Search

The corona virus outbreak in Wuhan, China, at the end of 2019 has rapidly evolved into a pandemic which is still virulent in many countries. An infection with SARS-CoV-2 can lead to corona virus disease (Covid-19). This paper presents an overview of the knowledge gained so far with regard to histopathological lung lesions in fatal courses of Covid-19. The main findings were diffuse alveolar damage and micro-angiopathies. These included the development of hyaline membranes, thrombi, endothelial inflammation, haemorrhages and angiogenesis. Overall, the vessel lesions seemed to be more lethal than the diffuse alveolar damage. There was obvious hyperreactivity and hyperinflammation of the cellular immune system. An expanded T-cell memory may explain the increased risk of a severe course in the elderly.

The purpose of this article is to understand the experience of workers’ perceptions of job insecurity and its relation to performance. To this end, we conducted semi-structured interviews with 38 workers in the retail, services, education, financial, construction, and pharmaceutical industries in Chile. Using content analysis based on workers’ accounts of their own experience, we identified two main categories: (a) the experience of job insecurity viewed in relation to the context of the COVID-19 pandemic and emotional aspects of job insecurity, and (b) the relation between job insecurity and performance. The possibility of job loss expresses itself in experiences and emotions that are related to the performance of workers in different ways. These findings are discussed in terms of stress theory and the motivation to preserve jobs.

OBJECTIVES The aim of this study was to compare the diagnostic accuracy of apparent diffusion coefficient (ADC) ratios as a monoparametric magnetic resonance imaging (MRI) protocol for the detection of prostate cancer (PCa) with the established multiparametric (mp) MRI at 3.0 T. MATERIALS AND METHODS According to power analysis, 52 male patients were included in this monocenter study with prospective data collection and retrospective, blinded multireader image analysis. The study was approved by the local ethics committee. Patients were recruited from January to December 2020. Based on mpMRI findings, patients underwent in-bore MR biopsy or prostatectomy for histopathologic correlation of suspicious lesions. Three readers, blinded to the histopathologic results and images of mpMRI, independently evaluated ADC maps for the detection of PCa. The ADC ratio was defined as the lowest signal intensity (SI) of lesions divided by the SI of normal tissue in the zone of origin. Predictive accuracy of multiparametric and monoparametric MRI were compared using logistic regression analysis. Moreover, both protocols were compared applying goodness-of-fit analysis with the Hosmer-Lemeshow test for continuous ADC ratios and Pearson χ2 test for binary decision calls, correlation analysis with Spearman ρ and intraclass correlation coefficients, as well as noninferiority assessment with a TOST ("two one-sided test"). RESULTS Eighty-one histopathologically proven, unique PCa lesions (Gleason score [GS] ≥ 3 + 3) in 52 patients could be unequivocally correlated, with 57 clinically significant (cs) PCa lesions (GS ≥ 3 + 4). Multiparametric MRI detected 95%, and monoparametric ADC detected ratios 91% to 93% of csPCa. Noninferiority of monoparametric MRI was confirmed by TOST (P < 0.05 for all comparisons). Logistic regression analysis revealed comparable predictive diagnostic accuracy of ADC ratios (73.7%-87.8%) versus mpMRI (72.2%-84.7%). Spearman rank correlation coefficient for PCa aggressiveness revealed satisfactory correlation of ADC ratios (P < 0.013 for all correlations). The Hosmer-Lemeshow test for the logistic regression analysis for continuous ADC ratios indicated adequate predictive accuracy (P = 0.55-0.87), and the Pearson χ2 test showed satisfactory goodness of fit (P = 0.35-0.69, χ2 = 0.16-0.87). CONCLUSIONS Normalized ADC ratios based on advanced DWI are noninferior to mpMRI at 3.0 T for the detection of csPCa in a preselected patient cohort and proved a fast and accurate assessment tool, thus showing a potential prospect of easing the development of future screening methods for PCa.

Although job insecurity has been shown to predict numerous adverse outcomes, more is yet to be known about the mechanisms that explain when and why these effects will occur. Using social exchange as our theoretical foundation and three-wave lagged survey data collected from N = 300 employees within the United States, the current study found support for the contention that individuals with greater job insecurity pursue fewer idiosyncratic deals (I-Deals) with their employers. Fewer I-Deals in turn were associated with lower affective commitment, and higher turnover intentions, perceived psychological contract breach, and psychological contract violation. However, these indirect effects were conditional upon employee levels of grit (specifically, perseverance of effort), such that higher grit attenuated the negative relationship between job insecurity and the pursuit of I-Deals. Given that employees often have little control over the source of their experienced job insecurity (e.g., impending layoffs, a poor economy, announced merger or acquisition), these findings hold promise in potentially alleviating the well-documented negative outcomes of job insecurity through interventions designed to increase employee levels of grit and facilitate the creation of I-Deals. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Background and Aims: Despite older-aged individuals accounting for most patients with pancreatic cancer, elderly patients are still underrepresented in the clinical trials. Our study aims to identify treatment differences as well as to analyze survival times in the younger and older patient group. Methods: We evaluated the data of 97 pancreatic cancer patients (72

Cervical cancer therapy is still a major clinical challenge, as patients substantially differ in their response to standard treatments, including chemoradiotherapy (CRT). During cervical carcinogenesis, T‐helper (Th)‐17 cells accumulate in the peripheral blood and tumor tissues of cancer patients and are associated with poor prognosis. In this prospective study, we find increased Th17 frequencies in the blood of patients after chemoradiotherapy and a post‐therapeutic ratio of Th17/CD4+ T cells > 8% was associated with early recurrence. Furthermore, Th17 cells promote resistance of cervical cancer cells toward CRT, which was dependent on the AKT signaling pathway. Consistently, patients with high Th17 frequencies in pretherapeutic biopsies exhibit lower response to primary CRT. This work reveals a key role of Th17 cells in CRT resistance and elevated Th17 frequencies in the blood after CRT correspond with early recurrence. Our results may help to explain individual treatment responses of cervical cancer patients and suggest evaluation of Th17 cells as a novel predictive biomarker for chemoradiotherapy responses and as a potential target for immunotherapy in cervical cancer., This study demonstrates that chemoradiotherapy‐treated patients with cervical cancer have increased frequencies of Th17 cells. Th17 frequency was associated with early cancer relapse. Additionally, high pretherapeutic Th17 amounts in situ were correlated to a failed response to chemoradiotherapy. Our work suggests that the evaluation of Th17 cells could serve as a novel predictive biomarker for chemoradiotherapy responses and a potential target for immunotherapy in patients with cervical cancers.

