Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens

This study describes the three-dimensional crystal structure of a non-specific lipid transport protein (ns-LTP) from Rosaceae. Whilst ns-LTPs from species other than Rosaceae, such as nuts, cereals, grape, oranges and vegetables are also responsible for plant food allergies, this is less frequent compared with ns-LTPs from Rosaceae in the Mediterranean area. In this heterologously expressed peach Pru p3, a ligand is present inside the central cavity of the protein, presumably a fatty acid that was present or produced in the culture medium of the expression organism Escherichia coli. Moreover, the two molecules of ns-LTP present in the asymmetric unit bind this ligand in a different way, suggesting a significant degree of plasticity for the peach ns-LTP binding cavity, despite the presence of four disulphide bridges. Two molecules are present in the asymmetric unit: molecule A is a fully liganded protein, while molecule B apparently represents a partially liganded state. Also, molecular dynamics simulation, along with other evidence, suggests that these two molecular conformations represent different states in solution. Comparison of the 3D models of different ns-LTPs justifies the evidence of a high degree of conservation of the putative IgE binding epitopes among proteins of the Rosaceae family and the presence of significant amino acid replacements in correspondence of the same regions in ns-LTPs of botanical species unrelated to Rosaceae.     

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D‐SDS‐PAGE, digested in‐gel based on molecular weight, and identified using nano‐LC‐MS/MS. In the postprandial rat intestinal lymph, nine bovine‐specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as β‐lactoglobulin and caseins. Seven of the nine identified bovine‐specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross‐barrier transport or translocation.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

Air cleaners can reduce the risk of allergic sensitization

Exposure to house-dust mite allergens is a factor in the development of allergic symptoms in atopic patients. Several measures can be proposed to control indoor allergen levels, inducing a clinical benefit. The use of an air cleaner is one simple way of achieving this goal. We conducted a simulation trial in a proper room, to verify the usefulness of a domestic cleaner, based on mechanical air filtration, to reduce the levels of environmental allergens. We checked the presence of mite components by different methods (Aclotest and Der p 1 ELISA), in dust recovered before and after using the air cleaner. Our results indicate that this approach could be useful in significantly lowering the levels of mite allergens.     

Penicillium andAspergillus species in the habitats of allergy patients in the Tulsa, Oklahoma area

Penicillium andAspergillus have been recognized as important aeroallergens for more than 30 years, and are especially significant in indoor environments. There are over 400 species ofPenicillium andAspergillus combined, but there is little information on which species occur most frequently in the environment, or if each exhibits unique allergenic properties. A preliminary study showed no overlap between those species isolated from an outdoor site in Tulsa, Oklahoma and the species used in immunotherapy at allergy clinics in the Tulsa area. Pursuing this line of research, air samples were collected as three seasonal samples (over a 6 month period) in the homes or offices of ten allergy patients known to be allergic toPenicillium and/orAspergillus. Twenty three species ofPenicillium and 12 species ofAspergillus were identified from these samples through isolation, macroscopic, and microscopic examination.Penicillium corylophilum, P. glabrum, Aspergillus niger, andA. flavipes were the most abundant species isolated, supporting the data obtained in a preliminary study. At least in the Tulsa area, it appears that atopic patients are being tested and treated with extracts ofPenicillium andAspergillus species that are either not present or not abundant in the local indoor or outdoor environments. Additional research is necessary to determine if the environmental isolates share allergens with those species used in immunotherapy.     

异位性皮炎患者血清变应原过筛检测结果及其临床意义

目的:探讨异位性皮炎(AD)患者的血清嗜酸细胞阳离子蛋白(ECP)、总IgE、吸入性变应原Phadiatop和食物性变应原fx5E间的相关性。方法:随机选取我院门诊就诊和住院治疗的AD患者60例,选取健康体检者30例为对照组,同期检测ECP、总IgE、Phadiatop和fx5E血清浓度并比较。结果:AD患者血清ECP和总IgE水平均明显高于正常对照组(P<0.05);患者组中血清ECP与总IgE之间无相关性;Phadiatop在患者组中阳性率为86.67%,fx5E阳性率为18.33%。结论:AD患者血清ECP水平是反映嗜酸粒细胞活化和过敏状态的敏感指标,能更有效地监测AD患者病情变化与疗效。吸入性和食物性变应原均可引起AD,尤以吸入性变应原为主。对AD患者进行CAP系统中变应原过筛检测,是AD防治的重要手段之一。     

