ELISA Inhibition Assay Research Articles

Content of Asthmagen Natural Rubber Latex Allergens in Commercial Disposable Gloves.

The use of natural rubber latex (NRL) gloves in many occupations may lead to latex sensitization, allergic asthma, and skin reactions. Due to their good properties and environmental safety NRL gloves are still being used in the healthcare setting, but also in the food industry, by hairdressers, cleaners, etc. The aim of our study was to assess the protein and NRL allergen content in commercial gloves by different methods, including a new assay. Twenty commercially available NRL gloves were analyzed. Protein extraction was performed according to the international standard ASTM D-5712. Total protein content was measured with a modified Lowry method, NRL content with the CAP Inhibition Assay, the Beezhold ELISA Inhibition Assay, and an innovative ELISA with IgY-antibodies extracted from eggs of NRL-immunized hens (IgY Inhibition Assay). We found a high protein content in a range of 215.0-1304.7μg/g in 8 out of the 20 NRL gloves. Seven of the 20 gloves were powdered, four of them with a high protein content. In gloves with high protein content, the immunological tests detected congruently high levels of NRL allergen. We conclude that a high percentage of commercially available NRL gloves still represent a risk for NRL allergy, including asthma. The modified Lowry Method allows to infer on the latex allergen content.

The antibody response to influenza vaccination is not impaired in type 2 diabetics

BackgroundDiabetics are considered to be at high risk for complications from influenza infection and type 2 diabetes is a significant comorbidity of obesity. Obesity is an independent risk factor for complications from infection with influenza. Annual vaccination is considered the best strategy for protecting against influenza infection and it's complications. Our previous study reported intact antibody responses 30 days post vaccination in an obese population. This study was designed to determine the antibody response to influenza vaccination in type 2 diabetics. MethodsSubjects enrolled were 18 or older without immunosuppressive diseases or taking immunosuppressive medications. A pre-vaccination blood draw was taken at time of enrollment, the subjects received the influenza vaccine and returned 28–32 days later for a post-vaccination blood draw. Height and weight were also obtained at the first visit and BMI was calculated. Antibody levels to the vaccine were determined by both ELISA and hemagglutination inhibition (HAI) assays. ResultsAs reported in our previous work, obesity positively correlates with the influenza antibody response (p=0.02), while age was negatively correlated with antibody response (p<0.001). In both year 1 and year 2 of our study there was no significant difference in the percentage of the type 2 diabetic subjects classified as seroprotected or a responder to the influenza vaccine compared to the non-diabetic subjects. ConclusionsThese data are important because they demonstrate that diabetics, considered a high risk group during influenza season, are able to mount an antibody response to influenza vaccination that may protect them from influenza infection.

Effects of Ag/adjuvant ratio on the systemic and mucosal antibody responses of BALB/c mice immunized intradermally with CfaEB, a minor-major subunit fusion of the enterotoxigenic E. coli CFA/I fimbriae, and LT(R192G/L211A) (VAC9P.1104)

Abstract The ratio of Ag/adjuvant in a vaccine can modulate the immune response as well as side effects. We studied the effects of different doses of CfaEB, a minor-major subunit fusion of the enterotoxigenic E. coli (ETEC) CFA/I fimbriae, given ID with the adjuvant LT(R192G/L211A), or dmLT, an attenuated form of the ETEC heat-labile enterotoxin. BALB/c mice were immunized with 0.1, 1, 10 or 100 mg of CfaEB alone or with 10 or 100 ng of dmLT on days 0 and 21. Sera from days 21 and 28 were tested by ELISA and hemagglutination inhibition assay. Mucosal responses were assessed in day 28 fecal pellets. Serum anti-CfaE IgG Ab titers directly correlated with the dose in groups receiving no adjuvant but the highest titers were seen in the two groups receiving 10 mg of CfaEB plus 10 or 100 ng dmLT. A similar pattern was observed with serum anti-CfaE IgA responses, although 100 mg of CfaEB plus 10 or 100 ng of adjuvant elicited the highest levels. Groups receiving 10 or 100 mg of CfaEB plus 100 ng dmLT manifested the highest functional Ab titers. The adjuvant at either dose was required to elicit a strong fecal anti-CfaE IgA response, and it directly correlated with CfaEB dose in the adjuvanted groups. Our results demonstrate the importance of dmLT in promoting mucosal Ab responses to our vaccine when given by the ID route. Since optimal mucosal vs. systemic functional Ab responses are elicited by different Ag/adjuvant ratios, the ratio should be selected to elicit the desired immune response.