Salvage lymph node dissection is a rescue treatment for patients with nodal recurrence after radical prostatectomy. Very limited data are available on robotic salvage lymph node dissection. Our purpose was to investigate perioperative and oncological outcomes of robotic salvage lymph node dissection in a large monocentric series.Perioperative data, complications within 30 days after surgery and oncological outcomes as assessed by histology, prostate specific antigen changes, prostate specific antigen nadir after salvage lymph node dissection, and time to further therapy were analyzed. To identify predictive factors for oncological outcome, Kaplan-Meier and Cox-regression analyses were performed. For cases with a mismatch between preoperative positron emission tomography/computed tomography and the number of histologically positive lymph nodes, prostate specific membrane antigen immunohistochemistry was performed on removed lymph nodes.A total of 68 patients underwent robotic salvage lymph node dissection with a median operation time of 126 minutes, a blood loss of 50 ml, and a length of stay of 4 days. No major complications (Clavien 3) occurred. Median followup was 12.1 months. Median time to further therapy was 12.4 months, 37% of patients experienced complete biochemical response (prostate specific antigen0.2 ng/ml) and 11% reached an undetectable prostate specific antigen, which was maintained for1 year in 3 cases. Lower preoperative prostate specific antigen, longer time between radical prostatectomy and salvage lymph node dissection, preoperative prostate specific membrane antigen positron emission tomography/computed tomography and complete biochemical response after salvage lymph node dissection were significant predictors of longer therapy-free survival (all p0.005). Prostate specific membrane antigen immunohistochemistry revealed that prostate specific membrane antigen positron emission tomography/computed tomography tends to miss small lymph node metastases5 mm.Robotic salvage lymph node dissection is a feasible approach with low perioperative morbidity and delays further systemic therapy in most patients. Prostate specific membrane antigen positron emission tomography/computed tomography detection is mostly limited to tumor foci5 mm.

IntroductionThe last two decades have seen a shift in former Soviet countries from highly specialized to more family medicine-focused systems. Medical education has slowly adjusted to these reforms, although the region is still at risk to have a chronic shortage of family doctors. This paper presents the evaluation of a new post-graduate family medicine program in Tajikistan, focused on competency-based training. The findings are relevant for policy makers, international organizations and practitioners participating in similar medical education reform programs.MethodsWe employed a quasi-experimental control group design and compared intervention residents, control group residents with traditional training, and 1st year residents with no training in two outcomes, clinical knowledge and competencies. We employed two objective measures, a written multiple-choice question test (MCQT) and an Objective Structured Clinical Examination (OSCE), respectively. We report reliability and validity of the measures along with ANOVA, planned contrasts and effect size estimates to examine differences across groups.ResultsWe found statistically significant differences in both clinical knowledge and competencies between intervention and control groups. We also detected a large intervention effect size. Participants in the intervention outperformed control group participants in the two measures. Our analysis suggests that intervention and control group participants are comparable in terms of initial knowledge and competencies, strengthening the argument that the intervention caused the improvement in the program outcomes.DiscussionReceiving tailored training and structured opportunities to practice knowledge and competencies in clinical settings have a positive effect on the education of family medicine doctors in Tajikistan. Our results support curriculum reform and investment in medical education in the form of longer and supervised on-the-job preparation designed to be more in line with international standards. We discuss suggestions for future studies and potential requirements to inform replicability in other countries.ConclusionFamily medicine is well recognized as central to health systems throughout the world, but high quality residency training lags behind in some countries. Our study showed that investing in family medicine residency programs and structured training is effective in increasing critical clinical competencies. We encourage promoting comprehensive post graduate family medicine doctor training so that the goals of a family medicine centered health system are attainable.

Costimulatory signals potently promote T‐cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4‐1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen‐specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA‐A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T‐cell responses. CD28 costimulation potently augmented the percentage and number of antigen‐reactive CD8 T cells, whereas eAPC expressing 4‐1BB‐ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4‐1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose‐dependent manner. However, the promotion of bystander CD8 T‐cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen‐specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy., Using a novel system of engineered antigen presenting cells (eAPC), we demonstrate that 4‐1BB signals generated by 4‐1BBL or agonistic 4‐1BB antibodies promote bystander activation of human CD8 T cells

Background Medical digital twins are computational disease models for drug discovery and treatment. Unresolved problems include how to organize and prioritize between disease-associated changes in digital twins, on cellulome- and genome-wide scales. We present a dynamic framework that can be used to model such changes and thereby prioritize upstream regulators (URs) for biomarker- and drug discovery. Methods We started with seasonal allergic rhinitis (SAR) as a disease model, by analyses of in vitro allergen-stimulated peripheral blood mononuclear cells (PBMC) from SAR patients. Time-series a single-cell RNA-sequencing (scRNA-seq) data of these cells were used to construct multicellular network models (MNMs) at each time point of molecular interactions between cell types. We hypothesized that predicted molecular interactions between cell types in the MNMs could be traced to find an UR gene, at an early time point. We performed bioinformatic and functional studies of the MNMs to develop a scalable framework to prioritize UR genes. This framework was tested on a single-cell and bulk-profiling data from SAR and other inflammatory diseases. Results Our scRNA-seq-based time-series MNMs of SAR showed thousands of differentially expressed genes (DEGs) across multiple cell types, which varied between time points. Instead of a single-UR gene in each MNM, we found multiple URs dispersed across the cell types. Thus, at each time point, the MNMs formed multi-directional networks. The absence of linear hierarchies and time-dependent variations in MNMs complicated the prioritization of URs. For example, the expression and functions of Th2 cytokines, which are approved drug targets in allergies, varied across cell types, and time points. Our analyses of bulk- and single-cell data from other inflammatory diseases also revealed multi-directional networks that showed stage-dependent variations. We therefore developed a quantitative approach to prioritize URs: we ranked the URs based on their predicted effects on downstream target cells. Experimental and bioinformatic analyses supported that this kind of ranking is a tractable approach for prioritizing URs. Conclusions We present a scalable framework for modeling dynamic changes in digital twins, on cellulome- and genome-wide scales, to prioritize UR genes for biomarker and drug discovery. Funding Agencies|Linkoping University; NIH [HG007690, HL108630, HL155107, HL155096, HL119145]; American Heart Association [D700382]; Vinnova; Research Council of Norway [90825]; European Commission [848028.2]; Swedish Cancer Society [CAN 2017/411]; National Research Foundation of Korea (NRF) - Korea government (Ministry of Science and ICT) [2021R1A2C1010082]; Austrian Science Funds (FWF) [P32953, I4437]; Swedish Research Council, VR [2019-01165]; Cocozza Foundation; Innovationsfonden

