New light on an old syndrome: Role of Api g 7 in mugwort pollen-related celery allergy.

BACKGROUND: Celery root is known to cause severe allergic reactions in patients sensitized to mugwort pollen. OBJECTIVE: We studied clinically well-characterized patients with celery allergy by IgE testing with a comprehensive panel of celery allergens to disentangle the molecular basis of what is known as the celery-mugwort syndrome. METHODS: Patients with suspected food allergy to celery underwent a standardized interview. Main inclusion criteria were a positive food challenge with celery or an unambiguous case history of severe anaphylaxis. IgE to celery allergens (rApi g 1.01, rApi g 1.02, rApi g 2, rApi g 4, nApi g 5, rApi g 6, rApi g 7) and to mugwort allergens (rArt v 1, rArt v 3, rArt v 4) were determined. IgE levels ≥0.35 kUA/L were regarded positive. RESULTS: Seventy-nine patients with allergy to celery were included. Thirty patients had mild oral or rhinoconjunctival symptoms, and 49 had systemic reactions. Sixty-eight percent had IgE to celery extract, 80% to birch pollen, and 77% to mugwort pollen. A combination of Api g 1.01, 1.02, 4, 5, and 7 increased the diagnostic sensitivity for celery allergy to 92%. The lipid transfer proteins Api g 2 and Api g 6 were not relevant in our celery-allergic population. IgE to Api g 7, detected in 52% of patients, correlated closely (r = 0.86) to Art v 1 from mugwort pollen. Eleven of 12 patients with monosensitization to Api g 7 were IgE negative to celery extract. The odds ratio for developing a severe anaphylactic reaction rather than only mild oral symptoms was about 6 times greater (odds ratio, 5.87; 95% confidence interval, 1.08-32.0; P = .0410) for Api g 7-sensitized versus -nonsensitized subjects. CONCLUSION: There is an urgent need for routine diagnostic tests to assess sensitization to Api g 7, not only to increase test sensitivity but also to identify patients at risk of a severe allergic reaction to celery.

Anti-apoptotic genes and non-coding RNAs are potential outcome predictors for ulcerative colitis.

Due to the lack of clinical, immunologic, genetic, and laboratory markers to predict remission in ulcerative colitis (UC) without relapse, there is no clear recommendation regarding withdrawal of therapy. Therefore, this study was to investigate if transcriptional analysis together with Cox survival analysis might be able to reveal molecular markers that are specific for remission duration and outcome. Mucosal biopsies from patients in remission with active treatment-naïve UC and healthy control subjects underwent whole-transcriptome RNA-seq. Principal component analysis (PCA) and Cox proportional hazards regression analysis were applied to the remission data concerning duration and status of patients. A randomly chosen remission sample set was used for validation of the applied methods and results. The analyses distinguished two different UC remission patient groups with respect to remission duration and outcome (relapse). Both groups showed that altered states of UC with quiescent microscopic disease activity are still present. The patient group with the longest remission duration and no relapse revealed specific and increased expression of antiapoptotic factors belonging to the MTRNR2-like gene family and non-coding RNAs. In summary, the expression of anti-apoptotic factors and non-coding RNAs may contribute to personalized medicine approaches in UC by improving patient stratification for different treatment regimens.

Plant community responses to experimental climate manipulation in a Welsh ombrotrophic peatland and their palaeoenvironmental context.

We test whether vegetation community composition from a 10-year climate manipulation experiment on a Welsh peat bog resembles vegetation communities during periods of climate change inferred from a peat core. Experimentally warmed and combined warmed and droughted treatments drove significant increases in ericaceous shrubs but Sphagnum was unaffected. Similarly, Calluna vulgaris seeds increase during inferred warmer periods in the palaeoecological record. Experimental short-term episodic drought (four 4-week drought treatments) did not affect vegetation. Plant community composition has undergone several abrupt changes throughout the past c. 1500 years, often in response to human disturbance. Only slight changes occurred during the Medieval Climate Anomaly (c. 950-1250 Common Era [CE]) in vegetation and hydrology, while abrupt changes occurred during the Little Ice Age (c. 1300-1850 CE) when water tables were highest, suggesting that these shifts were driven by changes in water table, modulated by climate. A period of water table drawdown c. 1800, synchronous with historical records of increased drainage, corresponds with the development of the present-day vegetation community. Modern analogues for fossil material, characterized by abundant Rhynchospora alba and Sphagnum pulchrum, are more common after this event. Vegetation changes due to climate inferred from the palaeo record differ from those observed in the experiments, possibly relating to differences in the importance of drivers of vegetation change over varying timescales. Whereas temperature is frequently identified as the dominant driver of plant community change in experiments, sustained changes in water table appear to be more important in the long-term record. We find evidence that recent climate change and other anthropogenic stressors (e.g. drainage, heavy metal and nitrogen pollution) may promote the development of novel plant communities without analogues in the fossil record. These communities may be poorer at sequestering carbon and may respond differently to future climate change.

