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Background CD36 isoantibodies are capable of inducing neonatal alloimmune thrombocytopenia (NAIT) and platelet refractoriness. As to now the CD36 type I deficiency has been reported in East Asian and African individuals. However, it is virtually unknown in Caucasians. The aim of this study was to display the prevalence of the CD36 deficiency within parts of the Arabian population in Germany. Secondly, we are presenting the case of a newborn suffering from NAIT which was induced by CD36 antibody. Methods Platelet (p) CD36 was determined by flow cytometry on 1328 samples mainly from individuals of Arabian origin and a family with a neonate affected by NAIT. DNA sequencing was performed on all pCD36-negative samples. Results Thirty-five (2.64%) of all donor samples were pCD36 negative, 19 (1.43%) had a weak expression. Including only individuals from the Arabian peninsula, frequencies were 3.39% and 1.75%, respectively. CD36 type I deficiency on both platelets and monocytes combined with a CD36 isoantibody were detected in the mother of the NAIT baby. The baby was successfully transfused with two HPA-unselected platelet concentrates. In case of need, two platelet units with a weak pCD36 expression were on hand. A total of 45 different CD36 mutations were detected within pCD36-negative individuals, some being homozygous, most of them only present on one allele. Conclusion The CD36-negative phenotype is present in a significant number of individuals of Arabian origin and enables CD36 isoimmunization in NAIT or refractoriness. Blood transfusion services should be aware of such cases.

Background and objectives Colloid osmotic pressure (COP) is a principal determinant of intravascular fluid homeostasis and a pillar of fluid therapy and transfusion. Transfusion-associated circulatory overload (TACO) is a leading complication of transfusion, and COP could be responsible for recruiting additional fluid. Study objective was to measure COP of blood products as well as investigate the effects of product concentration and storage lesion on COP. Materials and methods Three units of each product were sampled longitudinally. COP was measured directly as well as the determinants thereof albumin and total protein. Conventional blood products, that is red blood cell (RBC), fresh-frozen plasma (FFP) and platelet concentrates (PLTs), were compared with their concentrated counterparts: volume-reduced RBCs, hyperconcentrated PLTs, and fully and partially reconstituted lyophilized plasma (prLP). Fresh and maximally stored products were measured to determine changes in protein and COP. We calculated potential volume load (PVL) to estimate volume recruited using albumin's water binding per product. Results Colloid osmotic pressure varies widely between conventional products (RBCs, 1·9; PLTs, 7·5; and FFP, 20·1 mmHg); however, all are hypooncotic compared with human plasma COP (25·4 mmHg). Storage lesion did not increase COP. Concentrating RBCs and PLTs did not increase COP; only prLP showed a supraphysiological COP of 47·3 mm Hg. The PVL of concentrated products was lower than conventional products. Conclusion Colloid osmotic pressure of conventional products was low. Therefore, third-space fluid recruitment is an unlikely mechanism in TACO. Concentrated products had a lower calculated fluid load and may prevent TACO. Finally, storage did not significantly increase oncotic pressure of blood products.

ZusammenfassungIm Rahmen der präklinischen Versorgung von schwer verletzten Patienten wird der Nutzen einer Transfusion von Erythrozytenkonzentraten (EK) schon am Unfallort diskutiert. Hierbei kommt der Luftrettung hinsichtlich Vorhaltung und Transport eine erhebliche Bedeutung zu. Unklar ist, ob die Vibrationen im Hubschrauber eine Hämolyse induzieren und so eine richtlinienkonforme und damit sichere Anwendung verhindern könnten. Ziel dieser Untersuchung ist es, die Hämolyseraten von standardmäßig gelagerten mit denen in einem Hubschrauber transportierten EK zu vergleichen. Es erfolgte die Herstellung von identischen Paaren von EK. Während die EK der Kontrollgruppe richtlinienkonform gelagert wurden, erfolgte für die EK der Interventionsgruppe die Mitnahme im Hubschrauber während Realeinsätzen. Danach erfolgte die Bestimmung der Hämolyserate zu 3 verschiedenen Zeitpunkten. In beiden Gruppen ließ sich ein Anstieg der Hämolyserate über die Zeit nachweisen, was dem üblichen Alterungsprozess der Erythrozytenkonzentrate entspricht. Alle Werte blieben deutlich unter der von der Richtlinie geforderten maximalen Hämolyserate. Ein relevanter Unterschied zwischen beiden Gruppen ließ sich zu keinem Zeitpunkt zeigen. Ein sicherer Transport von EK im Rettungshubschrauber hinsichtlich der Qualität im Sinne einer im Rahmen liegenden Hämolyserate und damit die Voraussetzung zu deren sicherer Anwendung im präklinischen Setting ist daher problemlos möglich.