In this Letter to the Editor supportive data were presented to a recent paper published in this journal reporting the involvement of TRP channels in COVID-19 pneumonia and its role for new therapies. Since gene expression of TRP channels was found in human lung tissues the protein was not being reported so far. TRP channels are supposed to be involved in the pulmonary inflammation and its symptoms such as fever, cough and others. Here, TRPC6 was investigated in tissues of normal human lungs and of SARS-Cov-2 infected lungs in a preliminary study. Tissue was obtained post mortem from anatomical body donations during dissections and during pathological dissections (13 normal, 4 COVID-19 pneumoniae) and processed for immunohistochemistry. In normal lungs TRPC6 was found in the ciliated epithelium, in the wall of larger lung vessels and in the alveolar septa. In COVID-19 pneumonia the distribution of TRPC6 was different. Inflammatory lesions, cellular infiltrates, hyaline membranes and fibrosis were labelled intensively as well as dilated capillaries. These observations are from four patients with COVID-19 pneumonia.The observations do not elucidate the molecular mechanisms but support the view that TRPC6 channels are involved in normal physiology of normal human lungs and in COVID-19 pneumonia. TRPC6 might aggravate SARS-2 induced inflammation and could be a target for inhibiting drugs.

Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so‐called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)‐adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET‐formation. Moreover, we characterized the mechanism leading to alum‐induced NET‐release in human neutrophils as rapid, NADPH oxidase‐independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+‐flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET‐release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum‐containing vaccines in humans and provides novel insights into bioenergetic requirements of NET‐formation.

This prospective multicenter study funded by the DEGUM assesses the diagnostic accuracy of standardized contrast-enhanced ultrasound (CEUS) for the noninvasive diagnosis of hepatocellular carcinoma (HCC) in high-risk patients. Patients at high risk for HCC with a histologically proven focal liver lesion on B-mode ultrasound were recruited prospectively in a multicenter approach. Clinical and imaging data were entered via online entry forms. The diagnostic accuracies for the noninvasive diagnosis of HCC were compared for the conventional interpretation of standardized CEUS at the time of the examination (= CEUS on-site) and the two CEUS algorithms ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) and CEUS LI-RADS (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System). 321 patients were recruited in 43 centers; 299 (93.1 %) had liver cirrhosis. The diagnosis according to histology was HCC in 256 cases, and intrahepatic cholangiocarcinoma (iCCA) in 23 cases. In the subgroup of cirrhotic patients (n = 299), the highest sensitivity for the diagnosis of HCC was achieved with the CEUS algorithm ESCULAP (94.2 %) and CEUS on-site (90.9 %). The lowest sensitivity was reached with the CEUS LI-RADS algorithm (64 %; p 0.001). However, the specificity of CEUS LI-RADS (78.9 %) was superior to that of ESCULAP (50.9 %) and CEUS on-site (64.9 %; p 0.001). At the same time, the negative predictive value (NPV) of CEUS LI-RADS was significantly inferior to that of ESCULAP (34.1 % vs. 67.4 %; p 0.001) and CEUS on-site (62.7 %; p 0.001). The positive predictive values of all modalities were high (around 90 %), with the best results seen for CEUS LI-RADS and CEUS on-site. This is the first multicenter, prospective comparison of standardized CEUS and the recently developed CEUS-based algorithms in histologically proven liver lesions in cirrhotic patients. Our results reaffirm the excellent diagnostic accuracy of CEUS for the noninvasive diagnosis of HCC in high-risk patients. However, on-site diagnosis by an experienced examiner achieves an almost equal diagnostic accuracy compared to CEUS-based diagnostic algorithms. Diese prospektive, multizentrische, DEGUM-geförderte Studie untersucht die diagnostische Genauigkeit standardisierter Algorithmen für die Kontrastmittelsonografie (CEUS-Algorithmen) in der nichtinvasiven Diagnostik des hepatozellulären Karzinoms (HCC) bei Hochrisikopatienten. HCC-Hochrisikopatienten mit histologisch gesicherter Leberläsion im B-Bild-Ultraschall wurden prospektiv multizentrisch eingeschlossen. Klinische Daten und Bildgebungsbefunde wurden über Online-Eingabemasken erfasst. Es erfolgte ein direkter Vergleich der diagnostischen Genauigkeiten für die konventionelle CEUS-Befundung zum Untersuchungszeitpunkt (CEUS-on-site) und die CEUS-Algorithmen ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) und CEUS LI-RADS (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System). 321 Patienten an 43 Zentren wurden eingeschlossen (93,1 % Leberzirrhose). Der histologische Befund ergab 256 HCCs und 23 intrahepatische cholangiozelluläre Karzinome (iCCA). Die höchste Sensitivität bei Zirrhose-Patienten (n = 299) erzielten der CEUS-Algorithmus ESCULAP (94,2 %) und CEUS-on-site (90,9 %), die geringste Sensitivität der CEUS LI-RADS-Algorithmus (64 %; p 0,001). Die Spezifität war höher für CEUS LI-RADS (78,9 %) versus ESCULAP (50,9 %) und CEUS on-site (64,9 %; p 0,001). Der negativ prädiktive Wert (NPW) war für CEUS LI-RADS niedriger als für ESCULAP (34,1 % vs. 67,4 %; p 0,001) und CEUS-on-site (62,7 %; p 0,001). Der positiv prädiktive Wert (PPW) war für alle Modalitäten hoch (rund 90 %). Dies ist die erste prospektive, multizentrische Studie zum Vergleich der standardisierten Kontrastmittelsonografie mit den kürzlich entwickelten CEUS-Algorithmen in histologisch gesicherten Leberläsionen bei Zirrhose-Patienten. Unsere Ergebnisse bestätigen die exzellente diagnostische Genauigkeit der Kontrastmittelsonografie in der nichtinvasiven HCC-Diagnostik bei Hochrisikopatienten. Die On-site-Diagnose eines erfahrenen Untersuchers erzielt dabei eine beinahe ebenso gute diagnostische Genauigkeit wie die CEUS-basierten Diagnosealgorithmen.