Pharmacodynamic mechanisms behind a refractory state in inflammatory bowel disease.

BACKGROUND AND AIMS: Biological therapy for inflammatory bowel disease is efficient in many cases but not all. The underlying molecular mechanisms behind non-response to biological therapy in inflammatory bowel disease are poorly described. Therefore, we aimed to characterize the mucosal cytokine transcript profile in non-immunogenic, non-responder patients with adequate trough level. MATERIAL AND METHODS: Patients with ulcerative colitis (UC) (n = 21) and Crohn's disease (CD) (n = 12) with non-response to biological therapy (anti-tumor necrosis factor (TNF) or vedolizumab) were included. Reference groups were A: untreated patients with UC or CD at debut of disease who had severe 1-year outcome, B: patients with UC or CD treated to endoscopic remission with biological agents, and C: healthy normal controls. Mucosal transcripts of TNF, interleukin (IL)17 and IL23 were measured by reverse transcription real-time quantitative polymerase chain reaction. Results Of the non-responders, 2 out of 12 CD and 1 out of 21 UC patients needed surgery during follow-up. Of the remaining non-responding patients, 8 out of 10 CD and 12 out of 20 UC patients switched biologic treatment. The remaining 2 CD and 8 UC patients continued treatment with the same biological agent with the addition of steroids, immunomodulators (AZA/MTX) and /or local steroids/5ASA. Twelve (8 UC/4 CD) out of 20 IBD patients were still non-responders after changing biological therapy to either anti-TNF (2), vedolizumab (9) or ustekinumab (1). The transcripts of IL17, IL23 and TNF were significantly upregulated in the non-response group compared to normal controls and patients in remission. In UC, 24% of the non-responders had normal mucosal TNF transcript indicating a non-TNF mediated inflammation. No obvious differences in gene expression were observed between primary and secondary non-responders, nor between anti-TNF and vedolizumab non-responders. CONCLUSIONS: Mucosal transcripts of IL17 and IL23 are highly associated with non-response to biological therapy, whereas some UC patients may also have a non-TNF mediated inflammatory pathway.

Prediction of long-term remission in patients following discontinuation of anti-TNF therapy in ulcerative colitis: a 10 year follow up study.

BACKGROUND: The long-term outcomes of Ulcerative colitis (UC) after discontinuation of biological therapy are largely unknown. There is also a lack of accurate and validated markers that can predict outcome after withdrawal accurately. The aims of this study were to describe the long-term outcomes in UC patients following cessation of anti-TNF therapy and explore potential biomarkers as an approach towards precision medicine. METHODS: Seventy-five patients with moderate to severe UC treated to remission with anti-tumor necrosis factor (TNF) were included in the study. This is a follow-up of previously reported UC outcomes. The patients were categorized as either "Remission" or "Relapse". The "Relapse" group was divided into subgroups determined by the highest treatment level needed to obtain remission the last 3 years of observation: non-biological therapy, biological therapy or colectomy. Remission were divided in long term remission (LTR), those using immunomodulating drugs (LTR + imids) and those using only 5-amino-salicylate (5-ASA) treatment (LTR) for the past 3 years. Analyses of mucosal gene expression by real-time PCR were performed. RESULTS: The median (IQR) observation time of all patients included was 121 (111-137) months. Of the 75 patients, 46 (61%) did not receive biological therapy, including 23 (31%) in LTR ± imids. Of these 23 patients, 16 (21%) were defined as LTR with a median observation time of (IQR) 95 (77-113) months. In total 14 patients (19%) underwent colectomy during the 10 years after first remission. Mucosal TNF copies/µg mRNA < 10 000 at anti-TNF discontinuation predicted long-term remission, biological free remission and lower risk of colectomy with a HR 0.36 (0.14-0.92) for long-term remission, HR 0.17 (0.04-0.78) for biological free remission and HR 0.12 (0.01-0.91) for colectomy. IL1RL1 was normalized in LTR phenotype and higher in relapsing UC. CONCLUSION: In this 10-year follow-up of UC of patients with moderate to severe disease, 61% of patients experience an altered phenotype to a milder disease course without need of biological therapy. Twenty-one percent of the patients were LTR without any medication except of 5-ASA. Mucosal TNF gene expression and IL1RL1- transcripts may be of clinical utility for long term prognosis in development of precision medicine in UC.