Background and Aims: Only little is known about blood groups other than ABO blood groups and Rhesus factors in Arabian countries and Iran. During the last years, increased migration to Central Europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. Therefore, blood group allele frequencies should be determined in individuals from Arabian countries and Iran by molecular typing and compared to a German rare donor panel. Methods: 1,111 samples including 800 individuals from Syria, 147 from Iran, 123 from the Arabian Peninsula, and 41 from Northern African countries were included in a MALDI-TOF MS assay to detect polymorphisms coding for Kk, Fy(a/b), Fynull, Cw, Jk(a/b), Jo(a+/a–), Lu(a/b), Lu(8/14), Ss, Do(a/b), Co(a/b), In(a/b), Js(a/b), Kp(a/b), and variant alleles RHCE*c.697C>G and RHCE*c.733C>G. Yt(a/b), S–s–U–, Velnull, Conull, and RHCE*c.667G>T were tested by PCR-SSP. Results: Of the Arabian donors, 2% were homozygous for the FY*02.01N allele (Fynull), and 15.7% carried the heterozygous mutation. However, 0.8% of the German donors also carried 1 copy of the allele. 3.6% of all and 29.3% of Northern African donors were heterozygous for the RHCE*c.733C>G substitution, 0.4% of the Syrian probands were heterozygous for DO*01/DO*01.-05, a genotype that was lacking in German donors. Whereas the KEL*02.06 allele coding for the Js(a) phenotype was missing in Germans; 0.8% of the Syrian donors carried 1 copy of this allele. 1.8% of the Syrian but only 0.3% of the German donors were negative for YT*01. One donor from Northern Africa homo­zygously carried the GYPB*270+5g>tmutation, inducing the S–s–U+w phenotype, and in 2 German donors a GYPB*c.161G>A exchange, which induces the Mit+ phenotype, caused a GYPB*03 allele dropout in the MALDI assay. The overall failure rate of the Arabian panel was 0.4%. Conclusions: Some blood group alleles that are largely lacking in Europeans but had been described in African individuals are present in Arabian populations at a somewhat lower frequency. In single cases, it could be challenging to provide immunized Arabian patients with compatible blood.

ZusammenfassungBei der hereditären Hämochromatose (HH) handelt es sich um eine genetisch bedingte Störung des Eisenstoffwechsels, die bei der Mehrzahl der Betroffenen zu einer Verminderung von Hepcidin führt, das durch Bindung an Ferroportin im Darm die Resorption von Eisen hemmt. Hieraus resultieren Eisenablagerungen in verschiedenen Organen. Die Therapie besteht in der Entfernung von in Hämoglobin gebundenem Eisen durch regelmäßigen Aderlass, weshalb Blutspenden den Krankheitsverlauf positiv beeinflussen.