Background and Aims: Self-expandable metal stents are used for the treatment of anastomotic leaks after gastro- esophageal surgery. Predictors for treatment failure and complications are unknown. In this observational retrospective study, we summarize our experience with self-expandable metal stents for the treatment of anastomotic leaks, in order to determine the predictors of treatment failure. Methods: Between 2009 and 2015, 34 patients with anastomotic leak after curative resection of gastro- esophageal cancer were treated with self-expandable metal stents. Gender, histology, comorbidity, body mass index, neoadjuvant therapy, previous surgery, leak size, and stent diameter were analyzed for their predictive value according to treatment success and complication rate. Results: Leak closure rate was 76%. Risk factors for treatment failure were neoadjuvant chemo-radiotherapy, squamous cell histology, and esophageal tumor location. Gender, comorbidity, body mass index, neoadjuvant chemotherapy, and previous surgery were not correlated with outcome. Mortality rate was 20%, most often due to uncontrolled leak. Severe stent-related complications occurred in 15% of patients, most of them following insertion of a large-sized stent. Conclusion: Squamous cell histology, neoadjuvant chemo-radiotherapy, and esophageal tumor location are predictors for treatment failure. Severe stent-related complications seem to be preferentially associated with the use of large-sized stents.

Purpose: To analyse the changing trends in penetrating keratoplasty indications between January 2011 and December 2018, at the Department of Ophthalmology, Saarland University Medical Center, Hombu...

Virchows Archiv (2022). doi:10.1007/s00428-022-03319-2, Published by Springer, Berlin

Background Despite the large-scale rollout of malaria rapid diagnostic tests (RDTs) in Tanzania, many healthcare providers (HCPs) continue using blood film microscopy (BFM) and clinical examination to diagnose malaria, which can increase the risk of mal-diagnosis and over-prescribing of anti-malarials. Patients disregarding negative test results and self-treating exacerbate the problem. This study explored the knowledge, attitudes and practices of HCPs and healthcare-seekers regarding RDTs in comparison to BFM testing. Methods A situational analysis was, therefore, conducted in Kondoa District, Dodoma Region, Tanzania. A multi-methodological approach was adopted including (i) a health facility inventory and screening of logbooks from May 2013 to April 2014 with 77,126 patient entries from 33 health facilities; (ii) a survey of 40 HCPs offering malaria services; and iii) a survey of 309 randomly selected household members from the facilities’ catchment area. Surveys took place in April and May 2014. Results Health facility records revealed that out of 77,126 patient entries, 22% (n = 17,235) obtained a malaria diagnosis. Of those, 45% were made with BFM, 33% with RDT and 22% with clinical diagnosis. A higher rate of positive diagnoses was observed with BFM compared with RDT (71% vs 14%). In the HCP survey, 48% preferred using BFM for malaria testing, while 52% preferred RDT. Faced with a negative RDT result for a patient presenting with symptoms typical for malaria, 25% of HCPs stated they would confirm the result with a microscopy test, 70% would advise or perform a clinical diagnosis and 18% would prescribe anti-malarials. Interviews with household members revealed a preference for microscopy testing (58%) over RDT (23%), if presented with malaria symptoms. For participants familiar with both tests, a second opinion was desired in 45% after a negative microscopy result and in 90% after an RDT. Conclusions Non-adherence to negative diagnostics by HCPs and patients continues to be a concern. Frequent training and supportive supervision for HCPs diagnosing and treating malaria and non-malaria febrile illnesses is essential to offer quality services that can instil confidence in HCPs and patients alike. The introduction of new diagnostic devices should be paired with context-specific behaviour change interventions targeting healthcare-seekers and healthcare providers.

BACKGROUND Recent studies showed that a single injection of human monoclonal allergen-specific IgG antibodies significantly reduced allergic symptoms in birch pollen-allergic patients. Since the production of full monoclonal antibodies in sufficient amounts is laborious and expensive, we sought to investigate if smaller recombinant allergen-specific antibody fragments, i.e. nanobodies, have similar protective potential. For this purpose, nanobodies specific for Bet v 1, the major birch pollen allergen, were generated to evaluate their efficacy to inhibit IgE-mediated responses. METHODS A cDNA-VHH library was constructed from a camel immunized with Bet v 1 and screened for Bet v 1 binders encoding sequences by phage display. Selected nanobodies were expressed, purified and analyzed in regards of epitope-specificity and affinity to Bet v 1. Furthermore, cross-reactivity to Bet v 1-homologues from alder, hazel and apple, and their usefulness to inhibit IgE binding and allergen-induced basophil activation were investigated. RESULTS We isolated three nanobodies that recognize Bet v 1 with high affinity and cross-react with Aln g 1 (alder) and Cor a 1 (hazel). Their epitopes were mapped to the alpha-helix at the C-terminus of Bet v 1. All nanobodies inhibited allergic patients´ polyclonal IgE binding to Bet v 1, Aln g 1 and Cor a 1 and partially suppressed Bet v 1-induced basophil activation. CONCLUSION We identified high-affinity Bet v 1-specific nanobodies that recognize an important IgE-epitope and reduce allergen-induced basophil activation revealing the first proof that allergen-specific nanobodies are useful tools for future treatment of pollen allergy.

IgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far.Several experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively.Topical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition.Collectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy.Austrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).

We report a case in whom aortic wrapping had been performed for aortic dilatation. Ten years later, further progression of aortic root diameter was documented (root size of 66 mm). We performed complete replacement of the proximal aorta. Macroscopically and microscopically, there was extreme degeneration and thinning of the aorta under the graft.

