between theproteins from Ostrya and Corylus extracts. The immunoblotting assay, developed withhuman IgE from subjects allergic to hop-hornbeampollen, displayed the major IgE reactivity for acomponent with a molecular weight of 17 kDa expressedin both Ostrya and Corylus extracts. This reactivity is consistent with the presence ofBet v 1 that is described as the major pollen allergenin the Betulaceae and Corylaceae families. Sera fromsubjects allergic to Ostrya were then preadsorbed with recombinant Bet v 1 immobilized in the Pharmacia CAP System; a significant reduction ofthe IgE binding activity was observed after thetreatment. We therefore suggest that Bet v 1 couldbe one of the allergenic proteins present in theOstrya pollen possibly being responsible forcross-reactivity with other members of taxonomicallyrelated families.     

重组红火蚁毒素致敏原Sol i1的表达及活性分析

【目的】为研究红火蚁Solenopsis invicta Buren毒素蛋白致病分子机理,制备用于红火蚁蜇伤防治的制剂。【方法】本研究采用反转录PCR（RT-PCR）与套式PCR（nPCR）扩增出红火蚁体内毒素蛋白Sol i1全基因及其活性基因片段Sol i1a,进行测序与序列分析,构建重组质粒Sol i1-pET43.1a 和 Sol i1a-pET43.1a, 经PCR、酶切和测序鉴定后转化BL21（DE3）进行IPTG诱导表达,对表达产物进行SDS-PAGE分析和Western blot检测后,用亲合层析法纯化,并将纯化的重组蛋白通过动物试验进行了致敏活性分析以及过敏体质治疗研究。【结果】本研究克隆的Sol i1基因序列与GenBank中红火蚁序列同源性为99%,原核表达预期大小的2种重组蛋白能与组氨酸单抗发生特异性反应,且具有较高的致敏活性和良好的免疫治疗效果。【结论】原核表达的2种重组蛋白Sol i1和 Sol i1a具有良好的生物活性,为红火蚁蜇伤的致病机理和防治研究奠定了基础。     

Malachite green treatment of industrial Penicillium chrysogenum protoplasts results in increased penicillin-V formation

We attempted protoplast fusion in order to generate gene transfer between an industrial strain of Penicillium chrysogenum and a fission yeast, Schizosaccharomyces pombe. The Penicillium strain was treated with malachite green. The S. pombe strain was auxotrophic for lysine. The regenerated colonies showed Penicillium morphology. The number of Penicillium colonies was significantly higher when the inactivated Penicillium protoplasts were fused to S. pombe protoplasts than in the self-fusion control experiments. We randomly isolated colonies from the regeneration plates and measured beta-lactam formation in cultures from shaken flasks. Antibiotic production was increased in colonies originated from the malachite green-treated protoplasts. Received 2 June 1998/ Accepted in revised form 30 November 1998     

Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.     

Ragweed and mugwort pollen in Szczecin, Poland

Ragweed (genus Ambrosia) and mugwort (Artemisia vulgaris) pollen grains are known to be very potent aeroallergens, often noted to enter into cross reactions. The aim of the study was to analyse ragweed and mugwort pollen release in Szczecin (western Poland) during the period 2000–2003. Measurements were performed by the volumetric and gravimetric method. Pollen seasons were defined as the periods of 90% of the total catch. Of the 4 years studied, the lowest concentration of ragweed pollen was observed in 2000. In 2000, the annual ragweed pollen count was very high, threefold higher than in 2001. There was a high Ambrosia pollen count in 2003, with the highest daily value of 84 grains/m³. The mugwort pollen season started in the third 10-day period of July and lasted to the end of August in all of the years studied. Analysis of pollen deposition from different Szczecin city’s districts showed that the highest exposure to ragweed pollen allergens occurred in the Majowe district, which is related to the presence of numerous plants of Ambrosia in that district. The mugwort pollen deposition was more abundant in the Żelechowa district, which is an area with villas and gardens. Statistically significant correlations were found between the ragweed pollen count in the air and the maximum wind speed, air temperature and relative humidity and between the mugwort pollen count in the air and air temperature and relative humidity.     

Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.     

The Occurrence of Ferredoxin-sulfite Reductase in Barley Roots

A simple method was established for determining 10 preservatives, butylhydroxyanisole and dibutylhydroxytoluene in food. Steam distillation was carried out, and the distillate was trapped with dichloromethane and distilled water. After acidification and addition of sodium chloride, food additives were extracted from aqueous phase with dichloromethane. The food additives were analyzed with a gas Chromatograph equipped with a dual column system of 10% FFAP and 5% DEGS + 1% H₃PO₄. Column temperature was increased from 140 to 210°C at the rate of 3°C/min, Fluorene was used as an internal standard.

Ethyl p-hydroxybenzoate and isopropyl p-hydroxybenzoate were not separated with the 10% FFAP column, but other food additives were simultaneously determined with this column. With the 5% DEGS + 1% H₃PO₄ column, isobutyl p-hydroxybenzoate and propyl p-hydroxybenzoate were not separated, but the others were simultaneously determined.

Added recovery tests were carried out on about 38 foods.     

Conformation of Pinan-3-ol Derivatives

We report here the isolation and molecular characterization of single-chain Fv (scFv) antibodies raised against two major allergens, Cryj1 and Cryj2, in the pollen of Cryptomeria japonica by the phage display method. Recombinant phages that produced scFv antibodies that bound to Cryj1 or Cryj2 were isolated by selection with immobilized antigens in microtiter plates. After selection of six Cryj1- and four Cryj2-specific scFv antibodies with strong binding activity, we performed pairwise interaction analysis of them by surface plasmon resonance. The analysis revealed that the scFv antibodies against Cryj1 bound to only four non-overlapping epitopes, with dissociation constants that ranged from 4.84×10^?9 M to 1.62×10^?7 M. By contrast, four Cryj2-specific scFv antibodies inhibited each other’s binding to Cryj2, with dissociation constants from 1.11×10^?7 M to 4.21×10^?7 M. Our results indicate that recombinant technology provides a time-saving method for the production of antibodies against pollen allergens.     

Changes in atmospheric CO2 influence the allergenicity of Aspergillus fumigatus

Increased susceptibility to allergies has been documented in the Western world in recent decades. However, a comprehensive understanding of its causes is not yet available. It is therefore essential to understand trends and mechanisms of allergy‐inducing agents, such as fungal conidia. In this study, we investigated the hypothesis that environmental conditions linked to global atmospheric changes can affect the allergenicity of Aspergillus fumigatus, a common allergenic fungal species in indoor and outdoor environments and in airborne particulate matter. We show that fungi grown under present‐day CO₂ levels (392 ppm) exhibit 8.5 and 3.5 fold higher allergenicity compared to fungi grown at preindustrial (280 ppm) and double (560 ppm) CO₂ levels, respectively. A corresponding trend is observed in the expression of genes encoding for known allergenic proteins and in the major allergen Asp f1 concentrations, possibly due to physiological changes such as respiration rates and the nitrogen content of the fungus, influenced by the CO₂ concentrations. Increased carbon and nitrogen levels in the growth medium also lead to a significant increase in the allergenicity. We propose that climatic changes such as increasing atmospheric CO₂ levels and changes in the fungal growth medium may impact the ability of allergenic fungi such as A. fumigatus to induce allergies.