Background: Exposure to non-matching human platelet alloantigens (HPA) may result in alloimmunization. Antibodies to HPA can be responsible for post-transfusion purpura, refractoriness to donor platelets, and fetal and neonatal alloimmune thrombocytopenia. For the supply of compatible apheresis platelet concentrates, the HPA genotypes are determined in a routine manner. Methods: Here, we describe a novel method for genotyping twelve different HPA systems simultaneously, including HPA-1 to HPA-5, HPA-9w, HPA-10w, HPA-16w, HPA-19w, HPA-27w, and the novel HPA-34w by means of amplicon-based next-generation sequencing (NGS). Blood donor samples of 757 individuals with a migration background and 547 of Western European ancestry were genotyped in a mass-screening setup. An in-house software was developed for fast and automatic analysis. TaqMan assay and Sanger sequencing results served for validation of the NGS workflow. Finally, blood donors were divided in several groups based on their country of origin and the allele frequencies were compared. Results: For 1,299 of 1,304 samples (99.6%) NGS was successfully performed. The concordance with TaqMan assay and Sanger sequencing results was 99.8%. Allele-calling dropouts that were observed for two samples with the TaqMan assay caused by rare single nucleotide polymorphisms were resolved by NGS. Additionally, twenty rare and two novel variants in the coding regions of the genes ITGB3, GPB1A, ITGBA2, and CD109 were detected. The determined allele frequencies were similar to those published in the gnomAD database. Conclusions: No significant differences were observed in the distribution of allele frequencies of HPA-1 through HPA-5 and HPA-15 throughout the analyzed groups except for a lower allele frequency for the HPA-1b allele in the group of donors with Southern Asian ancestry. In contrast, other nucleotide variants that have not yet been phenotypically characterized occurred three times more often in blood donors with a migration background. High-throughput amplicon-based NGS is a reliable method for screening HPA genotypes in a large sample cohort simultaneously. It is easily upgradeable for genotyping additional targets without changing the setup or the analysis pipeline. Mass-screening methods will help building up blood donor registries to provide matched blood products.

Summary Background Hereditary factors may influence individual susceptibility to contact allergy (CA). Objectives To investigate genetic variants with impacts on early inflammatory reactions and T cell functions that possibly increase the risk of CA. Patients and Methods Three hundred and seventy two patients undergoing patch testing were recruited from the Information Network of Departments of Dermatology (IVDK). Of these, 133 were monosensitized and 239 were polysensitized, defined as reacting to three or more unrelated sensitizers. Within the polysensitized individuals, a subgroup with at least one particularly strong patch test reaction (strong reactors; n = 194) was considered. Three hundred and forty-seven blood bank donors served as controls. Fifteen genetic variants in 13 genes were analysed. Results The homozygous variant CXCL11 AA genotype (rs6817952) was significantly more frequent among polysensitized patients (10 of 239 = 4.2%; p = 0.0048; odds ratio 7.49; 95%CI: 1.7–36.1) than among monosensitized patients (2.2%) and in the control group (0.6%). None of the remaining genetic variants investigated were characterized by similarly strong associations. However, the significance was lost after correction for multiple comparisons. Conclusions The homozygous variant CXCL11 genotype is associated with an increased risk of CA. To confirm this exploratory finding, further independent studies are needed.

Background and Objectives Bacterial contamination represents the major infectious hazard associated with transfusion of platelet concentrates (PCs). As bacterial screening of PCs is not mandatory in Germany, the BactiFlow flow cytometry test has been introduced as a rapid detection method to increase product safety. Materials and Methods During a period of 25 months, a total of 34 631 PCs (26 411 pooled and 8220 apheresis-derived PCs) were tested at the end of day 3 of their shelf life using the BactiFlow system. PCs initially reactive in BactiFlow testing and expired PCs not reactive in BactiFlow on day 3 were also investigated by the BacT/ALERT system and by microbiological cultivation in order to identify the contaminating bacterial species and to confirm reactive BactiFlow results. Results Two hundred and twenty-eight PCs (0·7%) had an initially reactive result, 24 of them remained reactive in a second test run. Out of these reproducible reactive BactiFlow results, 12 could not be verified by parallel BacT/ALERT culturing, resulting in a confirmed false-positive rate of 0·03%. The bacterial species were identified as S. aureus, S. epidermidis, S. dysgalactiae ssp. equisimilis and B. cereus. In 10 out of 9017 expired PCs (0·11%), a confirmed-positive result was obtained in the BacT/ALERT system which had a negative result in the BactiFlow system. Conclusion Testing of PCs by BactiFlow was successfully implemented in our blood donation service and proved sufficient as a rapid and reliable screening method. False reactive results are in an acceptable range since the transfusion of 12 bacterially contaminated PCs was prevented.