To report a case of simultaneous bilateral ophthalmic artery occlusion in diagnosed giant cell arteritis (GCA).A 77-year-old male patient presented to the emergency department with simultaneous vision loss in both eyes for 3 hours. Headache at both temples and jaw claudication had been present for 3 weeks. Laboratory values demonstrated an initially increased C-reactive protein (CRP) of 202.0 mg/L and an erythrocyte sedimentation rate (ESR) of 100 mm within the first 20 minutes. Duplex sonography of the right and left temporal arteries revealed a "halo sign." A case of GCA was suspected, and intravenous high-dose methylprednisolone therapy was immediately administered. The clinical examination revealed a bilateral central retinal artery occlusion and fluorescein angiography showed a hot optic disc in the right eye and patchy choroidal hypoperfusion in both eyes. Biopsy of the left temporal artery was performed, which confirmed a florid temporal arteritis with complete thrombotic occlusion of the vascular lumen. Despite a good response to the administered therapy (CRP 17.0 mg/L 1 week after initiation), the visual prognosis was significantly limited through retinal and optic nerve involvement. By the follow-up examination 8 weeks later, the near visual acuity was 20/400 in the right and left eye at a distance of 16 inches.We hereby present a simultaneous bilateral ophthalmic artery occlusion as a rare complication of GCA. The combination of central retinal artery occlusion, arteritic anterior ischemic optic neuropathy, and choroidal hypoperfusion suggests an acute inflammatory involvement of the ophthalmic artery. In cases of the slightest suspicion of giant cell arteritis, an immediate high-dose steroid therapy initiation is of utmost importance.Wir berichten über einen Patienten mit gleichzeitig aufgetretenem Verschluss beider Aa. ophthalmicae bei diagnostizierter Riesenzellarteriitis.Ein 77-jähriger männlicher Patient stellte sich in der Notaufnahme mit gleichzeitigem Sehverlust auf beiden Augen vor, der seit 3 Stunden bestehe. Kopf- und Kauschmerzen waren seit 3 Wochen vorhanden. Die Laborwerte zeigten ein initial erhöhtes C-reaktives Protein (CRP) von 202,0 mg/l und eine Erythrozytensedimentationsrate (ESR) von 100 mm innerhalb der ersten 20 Minuten. Die Duplexsonografie der rechten und linken A. temporalis zeigte ein „Halo-Zeichen“ und bei Verdacht einer Riesenzellarteriitis wurde eine sofortige intravenöse hochdosierte Methylprednisolon-Therapie eingeleitet. Die klinische Untersuchung ergab einen beidseitigen Zentralarterienverschluss und die Fluoreszenzangiografie zeigte eine Leckage der Papille des rechten Auges sowie eine choroidale Hypoperfusion an beiden Augen. Es wurde eine Biopsie der linken Temporalarterie durchgeführt, die eine floride Arteriitis temporalis mit vollständigem thrombotischem Verschluss des Gefäßlumens bestätigte. Trotz eines guten Ansprechens auf die verabreichte Therapie (CRP 17,0 mg/l 1 Woche nach Beginn) war die Sehprognose durch Netzhaut- und Sehnervenbeteiligung deutlich eingeschränkt. Bei der Nachuntersuchung 8 Wochen später lag der Nahvisus bei 1/20 bei der Lesetafel am rechten und linken Auge.Wir stellen hier einen gleichzeitigen bilateralen Verschluss der A. ophthalmica als seltene Komplikation der Riesenzellarteriitis vor. Die Kombination aus Zentralarterienverschluss, arteriitischer anteriorer ischämischer Optikusneuropathie und choroidaler Hypoperfusion lässt auf eine akute entzündliche Beteiligung der A. ophthalmica schließen. Bei dem geringsten Verdacht auf eine Riesenzellarteriitis ist die sofortige Einleitung einer hochdosierten Steroidtherapie von größter Bedeutung.

IgE-mediated food allergies affect both children and adults and are associated with dramatic decreases in the quality of life. In the majority of cases, food allergens have to be avoided which may be difficult, particularly in patients who suffer from life-threatening symptoms following the ingestion of minimal doses of food allergens. Several novel therapeutic approaches have been studied during the recent past and are summarized in this review. Therapies with novel therapeutic monoclonal antibodies, innovative allergen-specific immunotherapies using subcutaneous, sublingual, or epicutaneous routes, and oral immunotherapies leading to increases of individual thresholds of tolerable foods upon their continuous ingestion showed promising results which may change future management strategies in moderate to severe food allergy.

The determination of anti-hepatitis E virus antibodies (anti-HEV) has a high variability depending on the assay used. In 2015, with a manual ELISA method, we reported anti-HEV IgG seroprevalence of 32.6% in patients under hepatitis study. There are few reports of anti-HEV IgM. Recently, it was developed the first automated method and in the present study, we report the experience using this new method. Between May 2018 and August 2020, the results of 272 patients with an anti-HEV IgG and/or IgM study were analyzed using the automated ELFA technique (VIDAS®). Seroprevalence was 25.8% (68/264) for anti-HEV IgG and 3.5% (9/259) for anti-HEV IgM. Four samples were positive for both antibodies. Anti-HEV IgG seropositivity increased with age. In conclusion, the seroprevalence of anti-HEV IgG obtained was similar to previously reported. Taking into account the advantages of these assays, anti-HEV IgM and IgG assays on VIDAS® system, seem to be valuable new tools in serological study of HEV.

Working time arrangements that require shift work or other non-standard working hours have significant potential to encroach on time that is highly valued for family, social and leisure activity. This can often result in workers experiencing poorer work-family or work-life balance. Based on an extensive literature search and expert knowledge, primary risk factors were identified including shift work; long, irregular and unpredictable working hours; and work on evenings and weekends (in combination and independent of shift work). On the other hand, flexibility, in the form of adequate worker control over work schedules, may be a protective factor. In addition, workers experiencing excessive work-life conflict are likely to reduce their working hours, reflecting a reciprocal relationship between working hours and work-life balance. Workers’ families are also affected by shift work and non-standard working hours. Parents’ shift work is associated with poorer emotional and developmental outcomes for their children, and to a greater likelihood of risky behavior in adolescence. Additionally, the risk of separation or divorce is increased, especially for parents working night shifts. Due to relationships such as those above, the consequences of shiftwork and non-standard working hours on family and social life are largely dependent on a complex interaction between specific work schedules, other aspects of work organization, and family and individual worker characteristics. This article provides an overview of current evidence regarding the relationships between working time arrangements and various social and family variables, and concludes with shift scheduling and intervention recommendations to improve work-life balance and social well-being.

This consensus report summarizes the negative impact of work-related psychosocial factors and job stressors on the health and wellbeing of shift workers. Psychosocial factors may (a) directly affect work schedules or (b) mediate or moderate relationships between work schedules, circadian factors, and health. In this paper, prominent psychosocial models (e.g. Job Strain and Effort-Reward Imbalance) are used to help assess detrimental effects, including pathophysiologic outcomes. Several studies indicate the psychosocial environment can be more problematic for shift workers compared to regular day workers. This is likely due to shift worker’s experiencing greater risks of low job control, high physical work demands, lower support from supervisors, and greater levels of over-commitment. Workplace violence is another frequently encountered psychosocial stressor for shift workers more likely to be in regular contact with the general public, such as police officers, security personnel, professional drivers, and other service employees being at elevated risk. A large body of literature confirms night and irregular shift schedules increase risk for injury. Non-diurnal schedules can trigger and worsen such incidents, especially under unsafe conditions. The problem of workplace violence for shift workers, in terms of severity and consequences, is probably underestimated, especially when present among other occupational stressors. Practical considerations and recommendations for action to mitigate the detrimental effects of psychosocial stressors on night and shift workers are presented.

