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.

Exploring the Impact of Subclinical Hypothyroidism on Hyperpigmentation: A Rare Presentation in a 42-Year-Old Woman.

Hyperpigmentation of the skin can occur due to internal and external causes. This case highlights an unusual presentation of generalized acute hyperpigmentation associated with subclinical hypothyroidism in a 42-year-old Indian American woman. After unsuccessful trials of various topical agents, the patient exhibited significant improvement in hyperpigmentation after levothyroxine treatment. Improvements included lightening in the bilateral antecubital fossa, axillae, and neck regions. This case underscores the importance of considering thyroid dysfunction as a potential factor that may contribute to atypical pigmentation disorders.

DNA methylation fine-tunes pro-and anti-inflammatory signalling pathways in inactive ulcerative colitis tissue biopsies.

DNA methylation has been implied to play a role in the immune dysfunction associated with inflammatory bowel disease (IBD) and the disease development of ulcerative colitis (UC). Changes of the DNA methylation and correlated gene expression in patient samples with inactive UC might reveal possible regulatory features important for further treatment options for UC. Targeted bisulfite sequencing and whole transcriptome sequencing were performed on mucosal biopsies from patients with active UC (UC, n = 14), inactive UC (RM, n = 20), and non-IBD patients which served as controls (NN, n = 11). The differentially methylated regions (DMRs) were identified by DMRseq. Correlation analysis was performed between DMRs and their nearest differentially expressed genes (DEGs). Principal component analysis (PCA) was performed based on correlated DMR regulated genes. DMR regulated genes then were functional annotated. Cell-type deconvolutions were performed based on methylation levels. The comparisons revealed a total of 38 methylation-regulated genes in inactive UC that are potentially regulated by DMRs (correlation p value < 0.1). Several methylation-regulated genes could be identified in inactive UC participating in IL-10 and cytokine signalling pathways such as IL1B and STAT3. DNA methylation events in inactive UC seem to be fine-tuned by the balancing pro- and anti- inflammatory pathways to maintain a prevailed healing process to restore dynamic epithelium homeostasis.

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.

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.

Development of a Factor VII Activating Protease (FSAP) generation assay and its application in studying FSAP in venous thrombosis.

Human genetic studies based on the Marburg I polymorphism in the factor VII activating protease (FSAP) encoding gene, analysis of FSAP activity in plasma and biochemical characterization of FSAP substrates indicate a possible causal link between FSAP activity and venous thrombosis. We hypothesized that a direct standardized assay to measure FSAP activity in plasma could provide convincing arguments for or against such a potential link. Using Ac-Pro-DTyr-Lys-Arg-AMC as a highly specific and sensitive substrate, histones as a trigger to activate pro-FSAP and plasma-purified active FSAP as a calibrator, we have developed a fluorogenic kinetic assay that reveals the FSAP generating potential in human plasma in real time. This assay is similar to the thrombin generation assay and allows analysis of lag phase, time to peak and velocity, as well as peak FSAP and the endogenous FSAP potential (EFP) of plasma samples. Carriers of the Marburg I polymorphism showed clearly delayed FSAP generation and lower peak FSAP and EFP level. There were no significant differences in all FSAP activity parameters between plasma from patients with a history of venous thrombosis and controls. When excluding Marburg I carriers, which were evenly distributed between groups, delayed FSAP generation significantly correlated with venous thrombosis in postmenopausal women. The novel FSAP activity assay is robust and easy to perform and will be a useful tool for analyzing plasma FSAP activity, also, in other pathophysiological conditions.

Recent climate change has driven divergent hydrological shifts in high-latitude peatlands.

High-latitude peatlands are changing rapidly in response to climate change, including permafrost thaw. Here, we reconstruct hydrological conditions since the seventeenth century using testate amoeba data from 103 high-latitude peat archives. We show that 54% of the peatlands have been drying and 32% have been wetting over this period, illustrating the complex ecohydrological dynamics of high latitude peatlands and their highly uncertain responses to a warming climate.

Hypo-osmotic stress induces the epithelial alarmin IL-33 in the colonic barrier of ulcerative colitis.

Epithelial alarmins are gaining interest as therapeutic targets for chronic inflammation. The nuclear alarmin interleukin-33 (IL-33) is upregulated in the colonic mucosa of acute ulcerative colitis (UC) and may represent an early instigator of the inflammatory cascade. However, it is not clear what signals drive the expression of IL-33 in the colonic mucosa, nor is the exact role of IL-33 elucidated. We established an ex vivo model using endoscopic colonic biopsies from healthy controls and UC patients. Colonic biopsies exposed to hypo-osmotic medium induced a strong nuclear IL-33 expression in colonic crypts in both healthy controls and UC biopsies. Mucosal IL33 mRNA was also significantly increased following hypo-osmotic stress in healthy controls compared to non-stimulated biopsies (fold change 3.9, p-value < 0.02). We observed a modest induction of IL-33 in response to TGF-beta-1 stimulation, whereas responsiveness to inflammatory cytokines TNF and IFN-gamma was negligible. In conclusion our findings indicate that epithelial IL-33 is induced by hypo-osmotic stress, rather than prototypic proinflammatory cytokines in colonic ex vivo biopsies. This is a novel finding, linking a potent cytokine and alarmin of the innate immune system with cellular stress mechanisms and mucosal inflammation.

Helicobacter pylori resistance to antibiotics before and after treatment: Incidence of eradication failure.