Potential changes in the allergenicity of three forms of peanut after thermal processing

This study aimed to analyze the influence of thermal processing on the IgE binding properties of three forms of peanut, its effects in the content of individual allergens and IgE cross-linking capacity in effector cells of allergy. Three forms of peanut were selected and subjected to thermal processing. Immunoreactivity was evaluated by means of immunoblot or ELISA inhibition assay. Specific antibodies were used to identify changes in the content of the main allergens in peanut samples. The ability of treated peanut to cross-link IgE was evaluated in a basophil activation assay and Skin Prick Testing (SPT). The results showed that thermal/pressure treatments at specific conditions had the capacity to decrease IgE binding properties of protein extracts from peanut. This effect went along with an altered capacity to activate basophils sensitized with IgE from patients with peanut allergy and the wheal size in SPT.

Protection of mice against pandemic H1N1 influenza virus challenge after immunization with baculovirus-expressed stabilizing peptide fusion hemagglutinin protein.

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein (2 μg/mouse) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

Analysis of cross-reactivity between the Fatty Acid Binding Proteins from Blomia tropicalis and Litopenaeus vannamei

Analysis of cross-reactivity between the Fatty Acid Binding Proteins from Blomia tropicalis and Litopenaeus vannamei

Glycated-H2A histone is better bound by serum anti-DNA autoantibodies in SLE patients: Glycated-histones as likely trigger for SLE?

Histones are the most abundant proteins associated with genomic DNA. Recent observations show that histones are quite susceptible to non-enzymatic glycation which results in the generation of free radicals causing structural perturbations. In this study, our aim is to define the role of deoxyribose-modified H2A histone in SLE initiation/progression. Glycation reaction was carried out by incubating H2A histone with 10 mM deoxyribose for 21 days at 37 °C. Structural changes in glycated-H2A were studied by various physico-chemical techniques. The antigen-antibody interaction was studied by direct binding, inhibition ELISA and mobility shift assay. Deoxyribose-modified-H2A histone showed increased hyperchromicity and increased fluorescence intensity. CD results demonstrated almost 50% loss in alpha helix conformation as a consequence of glycation. This was supported by an increase in Tm value vis-à-vis thermal stability. Glycated-H2A showed cross linking in SDS-PAGE. SLE sera positive for anti-nDNA autoantibodies showed preference for deoxyribose-modified-H2A histone compared to native H2A histone or native DNA. Inhibition ELISA supported the above findings. Band shift assay further reiterated the preferential recognition of glycated-H2A over native H2A by SLE IgG autoantibodies. Deoxyribose-modified-H2A histone exhibited damage as revealed by various physico-chemical studies. Glycation of H2A has resulted in the generation of neo-epitopes on H2A histone, which are preferably bound by SLE anti-nDNA autoantibodies. It implies that deoxyribose-modified-H2A may trigger immune response resulting in the generation of anti-glycated H2A antibodies with DNA cross reacting properties.