Chronic obstructive lung disease (COPD) and lung cancer are both caused by smoking and often occur as comorbidity. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important canonic immunoregulatory pathway, and antibodies that specifically block PD-1 or PD-L1 have demonstrated efficacy as therapeutic agents for non-small cell lung cancer. The role of the PD-1/PD-L1 axis in the pathogenesis of COPD is unknown. Here, we analyzed the function of the PD-1/PD-L1 axis in preclinical COPD models and evaluated the concentrations of PD-1 and PD-L1 in human serum and bronchoalveolar lavage (BAL) fluids as biomarkers for COPD. Anti-PD-1 treatment decreased lung damage and neutrophilic inflammation in mice chronically exposed to cigarette smoke (CS) or nontypeable Haemophilus influenzae (NTHi). Ex vivo stimulated macrophages obtained from anti-PD-1-treated mice released reduced amounts of inflammatory cytokines. PD-L1 concentrations correlated positively with PD-1 concentrations in human serum and BAL fluids. Lung sections obtained from patients with COPD stained positive for PD-L1. Our data indicate that the PD-1/PD-L1 axis is involved in developing inflammation and tissue destruction in COPD. Inflammation-induced activation of the PD-1 pathway may contribute to disease progression.

BACKGROUND Seminomatous germ cell tumours (SGCT) are the most frequent malignancy in young men. Reliable prognostic biomarkers for the prediction of metastasis at diagnosis and the risk of relapse in clinical stage I (CSI) are lacking. Adjuvant therapies carry a risk of overtreatment, whereas salvage therapies have a risk of high toxicities. Thus, the identification of reliable prognostic biomarkers is highly desirable to identify patients who will benefit from early adjuvant treatment. MicroRNAs (miRNAs) regulate tumour development and progression, and their potential as biomarkers has already been proven in a variety of malignancies. OBJECTIVES The aim of our study was to define a specific miRNA expression pattern that discriminates metastatic from non-metastatic primary SGCT. MATERIALS AND METHODS Total RNA was isolated from 24 formalin-fixed paraffin-embedded (FFPE) primary SGCT tumours (10 non-metastatic, five metachronously and nine synchronously metastatic) and from 10 normal testicular tissue samples. Microarray analysis was performed for global miRNA expression profiling. The results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis was performed using SPSS. RESULTS Microarray analyses revealed a specific miRNA pattern that distinguishes metastatic from non-metastatic SGCT. Sixty-three miRNAs were differentially expressed in metastatic compared to non-metastatic tumours (P

BACKGROUND Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16 weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen. METHODS IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking. RESULTS Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance. CONCLUSIONS Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity.

Background Children are discussed as hidden SARS‐CoV‐2 virus reservoir because of predominantly mild or even asymptomatic course of disease. The objective of this cross‐sectional study in May‐July 2020 was to assess the prevalence of SARS‐CoV‐2 antibodies and virus RNA in schoolchildren, consistent with previous infection by contact tracing. Methods School authorities approached parents for voluntary participation. Interested families were contacted by the study team. A nasal and oropharyngeal swab, a blood sample, and a questionnaire were employed. Primary endpoint was the frequency of SARS‐CoV‐2 real‐time PCR (RT‐PCR) and antibody‐positive children. Antibody positivity was assessed by a highly sensitive first‐line ELISA, and a neutralization assay and two other immunoassays as confirmatory assays. Results Of 2069 children (median age 13 years, IQR 10‐15), 2 cases (0.1%) tested positive for SARS‐CoV‐2 RNA and 26 cases (1.3%) tested positive for specific antibodies. SARS‐CoV‐2‐specific antibodies exhibited detectable virus‐neutralizing activity in 92% (24 of 26 samples). Seropositivity was associated with a history of mild clinical symptoms in 14 children (53.8%), while 12 children (46.2%) remained asymptomatic. Among 13 seropositive children being tested concomitantly with their siblings, only one pair of siblings was seropositive. Contact tracing revealed adult family members and school teachers as potential index cases. Conclusion In schoolchildren, the infection rate with SARS‐CoV‐2 is low and associated with a mild or asymptomatic course of disease. Virus spreading seemed to occur more likely in intergenerational contacts than among siblings in the same household. The presence of neutralizing SARS‐CoV‐2 antibodies in children may reflect protective adaptive immunity.

Background A new two-year Post University Specialty Training (PUST) programme in family medicine was introduced to improve the quality of postgraduate speciality medical education in Tajikistan. Postgraduate education of family doctors (FDs) needs to be urgently scaled up, as 38% of FD positions in Tajikistan remained unfilled in 2018. Moreover, the international financial support for the PUST programme is ending. This investment case assesses the minimum funding needed for the continuation and scale-up of PUST and establishes the rationale for the investment in the light of a recent evaluation. Methods The costs of the programme were calculated for 2018 and a scale-up forecast made for the period 2019–2023. The impact of the scale-up on the shortage of FDs was assessed. An evaluation using a Multiple Choice Questionnaire and Objective Structured Clinical Examination (OSCE) assessed and compared theoretical knowledge, clinical skills and competencies of PUST trained and conventionally trained FDs. Results The annual costs of the programme were US$ 228,000 in 2018. The total investment needed for scaling up PUST from 31 new FDs in 2018 to 100 FD graduates each year by 2023 was US$ 802,000.However, when the retirement of FDs and population growth are considered, the scale-up will result only in maintaining the current level of FDs working and not solve the country’s FD shortage. The PUST FDs demonstrated significantly better clinical skills than the conventionally trained interns, scoring 60 and 45% of OSCE points, respectively. Theoretical knowledge showed a similar trend; PUST FDs answered 44% and interns 38% of the questions correctly. Conclusions The two-year PUST programme has clearly demonstrated it produces better skilled family doctors than the conventional one-year internship, albeit some enduring quality concerns do still prevail. The discontinuation of international support for PUST would be a major setback and risks potentially losing the benefits of the programme for family medicine and also other specialities. To guarantee the supply of adequately trained FDs and address the FD shortage, the PUST should be continued and scaled up. Therefore, it is essential that international support is extended and a gradual transition to sustainable national financing gets underway.