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Discovery and validation of mucosal TNF expression combined with histological score - a biomarker for personalized treatment in ulcerative colitis.

BACKGROUND: There are no accurate markers that can predict clinical outcome in ulcerative colitis at time of diagnosis. The aim of this study was to explore a comprehensive data set to identify and validate predictors of clinical outcome in the first year following diagnosis. METHODS: Treatment naive-patients with ulcerative colitis were included at time of initial diagnosis from 2004 to 2014, followed by a validation study from 2014 to 2018. Patients were treated according to clinical guidelines following a standard step-up regime. Patients were categorized according to the treatment level necessary to achieve clinical remission: mild, moderate and severe. The biopsies were assessed by Robarts histopathology index (RHI) and TNF gene transcripts. RESULTS: We included 66 patients in the calibration cohort and 89 patients in the validation. Mucosal TNF transcripts showed high test reliability for predicting severe outcome in UC. When combined with histological activity (RHI) scores the test improved its diagnostic reliability. Based on the cut-off values of mucosal TNF and RHI scores from the calibration cohort, the combined test had still high reliability in the validation cohort (specificity 0.99, sensitivity 0.44, PPV 0.89, NPV 0.87) and a diagnostic odds-ratio (DOR) of 54. CONCLUSIONS: The combined test using TNF transcript and histological score at debut of UC can predict severe outcome and the need for anti-TNF therapy with a high level of precision. These validated data may be of great clinical utility and contribute to a personalized medical approach with the possibility of top-down treatment for selected patients.

Transferability study of the BINACLE (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity.

The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the "binding and cleavage" (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market. Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products.

Individual combinations of danger signals synergistically increase FVIII product immunogenicity.

INTRODUCTION: The most severe side effect in haemophilia A treatment is the development of antifactor VIII antibodies, also called inhibitors. Why inhibitors develop in a proportion of treated patients while others are unaffected still remains unanswered. The presence of immunological danger signals, associated with events such as infection or surgery, has been proposed to play a role. Previous studies demonstrated that the presence of the bacterial molecule lipopolysaccharide (LPS) can synergistically increase the activation of human DC and subsequent T cell activation by FVIII. AIM AND METHODS: In the present study, we investigated whether a combination of two danger signals can further increase immune cell activation by FVIII. For this, human in vitro differentiated DC that were treated with combinations of danger signals were co-cultured with autologous primary T cells, and T cell proliferation was analysed. RESULTS: Interestingly, by combining LPS with a second danger signal, lower LPS concentrations were sufficient to synergistically increase DC and subsequent T cell activation by FVIII. Of note, a combination of LPS and the double-stranded RNA, polyinosinic-polycytidylic acid (poly(I:C)), was most potent in increasing FVIII immunogenicity, followed by LPS + R848 (resiquimod). However, a combination of LPS and the bacterial lipopeptide Pam3CysSK4 did not induce increased immune cell activation by FVIII. CONCLUSION: Thus, individual combinations of danger signals can increase FVIII product immunogenicity. This should be considered in the treatment routine of haemophilia A patients.

Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies.