Background Due to the ageing population, blood donation by the elderly is necessary to maintain blood supply. We initiated a prospective study, to assess whether there is an increased risk of donor reactions in elderly donors. Study Design and Methods In this prospective study, regular donors aged from 66 to 68 and 69 to 71 years were invited to continue blood donation on mobile collection sites of the German Red Cross Blood Service West. A control group (50–52 years) was established. Admission of donors in all groups followed the German national guidelines for blood donation. Donor deferrals and all kinds of donor reactions during donation (on-site) and in the 48 h following donation (off-site) were monitored. Results A total of 64 260 valid cases were entered in the study. Donor deferrals increased with age from 1·12% in the control group up to 8·74 in female donors aged 69–71 years. Adverse reactions to blood donation were rare with an overall reaction rate of 0·63% (0·05% on-site; 0·58% off-site). Off-site reactions significantly decreased with increasing age. The relative risk (RR) for adverse reactions in elderly donors compared to the control group (50–52 years) was slightly increased for on-site reactions in the 69- to 71-year-old donors (RR 1·0309; 95% CI 1·0292–1·0325). In all other comparisons, the RR for adverse reactions was distinctively lower in elderly donors (RR 0·3785 – 0·7778). Conclusions Our data confirm that elderly regular blood donors may safely continue blood donation at least to the age of 71. Based on these data, we increased the upper age limit.

Hereditary factors may influence individual susceptibility to contact allergy.To investigate genetic variants with impacts on early inflammatory reactions and T cell functions that possibly increase the risk of contact allergy.Three hundred and seventy two patients undergoing patch testing were recruited from the Information Network of Departments of Dermatology (IVDK). Of these, 133 were monosensitized and 239 were polysensitized, defined as reacting to three or more unrelated sensitizers. Within the polysensitized individuals, a subgroup with at least one particularly strong patch test reaction (strong reactors; n = 194) was considered. Three hundred and forty-seven blood bank donors served as controls. Fifteen genetic variants in 13 genes were analysed.The homozygous variant CXCL11 AA genotype (rs6817952) was significantly more frequent among polysensitized patients (10 of 239 = 4.2%; p = 0.0048; odds ratio 7.49; 95%CI: 1.7-36.1) than among monosensitized patients (2.2%) and in the control group (0.6%). None of the remaining genetic variants investigated were characterized by similarly strong associations. However, the significance was lost after correction for multiple comparisons.The homozygous variant CXCL11 genotype is associated with an increased risk of contact allergy. To confirm this exploratory finding, further independent studies are needed.

Summary Background Provocation tests are invaluable in establishing threshold levels and a causal relationship between atopic asthma and a certain allergen source, especially in relation to work-associated exposure. Purified major allergens open possibilities for a more accurate assessment of sensitization. Objective To determine the threshold dose of purified major bovine dander allergen Bos d 2 in bronchial provocation in comparison with the standard allergen and a set of other parameters of allergy. Method Nine consecutive patients referred to hospital for confirming the bovine origin of their occupational asthma were subjected to bronchial provocation tests with purified natural Bos d 2 and a standard bovine dander allergen. Additional tests included bronchial histamine challenge, measurements of total IgE, specific IgE antibody determinations and skin prick tests (SPT) with both allergens. Results In the provocation tests with Bos d 2, a 15% decrease in the forced expiratory volume in 1 s (FEV1) and/or peak expiratory flow (PEF) values in eight out of nine patients confirmed the predominant role of Bos d 2 in the sensitization. The threshold dose of Bos d 2 varied from 0.1 µg to > 100 µg (median ± median absolute deviation = 4.5 ± 3.9 µg). A positive SPT was induced by a median dose of 13.9 ± 9.8 µg of Bos d 2. Bronchial response to histamine and IgE antibodies against Bos d 2 showed the highest correlations to the provocations results. Conclusions The efficacy of Bos d 2 in bronchial provocation in patients with occupational cattle-associated asthma was confirmed and the range of the threshold level was determined. There were individual variations, but the response in provocation remains the reference method for identification of the cause of occupational atopic asthma. SPT and the measurement of specific IgE antibodies, preferably with purified or recombinant major allergens, increase the accuracy of the diagnosis.