Prevalence of canine influenza virus A (H3N8) in dogs in Germany

The aim of this study was to investigate the prevalence of CIV H3N8 in dogs in Germany. Blood samples from 272 clinically healthy dogs and 35 dogs with acute respiratory signs were screened for antibodies against influenza virus by ELISA and haemagglutination inhibition (HI) assay; positive samples were further tested by fluorescent antibody test (FAT) and subtype-specific HI assay. Nasal and pharyngeal swabs from all sick dogs were submitted for real-time (RT)-PCR for influenza virus RNA; PCR results were negative in all cases. One healthy and one sick dog tested positive for antibodies against influenza virus nucleoprotein by ELISA, but both were negative by subtype-specific HI assay and FAT, and were therefore considered false positive results. Thus, antibody prevalence against CIV H3N8 was 0% (0–1.24%, 95% CI). Currently, the risk for CIV H3N8 infection in the German dog population seems very low.

Inhibition of Oxidative Stress and Lipid Peroxidation by Anthocyanins from Defatted Canarium odontophyllum Pericarp and Peel Using In Vitro Bioassays

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD+ and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection.

Occupational Rhinoconjunctivitis due to Maize in a Snack Processor: A Cross-Reactivity Study Between Lipid Transfer Proteins From Different Cereals and Peach.

We report the case of a snack processor who developed occupational rhinoconjunctivitis due to maize brand exposure during the extrusion process, and who experienced abdominal pain upon drinking beer. The allergens implicated and the cross-reactivity between non-specific lipid transfer proteins (LTPs) from different cereals and peach were investigated. Skin prick tests and specific IgE to cereal flours, pulmonary functions tests and specific conjunctival and inhalation challenges to maize extract were performed. In vitro studies included IgE immunoblotting and ELISA inhibition assays. Skin prick tests with maize flour, maize brand and wheat flour extracts were positive, whereas serum specific IgE was positive only to maize flour. Specific inhalation challenge (SIC) to maize flour did not elicit an asthmatic reaction; however, conjunctival challenge test with the same extract was positive. Patient's serum recognized IgE-binding bands in the maize and beer extracts corresponding to LTPs. In the ELISA inhibition assays, a significant degree of allergenic cross-reactivity was found between maize and beer LTPs, whereas no cross-reactivity was observed between maize LTP and wheat and peach LTPs.

Role of Art v 3 in pollinosis of patients allergic to Pru p 3

Food allergy caused by lipid transfer protein (LTP) from peach (Pru p 3) is frequently associated with sensitization to mugwort LTP (Art v 3). Although in vitro cross-reactivity is already well known, it has yet to be elucidated whether a pollen LTP can induce rhinitis. The aim of this study was to investigate whether mugwort LTP could elicit respiratory symptoms and whether a primary food LTP allergy could lead to a respiratory allergy. Patients with confirmed Pru p 3 allergy and control subjects were selected. Immediate responses to nasal allergen provocation tests (NAPTs) with Art v 3, Pru p 3, and mugwort were assessed by using the visual analog scale score, total nasal symptom score, and acoustic rhinometry. Tryptase and cysteinyl leukotriene (cysLT) levels were measured in nasal lavage fluid. Immunoblotting, ELISAs, and ELISA inhibition assays were also performed. Fifteen patients and 9 control subjects were selected. NAPT results with Art v 3 and Pru p 3 showed significant changes in acoustic rhinometry, visual analog scale scores, total nasal symptom scores, and cysLT levels (P < .001). Tryptase levels were only increased in NAPTs with Pru p 3. NAPTs with mugwort were used in those patients who were only sensitized to Art v 3, with similar results (P < .05). No significant changes were detected in control subjects. The results demonstrated that a pollen LTP can elicit rhinitis in sensitized patients. Findings also suggest that a primary sensitization to Pru p 3 can lead to a respiratory allergy through cross-reactivity.