INTRODUCTION: Standard treatment of congenital haemophilia A is based on replacement therapy with coagulation factor VIII (FVIII) products. A major complication of FVIII therapy is the occurrence of IgG alloantibodies (inhibitors) that neutralize FVIII activity. AIM: The aim of the analysis was estimating the risk of high-titre inhibitor associated with the second-generation full-length product compared to third-generation full-length product and other recombinant FVIII (rFVIII). METHODS: We conducted a combined analysis of individual patient data from three large studies in previously untreated patients (PUPs) with severe haemophilia A. RESULTS: A total of 1109 PUPs were treated from 1993 to 2013 including 787 PUPs treated from 2004 onwards (primary analysis cohort). A total of 322 patients (29.0%) developed an inhibitor, of which 192 (17.3%) a high-titre inhibitor. In the primary analysis set, 29.9% of patients developed an inhibitor and 17.2% a high-titre inhibitor. The combined analysis indicated a lower risk of high-titre inhibitor development for the third-generation rFVIII product compared to the second-generation rFVIII product (primary analysis: adjusted hazard ratio (HR) = 0.72, 95% CI: 0.49 to 1.06). Adjusted HR for all inhibitor development was significantly lower for the third-generation product compared to the second-generation product. CONCLUSION: The trend of an increased risk of inhibitor development in PUPs for one recombinant product illustrates that extrapolation from one recombinant factor VIII product to other products might not be justified.

Aluminium in plasma and tissues after intramuscular injection of adjuvanted human vaccines in rats.

Aluminium (Al) toxicokinetics after intramuscular (IM) injection of Al-adjuvanted vaccines is unknown. Since animal data are required for modeling and extrapolation, a rat study was conducted measuring Al in plasma and tissues after IM injection of either plain Al-hydroxide (pAH) or Al-phosphate (pAP) adjuvant (Al dose 1.25 mg), single human doses of three Al-adjuvanted vaccines (V1, V2, and V3; Al doses 0.5-0.82 mg), or vehicle (saline). A significant increase in Al plasma levels compared to controls was observed after pAP (AUC(0-80 d), mean ± SD: 2424 ± 496 vs. 1744 ± 508 µg/L*d). Percentage of Al dose released from injected muscle until day 80 was higher after pAP (66.9%) and AP-adjuvanted V3 (85.5%) than after pAH and AH-adjuvanted V1 (0 and 22.3%, resp.). Estimated absolute Al release was highest for pAP (836.8 µg per rat). Al concentration in humerus bone was increased in all groups, again strongest in the pAP group [3.35 ± 0.39 vs. 0.05 ± 0.06 µg/g wet weight (ww)]. Extrapolated amounts in whole skeleton corresponded to 5-12% of the released Al dose. Very low brain Al concentrations were observed in all groups (adjuvant group means 0.14-0.29 µg/g ww; control 0.13 ± 0.04 µg/g ww). The results demonstrate systemically available Al from marketed vaccines in rats being mainly detectable in bone. Al release appears to be faster from AP- than AH-adjuvants. Dose scaling to human adults suggests that increase of Al in plasma and tissues after single vaccinations will be indistinguishable from baseline levels.

Aluminium toxicokinetics after intramuscular, subcutaneous, and intravenous injection of Al citrate solution in rats.

Knowledge of dose linearity, plasma clearance, rate and extent of subcutaneous (SC) and intramuscular (IM) absorption of soluble aluminium (Al) citrate is considered a prerequisite for evaluation of toxicokinetic data obtained from SC or IM administration of Al adjuvants in medicinal products. Therefore, total Al plasma kinetics was investigated after SC, IM, and IV administration of single Al doses (36 and 360 µg/kg IM or SC; 30 and 300 µg/kg IV) given as citrate solution in rats. Control groups receiving vehicle (saline) were run in parallel to monitor background plasma Al levels over time resulting from dietary intake. Evaluation of Al plasma profiles was done by both non-compartmental analysis of baseline-corrected data and simultaneous model fitting to the raw data using a population kinetics approach. High and dose-independent total plasma clearance (6.6 mL/min/kg) was observed after IV administration corresponding to 60-82% of normal rat GFR. This supports the previous assumptions that parenterally administered Al citrate is more rapidly cleared from plasma than other Al species (e.g., chloride or lactate). Furthermore, plasma exposure of Al (Cmax and AUC0-inf) increased dose-proportionally at all administration routes. Fast and complete absorption of Al was observed at each dose level after both SC and IM administration (bioavailability estimates: 88 and 110%). Estimates for the first-order absorption rate constant ka correspond to absorption half-lives of 36 min (SC) and ≤ 13 min (IM). There was no increase in tissue Al content (whole bone and brain) after 36 µg/kg IM compared to control rats.