BACKGROUND: Increasing prevalence of antibiotic resistance especially to clarithromycin and metronidazole has been observed in Helicobacter pylori (H. pylori). AIM: To characterize the antimicrobial resistance pattern of H. pylori before and after treatment in a cohort of patients accumulated over a period of 15 years after an unsuccessful eradication treatment had been given comparing sensitivity data from patients with newly diagnosed H. pylori infection. A specific objective was to look for resistance to levofloxacin. MATERIAL AND METHODS: Total of 50 patients newly diagnosed for H. pylori infection treated with omeprazole and amoxicillin/clarithromycin and 42 H pylori treatment-resistant patients treated with omeprazole and amoxicillin/levofloxacin were enrolled in this study. Cultures including antibiotic sensitivity testing were conducted according to standard laboratory routines and thus also in keeping with a European study protocol using E-test gradient strips or disc diffusion methods. RESULTS: Clarithromycin resistance was more frequently observed in the H. pylori resistant group than in newly diagnosed H. pylori group (39% versus 11%). Regarding metronidazole the distribution was 70% versus 38%, and 8% versus 12% were resistant to tetracycline. No resistance was observed for amoxicillin. After re-treatment of patients belonging to the H. pylori treatment-resistant group, just two patient strains were recovered of which one harbored metronidazole resistance. In the group of newly diagnosed H. pylori, seven patients were culture positive by control after treatment. Two and three patient strains showing resistance to clarithromycin and metronidazole, respectively. None of the strains in our material was classified as resistant to amoxicillin and levofloxacin. Whereas 12% was resistant to tetracycline in the newly diagnosed before treatment. CONCLUSION: Clarithromycin resistance was more frequent in the H. pylori treatment-resistant group than strains from patients with newly diagnosed H. pylori infection. No resistance was observed to amoxicillin and levofloxacin. In such cases Therefore levofloxacin may be used provided in vitro sensitivity testing confirms applicability. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT05019586.

Malignant Pilomatricoma of the Lower Extremity: A Difficult and Rare Diagnosis.

Malignant pilomatricoma is a rare cutaneous malignancy that is commonly found on the head and neck. We present a patient with malignant pilomatricoma of the lower extremity with intralesional calcification and giant cells, confirmed by histopathology. This patient's case represents a clinically important variation of malignant pilomatricoma in an uncommon anatomical location.

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.

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.

Clinical characterization of Helicobacter pylori infected patients 15 years after unsuccessful eradication.

BACKGROUND AND AIMS: Patients that have failed therapy for Helicobacter pylori (H. pylori) infection are incompletely characterized. The aim of this study was to characterize a H. pylori treatment resistant cohort compared to the cohorts of newly diagnosed, earlier eradicated and non-infected. MATERIAL AND METHODS: Patients were selected from routine referrals to the Endoscopy units at three different Norwegian hospitals. In all four cohorts, gastric biopsies were scored according to the Sydney classification, and symptoms according to the Gastrointestinal Symptom Rating Scale score, including sub-scores for upper gastrointestinal symptoms and functional bowel symptoms. Patients in the H. pylori resistant group were treated with a triple therapy regimen that consisted of levofloxacin, amoxicillin and a proton pump inhibitor. RESULTS: We included 185 patients, 42 H. pylori treatment resistant, 50 newly diagnosed, 61 previously H. pylori eradicated and 32 never infected. The treatment-resistant cohort had higher scores for upper gastrointestinal symptoms and functional bowel symptoms compared to the other groups except for the group being never H. pylori infected. The H. pylori resistant patients had lower Sydney scores than patients with newly diagnosed H. pylori infection. The triple combination showed a high efficacy of 91% to eradicate H. pylori. CONCLUSIONS: Patients with treatment-resistant H. pylori infection had more gastrointestinal symptoms, but a lower Sydney score than patients with newly diagnosed infection. A treatment regimen including levofloxacin showed a high efficacy in eradicating H. pylori in patients that previously had failed eradication treatment.

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.

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.

Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing.

BACKGROUND: Globally, hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Epidemiology and clinical presentation of hepatitis E vary greatly by location and are affected by the HEV genotype. Nucleic acid amplification technique (NAT)-based assays are important for the detection of acute HEV infection as well for monitoring chronic cases of hepatitis E. OBJECTIVES: The aim of the study was to evaluate a panel of samples containing different genotypes of HEV for use in nucleic NAT-based assays. STUDY DESIGN: The panel of samples comprises eleven different members including HEV genotype 1a (2 strains), 1e, 2a, 3b, 3c, 3e, 3f, 4c, 4g as well as a human isolate related to rabbit HEV. Each laboratory assayed the panel members directly against the 1st World Health Organization (WHO) International Standard (IS) for HEV RNA (6329/10) which is based upon a genotype 3 a strain. RESULTS: The samples for evaluation were distributed to 24 laboratories from 14 different countries and assayed on three separate days. Of these, 23 participating laboratories returned a total of 32 sets of data; 17 from quantitative assays and 15 from qualitative assays. The assays used consisted of a mixture of in-house developed and commercially available assays. The results showed that all samples were detected consistently by the majority of participants, although in some cases, some samples were detected less efficiently. CONCLUSIONS: Based on the results of the collaborative study the panel (code number 8578/13) was established as the "1st International Reference Panel (IRP) for all HEV genotypes for NAT-based assays" by the WHO Expert Committee on Biological Standardization. This IRP will be important for assay validation and ensuring adequate detection of different genotypes and clinically important sub-genotypes of HEV.