The immunological characteristics of an important group of animal-derived allergens, lipocalins, are poorly known. To explore the immunology of the lipocalin allergen Bos d 2, several mouse strains with different H-2 haplotypes were immunized with the allergen. Only the BALB/c mouse mounted a distinct humoral response against Bos d 2. The proliferative spleen cell responses of all mouse strains remained very weak. Further experiments with BALB/c mice confirmed that Bos d 2 is a weak inducer of both humoral and cellular responses, and that the responses were weaker than with the control antigens hen egg lysozyme (HEL) and tetanus toxoid. IgG subclass analyses showed that Bos d 2 was prone to favor the T(h)2 response. Although s.c. immunization using complete Freund's adjuvant favored the T(h)1-deviated immune response by lymph node cells, Bos d 2 was able to induce the production of IL-4 while the control antigen HEL did not. Epitope mapping revealed that BALB/c mice recognized one immunodominant epitope in Bos d 2, almost identical to that recognized by humans. The epitope was shown to be immunogenic in subsequent experiments. However, further studies are needed to clarify the significance of priming and stimulation doses of the immunodominant and other epitopes in Bos d 2 for the outcome of immune response against the allergen. The murine immune response against Bos d 2 closely resembled that observed in humans. The weak immunogenicity of Bos d 2 may be associated with its allergenicity.

Allergens from various sources have been shown to comprise several isoforms. In the present study, a series of chromatographic steps was carried out to separate the lipocalin allergen Bos d 2 isoforms present in cow dander. Subsequent HPLC-MS-MS analyses revealed two new Bos d 2 variants. In one of the proteins, tyrosine (Y83) was substituted by aspartic acid, and in the other protein valine (V102) was replaced by alanine. We propose the three Bos d 2 variants be named as Bos d 2.0101 (previously sequenced Bos d 2), Bos d 2.0102 and Bos d 2.0103. Our results suggest that molecular polymorphism is a common property among lipocalin allergens. Since allergen isoforms may show variation in their IgE binding and/or T-cell reactivity, all of the many allergen forms should be taken into account when planning preparations for immunotherapy.

Background About one in every four cases of occupational rhinitis recorded in Finland is animal-induced. Bovine allergens are the most important in this respect and the largest patient group consists of dairy farmers. Allergen immunotherapy, if proven effective, safe and feasible, would be ideal for their treatment. The development of recombinant allergens has offered new potential therapeutic prospects. Fragments of recombinant Bos domesticus (Bos d 2) allergen could be suitable for this purpose because they are recognized by T cells but their IgE-binding capacity is attenuated. Objective The aim of this study was to verify whether the potential of the two fragments of recombinant Bos d 2 (corresponding to amino acids 1–131 and 81–172) to induce immediate allergic reaction in a shock organ (nose) was decreased compared to the complete recombinant allergen, which would be an advantageous property for a preparation intended for allergen immunotherapy. Methods The study group consisted of 10 dairy farmers with cow-induced allergic rhinitis. We used the IgE titres against native Bos d 2 measured by indirect IgE ELISA to characterize the level of sensitization and compared the IgE titres in the rhinitis patients with 12 cow-sensitized asthmatic farmers and 12 healthy students. In vitro reactivity against recombinant Bos d 2 and its two fragments was studied by indirect IgE ELISA and in vivo reactivity by nasal provocation tests. Results The IgE titres against native Bos d 2 of patients with rhinitis tended to be lower than the titres of asthmatics. The healthy students did not exhibit any detectable IgE reactivity to native Bos d 2. In the patients with rhinitis, there was no statistically significant difference between IgE responses against native and recombinant Bos d 2, whereas with both in vitro and in vivo, the reactivity to both fragments of recombinant Bos d 2 was lower than the reactivity to the complete recombinant allergen. Conclusions Due to the decreased in vivo capacity to induce immediate allergic reactions, the fragments may be better tolerated in allergen immunotherapy than the complete allergen.