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

Background: Peanut flour is a high-protein, low-oil, powdered material prepared from roasted peanut seed. In addition to being a well-established food ingredient, peanut flour is also the active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a processing strategy to generate hydrolysates from peanut flour with reduced allergenicity. Methods: Soluble fractions of 10% (w/v) light roasted peanut flour dispersions were hydrolyzed with the following proteases: Alcalase (pH 8.0, 60°C), pepsin (pH 2.0, 37°C) or Flavourzyme (pH 7.0, 50°C) for 60 min. Western blotting, inhibition ELISA and basophil activation tests were used to examine IgE reactivity. Results: Western blotting experiments revealed the hydrolysates retained IgE binding reactivity and these IgE-reactive peptides were primarily Ara h 2 fragments regardless of the protease tested. Inhibition ELISA assays demonstrated that each of the hydrolysates had decreased capacity to bind peanut-specific IgE compared with nonhydrolyzed controls. Basophil activation tests revealed that all hydrolysates were comparable (p > 0.05) to nonhydrolyzed controls in IgE cross-linking capacity. Conclusions: These results indicate that hydrolysis of peanut flour reduced IgE binding capacity; however, IgE cross-linking capacity during hydrolysis was retained, thus suggesting such hydrolysates are not hypoallergenic.

Protective role of anti-viral human defensins during acute HIV infection (P4049)

Abstract Background Human defensins are small cationic peptides with anti-bacterial and anti-viral activity. Beta defensins are expressed by epithelial cells and innate immune cells in tissue and circulating leukocytes, respectively. They are part of the first line of innate immune defense against invading pathogens at mucosal sites. Methods We investigated anti-HIV activity and production of beta defensins in intestinal mucosal biopsies and PBMCs from individuals with chronic untreated HIV, immunologically controlled HIV, acute HIV and HIV uninfected individuals using ELISA, immunohistochemistry, quantitative PCR and viral inhibition assays. Results Human beta defensin expression in PBMCs was found exclusively in classical monocytes whereas epithelial cells were found to be the main source in the intestinal tract. Expression of human beta defensins was not altered in gut tissue or PBMCs from HIV progressors and controller compared to HIV negative control sample. However, we observed increased expression in PBMCs from HIV acute patient samples. Further, recombinant human beta defensins showed anti-HIV activity in vitro. Conclusions Our data suggest that beta defensins have a direct effect on HIV and might act as an intrinsic antiviral factor early during HIV infection. Specific delivery of recombinant defensins might be a powerful tool to reduce viral burden during acute infection.

2108 BLOOD GROUP ANTIGEN-TARGETING PEPTIDE SUPPRESSES ANTI-BLOOD GROUP ANTIBODY BINDING TO ANTIGEN IN RENAL GLOMERULAR CAPILLARIES AFTER ABO-INCOMPATIBLE BLOOD REPERFUSION