New immunodiagnostic system

We have developed a new immunodiagnostic system whichmeasures personal allergen exposure and which can beused to identify allergens.Personal exposure is directly sampled using inertialimpaction filters which fit just inside the nose andcollect particles (mainly >5 µm) inhaled duringnormal respiration. These samplers provide both anindex of personal exposure as well as being aninexpensive and portable sampling system.The particles are captured on adhesive tapes which arethen laminated with a protein-binding membrane. Theallergens eluting from the particles are bound by the membrane in theperiphery of each particle. The systemthen uses either allergen-specific monoclonalantibodies or the subject's IgE as primary probes toimmuno-label the `halo' of allergen around individualallergen-containing particles. Such an assay is verysensitive and can detect a single particle carryingallergens. In addition, the system providesinformation on the size, shape and allergen content ofthe particles. Because the particles carryingallergens can be seen, high resolution video images ofpollen grains and fungal spores can be subjected to atraditional morphological study or a range of featureextraction routines. This information can be comparedto a database of some known allergenic pollen grainsand fungal spores which we have also assembled tofacilitate their identification.When using monoclonal antibodies as the probe, thesystem determines the amount of allergen the subjectis exposed to and the characteristics of the particles(size, shape, etc). When using the subject's IgE as theprobe, the system allows visualisation of the allergensources that an individual is allergic to. The systemmay have clinical applications in quantifying personalexposure as well as identifying allergens anddetermining exposure to unsuspected allergens.     

β-Lactam drug allergens: Fine structural recognition patterns of cephalosporin-reactive IgE antibodies

Lack of experimental findings on the spectrum of cephalosporin allergenic determinants has hindered diagnosis of adverse reactions to these drugs and retarded understanding of allergenic cross-reactions between cephalosporins and between cephalosporins and penicillins. Subjects allergic to the widely used cephalosporin antibiotic cefaclor have serum immuno globulin (Ig) E antibodies that react with the drug. Quantitative hapten inhibition studies employing sera from subjects allergic to cefaclor revealed fine structual recognition differences between the combining site specificities of cefaclor-reactive IgE antibodies in the sera of different subjects. Unlike penicillins, where discrete side chain or thiazolidine ring determinants alone may be recognized, IgE binding determinants on cefaclor encompassed the entire molecule. Fine structural recognition specificity differences at positions R₁ (side-chain) and R₂ (substituent attached to dihydrothiazine ring) were detected between IgE antibodies in different sera. Some antibodies showed clear preferential recognition of the aminobenzyl group at position R₁ and Cl at R₂ while with others, a greater degree of recognition tolerance was seen at R₁ where, for example, the aminohydroxybenzyl or aminodihydrobenzyl groups were recognized, and at R₂ where a methyl or even an ester group was tolerated. As with the penicillins, cephalosporins as allergens cannot simply be considered as a group of compounds with a common allergenic determinant structure. IgE antibodies that bind to cefaclor show great heterogeneity indicated by clear, fine structural differences in recognition of the R₁ and R₂ groups on the drug.     

Effects of beta-lactam antibiotics on eukaryotic cells

A symposium on effects of beta-lactam antibiotics on eukaryotic cells was held as part of the 9th International Congress of Infectious and Parasitic Diseases (Munich, Germany, July 1986). This symposium provided an opportunity to review recent work on the effect of beta-lactam structures on mammalian cells in culture and to speculate on possible clinical implications. This paper is a comment on the subject matter covered by the symposium papers which follow.Abbreviations Ara-C cytosine arabinoside - BLA beta-lactam antibiotic     

Enzyme antigens associated with pigeon breeder's disease. II. Isolation and characterization of acidic hydrolases

The hydrolytic enzymes in pigeon dropping extracts (PDE) were separated into basic and acidic components by DEAE-cellulose chromatography. Six distinct hydrolytic activities were isolated from the acidic group of enzymes. Elastase, trypsin, and two forms of collagenase were the proteases identified. An esterase and a phospholipase were also detected. The sera of symptomatic pigeon breeders, analyzed by crossed immunoelectrofocusing techniques, were shown to contain antibodies to the enzymes trypsin, collagenase, and esterase in the acidic fractions of PDE by staining the immune precipitates with specific chromogenic substrates. These enzymes were purified by preparative isoelectric focusing, affinity chromatography, and gel filtration. The purified elastase hydrolyzed elastin and consisted of a single polypeptide chain (M _r =23,100). Trypsin hydrolyzed a synthetic arginine substrate, but not elastin or collagen. Its size (M _r =27,500) and subunit structure were similar to those reported here for elastase. Both enzymes were inhibited by -1-antitrypsin,N-p-tosyl-L-lysine chloromethylketone, and phenylmethylsulfonyl fluoride. Two distinct collagenases were found; both cleaved bovine collagen. The high-molecular-weight (HMW) collagenase was a glycoprotein (M _r =117,500) and consisted of three polypeptide chains (M _r =37,100); a low-molecular-weight (LMW) collagenase (M _r =12,500) was also isolated. The HMW collagenase was inhibited byEDTA,p-chloromercuribenzoic acid, and phenylmethylsulfonyl fluoride, but not by -1-antitrypsin. The source of these enzymes as well as their relationship to the basic hydrolytic enzymes of PDE are discussed.