Injection-molded samples of polypropylene were exposed to oxygen plasma and SACO (SAndblasting and COating) treatments. The pretreated surfaces were successively adhesively bonded or lacquered. The...

Background Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. Objective The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule. Methods Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones. Results In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15–38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones. Conclusions The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.

Background: Domestic mammals are important sources of indoor allergens. However, the origin at the tissue level and the biologic function of mammalian allergens are largely unknown. Objective: The aim of this study was to localize the source of the major bovine dander allergen, Bos d 2, in bovine tissues. Methods: Samples from several organs were tested for the presence of mRNA encoding Bos d 2 and Bos d 2 protein by using the reverse transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Results: Skin proved to be the only tissue where mRNA encoding Bos d 2 was detected. This observation was confirmed by immunohistochemistry with a monoclonal anti-Bos d 2 antibody as the primary antibody. In the skin sections, Bos d 2 was found in the secretory cells of apocrine sweat glands and the basement membranes of the epithelium and hair follicles. Bos d 2 belongs to the family of lipocalins comprising a number of pheromone carrier proteins that are present, for example, in the secretions of the apocrine sweat glands. Conclusion: Together with earlier data, our findings suggest that Bos d 2 is produced in sweat glands and transported to the skin surface as a carrier of the pheromone ligand. Because dander allergens of a number of mammalian species are lipocalins, the common biologic function of being pheromone carriers seems to be a common feature of an important group of aeroallergens. (J Allergy Clin Immunol 1998;101:349-53.)

Injection moulded samples of six species of thermoplastic polymers were exposed to different surface treatments and successively glued together to form single lap shear joints Lap shear strength and failure mode of these specimens were examined The surfaces obtained after treatment were characterized by electron spectroscopy for chemical analysis, contact angle measurements and atomic force microscopy.

Allergens are characterized by their ability to be bound by gE. The Swiss-Prot protein database currently lists a partial or complete amino acid sequence of in excess of about 350 allergens. It is not clear how allergens participate in the process of allergic sensitization, the generation of specific T-helper type 2 (Th2) lymphocytes, which play a crucial role in stimulating B lymphocytes to produce allergen-specific IgE.T-helper (Th) cells play a key role in the regulation of immune responses. The recognition of antigen by T cells is complex and it can trigger qualitatively differential signaling. Therefore, it is conceivable that epitopes or antigenic determinants recognized by Th cells may influence the quality of immune response. The aim of this chapter is to describe the way in which T-cell epitopes can be identified (mapped). This is particularly important because knowledge of the precise T-cell epitopes of allergens can give important information on the pathogenesis of allergy and can help to develop better preparations for the diagnostics and/or immunotherapy of allergy.

Introduction: Regulated by German legal authorities quality control of stem cell products includes product volume, number and viability of CD34+ (PBSC products) or nucleated cells (bone marrow products), number of CD3+ cells (PBSC products), hematocrit and sterility testing. Material and Method: We performed a retrospective analysis of microbiological contamination of allogeneic stem cell products. Over the last seven years we performed 1051 peripheral blood stem cell collections and 300 bone marrow collections. For all products sterility testing was performed using BactAlert device. If result of screening was positive, differentiation of germs was done in a specialized microbiological laboratory. Results: 96 out of 1051 peripheral stem cell products (9,1%) and 1 out of 300 bone marrow products (0,3%) showed positive signal in BactAlert screening test. 14 out of 1051 peripheral stem cell products (1,3%) and 1 out of 300 bone marrow products (0,3%) were contaminated with bacteria. Fungi were not detected. In PBSC products Staphylococcus species, Propionibacteria and Streptococcus species were detected. In BM product Staphylococcus capitis was found. Discussion: Because of higher cell count rate of false positive BactAlert results is nearly 10% in PBSC products. But only 1,3% of all products is really contaminated with bacteria. All detected germs belong to normal skin flora and went into apheresis product via vein puncture. Even if skin disinfection is done properly sometimes not all bacteria are devitalized. You could expect same constellation in bone marrow collection procedure. But surprisingly we found lower contamination rate in BM product. Possibly complete shaving of puncture area contributes to this finding. Nevertheless allogeneic stem cell products extremely seldom cause dangerous bacteremia in patients, because detected bacteria respond to antibiotics, patient are given during cytopenia.