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation, Renal Vascular Surgery1 Apr 20132108 BLOOD GROUP ANTIGEN-TARGETING PEPTIDE SUPPRESSES ANTI-BLOOD GROUP ANTIBODY BINDING TO ANTIGEN IN RENAL GLOMERULAR CAPILLARIES AFTER ABO-INCOMPATIBLE BLOOD REPERFUSION Tohru Yoneyama, Shingo Hatakeyama, Yuki Tobisawa, Hayato Yamamoto, Kengo Imanishi, Teppei Okamoto, Noriko Tokui, Naoki Sugiyama, Yuichiro Suzuki, Shigemasa Kudo, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Noritaka Kamimura, Michiko N Fukuda, and Chikara Ohyama Tohru YoneyamaTohru Yoneyama Hirosaki, Japan More articles by this author , Shingo HatakeyamaShingo Hatakeyama Hirosaki, Japan More articles by this author , Yuki TobisawaYuki Tobisawa Hirosaki, Japan More articles by this author , Hayato YamamotoHayato Yamamoto Hirosaki, Japan More articles by this author , Kengo ImanishiKengo Imanishi Hirosaki, Japan More articles by this author , Teppei OkamotoTeppei Okamoto Hirosaki, Japan More articles by this author , Noriko TokuiNoriko Tokui Hirosaki, Japan More articles by this author , Naoki SugiyamaNaoki Sugiyama Hirosaki, Japan More articles by this author , Yuichiro SuzukiYuichiro Suzuki Hirosaki, Japan More articles by this author , Shigemasa KudoShigemasa Kudo Hirosaki, Japan More articles by this author , Takahiro YoneyamaTakahiro Yoneyama Hirosaki, Japan More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto Hirosaki, Japan More articles by this author , Takuya KoieTakuya Koie Hirosaki, Japan More articles by this author , Noritaka KamimuraNoritaka Kamimura Hirosaki, Japan More articles by this author , Michiko N FukudaMichiko N Fukuda La Jolla, CA More articles by this author , and Chikara OhyamaChikara Ohyama Hirosaki, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Antibody (Ab)-mediated rejection after ABO-incompatible kidney transplantation (ABO-I KTx) is a major barrier to transplantation success. The advent of immunosuppressive therapy has markedly improved graft survival in ABO-I KTx. However, compared with normal KTx, clinical conditions during ABO-I KTx are difficult to control because of overimmunosuppression. To reduce the need for immunosuppression, we aimed to develop a novel blood group antigen-neutralizing therapy. METHODS We screened for an ABO blood group antigen-targeting peptide (BATP) by screening of T7 phage-displayed peptide library. After screening, hemagglutination (HA) inhibition assays, ELISA and cytotoxicity assay were used to analyze the blood group antigen-blocking effect and toxicity of BATP. We also tested the inhibitory effects on anti-blood group Ab binding in normal human kidney tissues blocked with BATP and excised kidneys perfused ex vivo with BATP. RESULTS We identified 6 peptide sequences that efficiently suppressed HA of red blood cells by anti-A or -B antigen Abs. Most effective peptide is RPRNPNK and named as BATP. Cytotoxicity of BATP against primary culture of blood type O human kidney glomerular capillary microvascular endothelial cells (HGMEC) was very low. BATP bound to peritubular and microvascular endothelia in type A and B kidney tissues but not in type O kidney tissues. BATP significantly inhibited the reactivity of anti-A and B Abs against A and B histo-blood group antigens expressed in glomerular capillaries as well as peritubular and microvascular endothelia. Surprisingly, ex vivo perfusion of BATP in kidneys excised from renal cell carcinoma patients caused significant suppression of anti-blood group antibody binding to antigen, IgG and IgM deposition in renal glomerular capillaries after ABO-I blood reperfusion (shown in figure). CONCLUSIONS These data indicate that A/B blood group antigens on red blood cells and in kidney tissues may be neutralized by BATP. This approach may enable the development of a novel blood group antigen-neutralizing therapy to overcome the challenges of ABO-I KTx. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e864 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tohru Yoneyama Hirosaki, Japan More articles by this author Shingo Hatakeyama Hirosaki, Japan More articles by this author Yuki Tobisawa Hirosaki, Japan More articles by this author Hayato Yamamoto Hirosaki, Japan More articles by this author Kengo Imanishi Hirosaki, Japan More articles by this author Teppei Okamoto Hirosaki, Japan More articles by this author Noriko Tokui Hirosaki, Japan More articles by this author Naoki Sugiyama Hirosaki, Japan More articles by this author Yuichiro Suzuki Hirosaki, Japan More articles by this author Shigemasa Kudo Hirosaki, Japan More articles by this author Takahiro Yoneyama Hirosaki, Japan More articles by this author Yasuhiro Hashimoto Hirosaki, Japan More articles by this author Takuya Koie Hirosaki, Japan More articles by this author Noritaka Kamimura Hirosaki, Japan More articles by this author Michiko N Fukuda La Jolla, CA More articles by this author Chikara Ohyama Hirosaki, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3