First Demonstration of Laser-Assisted Charge Exchange for Microsecond Duration H

This Letter reports on the first demonstration of laser-assisted H^{-} charge exchange for microsecond duration H^{-} beam pulses. Laser-assisted charge exchange injection is a breakthrough technology that overcomes long-standing limitations associated with the traditional method of producing high intensity, time structured beams of protons in accelerators via the use of carbon foils for charge exchange injection. The central theme of this experiment is the demonstration of novel techniques that reduce the laser power requirement to allow high efficiency stripping of microsecond duration beams with commercial laser technology.

Harmonization of nucleic acid testing for Zika virus: development of the 1st World Health Organization International Standard.

BACKGROUND: With the ongoing public health emergency due to Zika virus (ZIKV), nucleic acid testing (NAT) is essential for clinical diagnosis and screening of blood donors. However, NAT for ZIKV has not been standardized, and this study was performed to establish a World Health Organization (WHO) International Standard (IS) for ZIKV RNA; WHO ISs have been used to improve detection and quantification of blood-borne viruses. STUDY DESIGN AND METHODS: The candidate IS (cIS), code number 11468/16, was prepared by heat inactivation and lyophilization of a ZIKV strain isolated from a patient in French Polynesia in 2013. The cIS was evaluated together with other reference materials, including both Asian and African ZIKV lineages as well as a panel of clinical samples and in vitro-transcribed RNAs. The samples for evaluation were distributed to 24 laboratories from 11 countries. The assays used consisted of a mixture of in-house developed and commercial assays (available or in development). RESULTS: The potencies of the standards were determined by quantitative and qualitative assays. In total, 37 sets of data were analyzed: 19 from quantitative assays and 18 from qualitative assays. Data demonstrated wide variations in reported potencies of the cIS and the other study samples. CONCLUSIONS: Assay variability was substantially reduced when ZIKV RNA concentrations from the biological reference materials and clinical samples were expressed relative to the cIS. Thus, the WHO has established 11468/16 as the 1st IS for ZIKV RNA, with a unitage of 50,000,000 IU/mL.

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform.

UNLABELLED: In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR(-/-))-CD46Ge mice with 2 × 10(5) 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE: Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.

Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA.

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.

A Novel Multipeptide Microarray for the Specific and Sensitive Mapping of Linear IgE-Binding Epitopes of Food Allergens.

BACKGROUND: The identification of B-cell epitopes of food allergens can possibly lead to novel diagnostic tools and therapeutic reagents for food allergy. We sought to develop a flexible, low-tech, cost-effective and reproducible multipeptide microarray for the research environment to enable large-scale screening of IgE epitopes of food allergens. METHODS: Overlapping peptides (15-mer, 4 amino acid offset) covering the primary sequence of either peanut allergen Ara h 1 or all 3 subunits of the soybean allergen Gly m 5 were simultaneously synthesized in-house on a porous cellulose matrix. Identical peptide microarrays created with up to 384 duplicate peptide-cellulose microspots each were investigated for specificity and sensitivity in IgE immunodetection and in direct experimental comparison to the formerly established SPOT™ membrane technique. RESULTS: The in-house microarray identified with 98% reproducibility the same IgE-binding peptides as the SPOT™ membrane technique. Additional IgE-binding peptides were identified using the microarray. While the sensitivity was increased between 2- and 20-fold, the amount of human serum required was reduced by at least two thirds over the SPOT™ membrane technique using the microarray. After subtraction of the potential background, we did not observe non-specific binding to the presented peptides on microarray. CONCLUSIONS: The novel peptide microarray allows simple and cost-effective screening for potential epitopes of large allergenic legume seed storage proteins, and it could be adapted for other food allergens as well, to study allergenic epitopes at the individual subject level in large paediatric and adult study groups of food allergic subjects.