Preventing the activation of PLTs may ameliorate (or mitigate) the PLT storage lesion (PSL), which encloses all structural and biochemical changes caused by collection, processing, and storage of PLT concentrates (PCs). Partial inhibition of PLT function due to ingestion of aspirin (ASA) by blood donors reduces the functional activity of the collected PLTs, however, by preventing premature PLT activation, it might reduce the PSL as well.In a randomized crossover study, 10 healthy donors donated two single-donor PCs (SDPCs) each, taking 500 mg ASA 12 hours before one of the aphereses (Group A) and taking no medication before the other donation (Group B). In-vitro tests of PLT function were performed in donors before and after apheresis and in SDPCs during storage (Days 1, 3, and 5).ASA ingestion resulted in a significant decrease of induced PLT aggregation in donors (p0.005) and SDPCs on Day 1 (p0.01). TRAP-6-induced expression of p-selectin (CD62p) was significantly reduced in Group A SDPCs only on Day 1 (p0.02). There were no significant differences of in-vitro function (LDH, lactate, pH, morphology score, CD62p expression, fibrinogen binding) between Group A and B (SDPCs and donors). Apheresis did not result in a significant activation of PLTs in donors or SDPCs.These limited data do not show a detectable beneficial effect of ASA ingestion on the PSL but do suggest that ASA ingestion before apheresis may not be detrimental to the clinical effectiveness of the stored product.

A new cell separator (COM.TEC, Fresenius) was recently developed aimed at efficient collection of WBC-reduced single-donor PLT concentrates (SDPs).Five German centers collected 554 WBC-reduced SDPs with help of the COM.TEC cell separator. Two multicenter cell counting studies were performed at the beginning and at the end of the study to document uniform counting results among the participating centers.A total of 441 (79.6%) PLT collections were included in the study according to the protocol. A total of 342 single-dose and 99 double-dose SDPs were collected. For single-dose SDPs, an average blood volume of 2826 +/- 409 mL was processed in a donation time of 55 +/- 11 minutes. Mean PLT yield of these products was 3.11 x 1011+/- 0.40 x 1011 and the WBC contamination was 0.11 x 106+/- 0.20 x 106. For double-dose SDPs (PLT count, 5.29 +/- 0.93 x 1011), 3943 +/- 639 mL was processed. The average difference between the target and the collected PLT concentration was -2.8 +/- 12.0 percent for single-dose SDPs and -1.8 +/- 9.5 for double-dose SDPs, respectively. The collection efficiency was 53.7 +/- 5.8 percent for single-dose SDPs and 58.2 +/- 6.2 percent for double-dose SDPs. If all results of each sample from the counting study were set to unity (to the mean over all centers), most PLT determinations were very similar to the mean, for example, near or 1 if set to unity.The COM.TEC machine makes it possible to obtain WBC-reduced SDPs that comply with current standards.

Major respiratory allergens of dogs, mice, rats, horses and cows belong to the lipocalin group of proteins. The sequence identity of lipocalins is often less than 20%, but they contain between one and three structurally conserved regions and their three–dimensional structures are similar. Lipocalins share common biological functions, predominantly related to the transport of small hydrophobic molecules, such as vitamins and pheromones. Immune reactivity to lipocalin allergens is not well known. In Bos d 5, the IgE–binding epitopes are spread along the molecule, whereas in Bos d 2, the C terminus appears to contain the human B cell epitopes. Bos d 5 contains several murine T cell epitopes. No information is available on human T cell epitopes. The maximal number of epitopes an allergic patient’s T cells could recognize in Bos d 2 was five. Three of the epitopes were colocalized in the structurally conserved regions of lipocalins. Interestingly, one of the epitopes was recognized by the T cells of all patients and the computer predictions suggested that there would be an epitope in the corresponding parts of human endogenous lipocalins. The proliferative responses of peripheral blood mononuclear cells of Bos d 2–allergic subjects to Bos d 2 were weak. The T cell response was Th2–dominated. To explain these observations, we have proposed that the allergenicity of lipocalins may be a consequence of molecular mimicry between lipocalin allergens and endogenous lipocalins at the T cell level.