Background Birch pollen is an important elicitor of respiratory allergy. The major allergen, Bet v 1, binds IgE exclusively via conformational epitopes. Objective To identify Bet v 1-specific epitope repertoires of IgE and IgG from birch pollen-allergic and non-allergic subjects. Methods Chimeric proteins were created by grafting individual epitope-sized, contiguous surface patches of Bet v 1 onto a non-allergenic structural homologue and expressed in Escherichia coli. Binding of IgE, IgG1 and IgG4 from sera of 30 birch pollen-allergic and 11 non-allergic subjects to Bet v 1, 13 chimeric proteins and four bacterial Bet v 1 homologues were measured by ELISA. The proportion of epitope-specific in total Bet v 1-specific IgE and the cross-reactivity of Bet v 1-specific IgE with bacterial homologues were determined by competitive ELISA. Results Thirteen soluble, correctly folded chimeric proteins were produced. IgE from 27/30 birch pollen-allergic patients bound to 1-12 chimeric proteins (median 4.0) with patient-specific patterns. Three chimeras binding IgE from the majority of sera were identified, whose pgrafted patches overlapped with previously published epitopes. Patterns of IgG1 and IgG4 binding to the chimeric proteins did not correspond to the binding patterns of IgE. Sera of 19/30 birch pollen-allergic patients contained low amounts of IgE to bacterial homologues. Bacterial proteins were able to partially inhibit IgE binding to Bet v 1. Conclusion Epitopes recognized by Bet v 1-specific antibodies from birch pollen-allergic patients are specific to each patient and differ between IgE, IgG1 and IgG4.

Background The petrochemical industry is hazardous, in part because of the inherently dangerous nature of the work conducted, and incidents frequently result in significant financial and social losses. The most common immediate cause of incidents and injuries in this industry is unsafe worker behaviour. Identifying the factors encouraging unsafe work behaviours is the first step in taking action to discourage them. The aim of this study was to (a) explore workers’, supervisors’ and safety managers’ attitudes and perceptions of safety in a petrochemical company in Iran, and (b) identify the factors that discourage safe work behaviours. Methods A qualitative study was conducted by applying the steps described by Graneheim and Lundman (2004). Twenty participants were recruited from an Iranian petrochemical company using a multi-stage approach, with initial purposive sampling followed by snowball sampling to enhance recruitment. Individual face-to-face and semi-structured interviews were conducted to gain an in-depth understanding of factors acting as barriers to safe behaviour. The interviews were recorded and transcribed in Persian and then translated into English. Conventional content analysis was performed. Results The main themes emerging from the interviews were: (i) poor direct safety management and supervision; (ii) unsafe workplace conditions; (iii) workers’ perceptions, skills and training; and (iv) broader organisational factors. Conclusions The findings give insights into practical organisational measures that can be implemented by management to promote workers’ commitment to safety and engage in safe behaviours in their workplace. Trial registration Iranian Registry of Clinical Trials: IRCT20170515033981N2. Retrospectively registered 19 June 2018.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma of B-cell origin with frequent expression of functional B-cell receptors (BCRs). Here we report that expression cloning followed by antigen screening identifies DNA-directed RNA polymerase beta’ (RpoC) from Moraxella catarrhalis as frequent antigen of BCRs of IgD+ LP cells. Patients show predominance of HLA-DRB1*04/07 and the IgVH genes encode extraordinarily long CDR3s. High-titer, light-chain-restricted anti-RpoC IgG1/κ-type serum-antibodies are additionally found in these patients. RpoC and MID/hag, a superantigen co-expressed by Moraxella catarrhalis that is known to activate IgD+ B cells by binding to the Fc domain of IgD, have additive activation effects on the BCR, the NF-κB pathway and the proliferation of IgD+ DEV cells expressing RpoC-specific BCRs. This suggests an additive antigenic and superantigenic stimulation of B cells with RpoC-specific IgD+ BCRs under conditions of a permissive MHC-II haplotype as a model of NLPHL lymphomagenesis, implying future treatment strategies., Nodular lymphocyte-predominant Hodgkin lymphoma with IgD+ lymphocyte-predominant (LP) cells is a rare clinical distinct lymphoma subset of B-cell origin. Here the authors show that antigens expressed by Moraxella catarrhalis are recognized by B cell receptors of IgD+ LP cells, suggesting the contribution of chronic antigen stimulation to lymphomagenesis.

To evaluate the prognostic potential of vimentin, p53, EGFR, CK5/6, CK 14, and CK 17 in patients with triple-negative breast cancer (TNBC). Tumor specimens of 60 patients with histologically confirmed TNBC were retrospectively analyzed. Formalin-fixed paraffin-embedded blocks of the tumor tissue were used to prepare tissue microarrays (TMAs). After immune-histochemical staining, protein expression of vimentin, p53, EGFR, CK5/6, CK 14, and CK 17 was determined and the immunoreactive score (IRS) was calculated. The protein expression was correlated to overall (OS) and disease-free survival (DFS). Ninety percent of patients suffered from an invasive ductal carcinoma T1 or T2, 66.7% were N0, and 70% had a G3 tumor with Ki67 of > 14%. Vimentin expression was found in 28/60 patients (46.7%), p53 expression in 30/60 patients (50%), and EGFR expression in 3/60 patients (5%). CK5/6, CK14, and CK17 expression was found in 60.0%, 63.3%, and 66.7%, respectively. Vimentin expression vs no expression was associated with significantly higher mean Ki67 values (52.5% vs. 31.1%; p = 0.0013) and significantly higher p53 expression (67.9% vs. 34.4%; p = 0.0097). No significant association between vimentin expression and OS (p = 0.7710) or DFS (p = 0.5558) was found during a mean follow-up of 92 months. None of the six proteins proved to be suitable prognostic factors for OS and DSF in patients with TNBC.

Background: A proportion of patients allergic to birch pollen are also allergic to pit fruit. The objective of this study was to investigate the effect of immunotherapy with birch pollen on birch-pollen-related apple allergy. Method: Patients with birch pollen immunotherapy underwent a skin-prick test with birch pollen, apple and rMal d 1, global assessments and nasal challenges with birch pollen, open food challenge with apple and a double-blind, placebo-controlled test with rMal d 1 at the start of and during the immunotherapy. Measurements of specific IgE in response to Bet v 1 and rMal d 1 and IgG4 in response to Bet v 1 and rMal d 1 took place. Results: Six of eight patients demonstrated an improvement of nasal challenge test results and all patients improved on global assessment during the immunotherapy. The median oral dose of apple required to elicit a reaction increased but was not statistically significant. The patients showed a decrease in skin-prick test values in response to birch pollen (1.05 to 0.36), apple (0.78 to 0.25) and rMal d 1 (0.51 to 0.10) with p-values of 0.04, 0.03 and 0.06, respectively and a decrease of specific IgE in response to Bet v 1 (10.66 kU/L to 5.19 kU/L) and rMal d 1 (0.99 to 0.61 kU/L) with p-values of 0.01 and 0.05, respectively. Only the median specific IgG4 value to Bet v 1 increased from 0.05 to 1.85 mg/L (p-value of 0.02) and not to IgG4 rMal d 1 (0.07 to 0.08 kU/L). Conclusion: The beneficial effects of immunotherapy for birch pollen were accompanied by a limited effect on apple allergy.