Ara h 1, a vicilin; Ara h 2, a 2S albumin; and Ara h 3, a legumin, are major peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but cosensitization to all 3 allergens is correlated with the severity of patients' symptoms. We investigated whether cosensitization to these 3 allergens is caused by IgE cross-reactivity, despite the fact that they do not display obvious structural or sequence similarities. IgE cross-inhibitions were performed with purified Ara h 1, Ara h 2, and Ara h 3 and IgG-depleted sera from 10 patients with peanut allergy. After an in silico search for similar peptides, IgE ELISA inhibition assays with synthetic peptides were performed. Ara h 2 inhibited IgE binding to Ara h 1 (average, 86% ± 13%) and Ara h 3 (average, 96% ± 6%). IgE binding to Ara h 2 was inhibited by Ara h 1 by 78% ± 15% and by Ara h 3 by 80% ± 6%. A subsequent sequence comparison showed that these nonhomologous allergens contained several similar surface-exposed peptides. IgE binding to Ara h 2-derived peptides was completely inhibited by Ara h 1 and Ara h 3. A mixture of these peptides reduced IgE binding to Ara h 1 and Ara h 3 by 20% to 60% and to Ara h 2 by 49% to 89%. Occurrence of similar sequences in the 3 major peanut allergens accounts for the high extent of cross-reactivity among them.

Dasatinib Therapy Affects Bone Homeostasis in Patients with Chronic Myelogenous Leukemia in Chronic Phase Independently of Molecular Response

AbstractAbstract 1682 BackgroundWith the success of targeted oral tyrosine kinase inhibitor (TKI) therapy, long-term/life-long therapy for patients with chronic myelogenous leukemia (CML) can now be envisaged. Dasatinib is a second generation TKI, which in a phase I clinical trial was found to be 100–300 times more potent at inhibiting tyrosine kinases than imatinib. In vitro data suggest that dasatinib therapy also may have a direct effect on bone metabolism by interfering with c-FMS, c-SRC and the PDGF-R pathways (Boufker et al, BMC Cancer 2010, 10:298). In this study, we evaluated the effects of dasatinib monotherapy on bone homeostasis of CML patients treated at our center between 2006 and 2011. The levels of traditional biomarkers of bone health, namely serum bone specific alkaline phosphatase isoenzyme (BAP) and osteoprotegerin (OPG) were measured and were correlated with morphometric assessment of bone marrow biopsy specimens (BM Bx). We also assessed for any correlations between bone homeostasis and the results of conventional cytogenetics and molecular analysis with respect to treatment response. MethodsThe study group included 23 patients with chronic phase CML without evidence of clonal evolution who were treated with dasatinib. The median age was 51 years (range, 25–63) with a male to female ratio of 1.5: 1. Four different treatment groups were assessed (20mg/day, 40 mg/day, 80 mg/day and 100 mg/day). We compared paired BM specimens, obtained at initial diagnosis and 12–51 months (mos) after commencing dasatinib therapy. We performed whole slide imaging (Philips) on routinely stained BM Bx and specimen compartments were quantified using an area pixel count algorithm (Image Pro Plus system Version 6.3, MediaCybernetics, Bethesda, MD, USA) to calculate trabecular bone volume as a percentage area. The morphometric results were correlated with the results of conventional cytogenetics and molecular analysis obtained every 3 mos after enrollment. We also performed competitive inhibition ELISA assays to measure serum levels of OPG (baseline and after 12/18 mos) and BAP (every 4 mos), respectively. ResultsWith a median follow up of 24 mos (range 12–51mo), a significant increase in trabecular bone volume (TBV) was noted (p=0.022, Figure 1); 74% (17 pts) showed an increase in TBV on therapy, with an absolute increase of 5.2% (mean). Twelve of 16 pts (75%) evaluated had a decrease in OPG after 18 mos, which correlated in 90% of pts with an increase in TBV using a linear regression model (p= 0.0017, Figure 2). No significant difference was detected in TBV changes among the four dose groups. There was no significant trend in serum BAP levels, thus no statistical correlation was performed at this time point. All patients treated with dasatinib showed cytogenetic and molecular BCR-ABL1 response. There was no correlation between the degree of TBV change and either cytogenetic or molecular response (Figure 3) Conclusions:Our results indicate that within the therapeutic range for CML patients, dasatinib therapy promotes bone formation. However, there was no correlation between the dose levels studied and degree of bone formation and no correlation between TBV change and either cytogenetic or molecular response. The combination of digital image analysis and bone biomarkers enhances our understanding of bone homeostasis in patients on dasatinib. Further studies in a larger cohort of patients will be helpful to further validate our results.Figure 1: mean and standard deviation of TBV for each visit.Figure 2: correlation between “percent change TBV between visit 1 and visit 2” and 18 months OPG levels (linear regression model R2 =0.292, p= 0.0017)Figure 3: association between “percent change in TBV between visit 1 and visit 2” and “Rate of PCR response change between 3 and 12 months” (Spearman correlation coefficient = −0.05; p =0.81) [Display omitted] [Display omitted] [Display omitted] Disclosures:Cortes:Ariad: research support and consulting, research support and consulting Other; Pfizer: research support and consulting, research support and consulting Other; Novartis: research support and consulting, research support and consulting Other; BMS: research support and consulting Other.