The relationship between the molecular structure of allergenic proteins and the allergenic determinants is one of the central issues in allergology. We report here that the natural preparation of Bos d 2, a mammalian lipocalin allergen, comprises three molecular variant proteins of 17,829, 17,781, and 17,800 Da. When cDNA of Bos d 2 (Genome Sequence Data Base No. L42867) was recloned and expressed in Pichia pastoris, two proteins were produced. One of the proteins (17,831 Da) and the proteins in the natural preparation had pyroglutamate as the N-terminal residue; in the other (17,849 Da) the N-terminal residue was glutamine. Recombinant Bos d 2 protein was crystallized and the native data set was collected at 1.8 A resolution.

Background: Lately, renewed interest has arisen in the new forms of allergen immunotherapy because they may offer alternatives for drug treatment. Objective: The purpose of this study was to develop a well-characterized preparation of the main respiratory cow dander allergen, Bos d 2, with attenuated allergenic activity. Methods: The immunologic characteristics of Bos d 2 preparations were studied by indirect IgE ELISA, ELISA inhibition, Western blotting, histamine release, skin prick tests, and the proliferation tests of allergen-specific T-cell clones. Results: The complete recombinant Bos d 2 was observed to bind effectively, IgE of cow-allergic patients in indirect ELISA. In other experiments, the IgE-binding capacity of recombinant Bos d 2 proved to be lower compared with native Bos d 2. When the two overlapping recombinant fragments of Bos d 2 (corresponding amino acids 1-131 and 81-172, respectively) covering the whole molecule were compared with the complete recombinant Bos d 2 with several methods, only a low level of residual reactivity was observed. For example, recombinant fragments could not bind antibody at all in ELISA inhibition tests retaining, however, some reactivity in skin prick tests. In contrast, the fragments were able to stimulate vigorously Bos d 2-specific T-cell clones. Conclusion: The approach we have taken may offer a simple and reproducible way to produce hypoallergenic preparations for immunotherapy, circumventing simultaneously some of the problems of other experimental methods such as individual T-cell epitope recognition in peptide-based immunotherapy. (J Allergy Clin Immunol 1997;100:721-7.)

BACKGROUND: A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE: The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS: Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS: In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS: The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research. (J ALLERGY CLIN IMMUNOL 1996;97:1297-1303.)

Summary Background Cow dust is one of the most important inducers of occupational allergic diseases in Finland. For example, in 1991 it accounted for almost 40% of the new occupational asthma cases. Objective This study compares the performance of the purified major cow allergen (BDA20) and crude bovine epithelial extract (BEA) in diagnostic tests and examines the role of milk allergy-associated bovine proteins (bovine serum albumin, α-lactalbumin, β-lactoglobulin, casein) in respiratory cow allergy. Methods The humoral responses of cow-asthmatic and healthy farmers to the various components of BEA were analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The levels of specific Ige and IgG antibodies were quantifieated with enzyme-linked inimunosorbent assays (FLISAs). The cellular responses were analysed with antigen-specific lymphoeyte proliferation tests. Results The specific anti-BDA20 IgE measurement was found to be best in distinguishing between the asthmatic farmers and their healthy colleagues. It proved possible to determine a cut-off value that gave the analysis a specificity and sensitivity of 100%; the distinction between the two groups was highly significant (P 0.0001). In the lymphocyte proliferation analysis, cow asthma was more closely associated with reactivity to BDA20 than to BEA. In the measurement of anti-BDA20 and anti-BEA IgG antibody levels, considerable overlap between the groups was observed, suggesting that these antibodies are not directly involved in cow allergy. When proteins associated with milk allergy were used as test reagents, no statistically significant differences could be observed between the groups, except for anti-casein IgE antibodies the level of which, however, overlapped considerably between the farmer groups. Conclusion These findings suggest that purified BDA20 is better than BEA for diagnosing cow asthma and that proteins associated with milk allergy are of only marginal significance in this disease.