Structural and immunological characterization of sulphatides: relevance of sulphate moieties inTrypanosoma cruziglycoconjugates

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.

Human intestinal beta defensins inhibit viral replication and are diminished in chronic untreated HIV infection

Background One of the hallmarks of HIV infection is an early and dramatic depletion of CD4 T cells in the intestinal lamina propria. This loss of T cells is accompanied by intestinal epithelial cell disruption resulting in the translocation of luminal bacterial products, persistent systemic immune activation and resultant disease progression. Beta defensins are cationic antimicrobial polypeptides produced by intestinal epithelial cells and phagocytes that are a part of the innate immune response. They have ap ivotal role in maintenance of immune homeostasis in the gut. Although beta defensins have been reported to inhibit bacterial and viral replication in vitro, their role in HIV infection has been incompletely characterized. Methods We investigated anti-viral activity and production of defensins in intestinal mucosal biopsies, stool and plasma from individuals with chronic untreated HIV (chronic progressors), immunologically controlled HIV (elite controllers) and HIV uninfected individuals using ELISA, immunohistochemistry, quantitative PCR and viral inhibition assays. Results Plasma levels of beta defensins were increased in the HIV controller group compared to HIV progressors, whereas levels of beta defensins were significantly decreased in biopsies from chronic progressors. Beta defensin release was induced by HIV in ex vivo cultured intestinal cells. Recombinant beta defensins inhibited HIV replication in vitro. Conclusion Beta defensins are upregulated by HIV and inhibit viral replication in vitro. Chronic progressors, however, had diminished levels of beta defensins in gut biopsies and in the plasma. We propose that beta defensins are induced in HIV infection and have a role in inhibiting viral replication and preventing intestinal epithelial cell disruption. In chronic infection, however, their release is diminished, likely due to epithelial damage in the setting of persistent viremia and mucosal inflammation.

Production and characterisation of monoclonal antibodies for the quantification of potentially allergenic xylanase from Aspergillus niger

Xylanase from Aspergillus niger (ANX) is widely used in bakeries as a processing aid since it stabilises and improves dough quality. An association between allergic symptoms among bakery workers and sensitisation to ANX has been reported, indicating that this enzyme is an occupational allergen. The presence of ANX in dough improvers and semi-finished goods is often hidden due to incomplete and unclear labelling. The quantification of microbial enzymes in these products is necessary and the determination of the actual concentration of ANX in workplaces is therefore essential to assess the occupational risk. To this purpose we have developed and characterised monoclonal antibodies to ANX. The monoclonal antibodies do not show any cross-reaction with other commonly used microbial enzymes, and they allow the detection of ANX in complex mixtures by ELISA inhibition assays down to the concentration limit of approximately 10 µg kg−1. These mAbs are a valuable tool to detect and quantify ANX and to investigate its allergenic potential in the workplace.

Consecutive Natural Influenza A Virus Infections in Sentinel Mallards in the Evident Absence of Subtype-Specific Hemagglutination Inhibiting Antibodies

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.