Non-alcoholic steatohepatitis (NASH) represents a risk factor for the development of hepatocellular carcinoma (HCC) and is characterized by quantitative and qualitative changes in hepatic lipids. Since elongation of fatty acids from C16 to C18 has recently been reported to promote both hepatic lipid accumulation and inflammation we aimed to investigate whether a frequently used mouse NASH model reflects this clinically relevant feature and whether C16 to C18 elongation can be observed in HCC development. Feeding mice a methionine and choline deficient diet to model NASH not only increased total hepatic fatty acids and cholesterol, but also distinctly elevated the C18/C16 ratio, which was not changed in a model of simple steatosis (ob/ob mice). Depletion of Kupffer cells abrogated both quantitative and qualitative methionine-and-choline deficient (MCD)-induced alterations in hepatic lipids. Interestingly, mimicking inflammatory events in early hepatocarcinogenesis by diethylnitrosamine-induced carcinogenesis (48 h) increased hepatic lipids and the C18/C16 ratio. Analyses of human liver samples from patients with NASH or NASH-related HCC showed an elevated expression of the elongase ELOVL6, which is responsible for the elongation of C16 fatty acids. Taken together, our findings suggest a detrimental role of an altered fatty acid pattern in the progression of NASH-related liver disease.

Some effector CD4+ T cell subsets display cytotoxic activity, thus breaking the functional dichotomy of CD4+ helper and CD8+ cytotoxic T lymphocytes. However, molecular mechanisms regulating CD4+ cytotoxic T lymphocyte (CD4+ CTL) differentiation are poorly understood. Here we show that levels of histone deacetylases 1 and 2 (HDAC1-HDAC2) are key determinants of CD4+ CTL differentiation. Deletions of both Hdac1 and 1 Hdac2 alleles (HDAC1cKO-HDAC2HET) in CD4+ T cells induced a T helper cytotoxic program that was controlled by IFN-γ–JAK1/2–STAT1 signaling. In vitro, activated HDAC1cKO-HDAC2HET CD4+ T cells acquired cytolytic activity and displayed enrichment of gene signatures characteristic of effector CD8+ T cells and human CD4+ CTLs. In vivo, murine cytomegalovirus–infected HDAC1cKO-HDAC2HET mice displayed a stronger induction of CD4+ CTL features compared with infected WT mice. Finally, murine and human CD4+ T cells treated with short-chain fatty acids, which are commensal-produced metabolites acting as HDAC inhibitors, upregulated CTL genes. Our data demonstrate that HDAC1-HDAC2 restrain CD4+ CTL differentiation. Thus, HDAC1-HDAC2 might be targets for the therapeutic induction of CD4+ CTLs., Histone deacetylases 1 and 2 restrict naive CD4+ T cell differentiation into CD4+ cytotoxic T lymphocytes and might be targets for the therapeutic induction of CD4+ cytotoxic T lymphocytes.

In the previous studies, we demonstrated that Sec62 is essential for tumor cell migration, epithelial-to-mesenchymal transition, and intracellular stress tolerance. An increase in Sec62 expression correlated with an increase in cervical dysplasia severity in liquid-based cytology specimens. Ki67 is an established proliferation marker. Thus, in this study, we examined a method of Sec62/Ki67 dual staining for the detection of high-grade dysplasia and cancer in cervical liquid-based cytology specimens. Sec62/Ki67 dual staining was performed on 100 cervical liquid-based cytology specimens. The staining results were correlated with cytological, immunocytological (p16/Ki67), colposcopic, and histological findings. All 56 (n = 56, 100%) cases of cervical intraepithelial neoplasia grade 3 and cervical cancer (CIN3+ lesions) were positive for Sec62/Ki67 staining, while low-grade lesions and normal cells were negative. Sec62/Ki67 staining was highly sensitive and specific for the detection of CIN2+ and CIN3+ lesions (94.37%; 100% and 100%; 84.09%, respectively). Sec62/Ki67 dual-staining immunocytochemistry is a promising cytological tool for interpreting high-grade squamous lesions in cytological specimens and for assessing the risk of progression to cancer.

To generate and characterize 3D spheroid suspension cultures from radical prostatectomy (RP) specimens as a versatile model system for organ-confined prostate cancer (PCa). Cancerous tissue samples from RP specimens were excised by a uropathologist. Preparation of 3D spheroids was done by mechanical disintegration and limited enzymatic digestion followed by serial filtration through 100 μm- and 40 μm-cell strainers. Thereafter, spheroids were cultured in a modified stem cell medium and characterized by a live/dead assay, whole-spheroid immunohistochemistry (IHC; CK5, CK8, AMACR, PSA, Ki67, AR, αSMA, Vimentin, E-Cadherin) and PSA-measurements in culture medium. Furthermore, their response to pharmaceutical treatment with docetaxel, bicalutamide, enzalutamide and abiraterone was tested. 173 RP cases were included. The median preoperative PSA-level was 16.12 ng/ml [range 0.99;345], the median Gleason score was 7b [6;10]. 64 cases were excluded due to low tumor content in frozen sections (43) or to insufficient spheroid formation (21). In the remaining 109 cases, spheroids formed successfully and stayed viable for up to several months. IHC analysis revealed AR-, CK8-, and AMACR-positivity in nearly all cases, while CK5-positive cells were detectable only occasionally as were α-SMA and Vimentin. E-Cadherin was positive in most cases. Furthermore, spheroids proved to be amenable to cryopreservation. While abiraterone had no effect and docetaxel only a moderate effect, spheroid viability was markedly reduced upon bicalutamide and enzalutamide treatment. Multicellular 3D spheroids can be generated from patient-derived RP tissue samples and serve as an innovative in vitro model of organ-confined PCa.

Dysregulation of the Wnt inhibitor dickkopf-1 protein (Dkk1) has been reported in a variety of cancers. In addition, it has been linked to the progression of malignant bone disease by impairing osteoblast activity. This study investigated serum- and tissue levels of Dkk1 in breast cancer patients with- or without bone metastases. Serum Dkk1 levels were measured by ELISA in 89 breast cancer patients and 86 healthy women. Tissue levels of Dkk1 and β-catenin, a major downstream component of Wnt transduction pathway, were tested with immunohistochemical staining in 143 different tissues, including adjacent non-tumoral breast tissues, primary breast tumours, lymph nodes metastases, and bone metastases. Serum levels of Dkk1 were significantly increased in breast cancer patients without metastases compared with healthy controls and even more increased in patients with bone metastases. Tissue expression of Dkk1 was positive in 70% of tested primary breast cancer tissues and demonstrated significant correlation with histological type and PR status. Less frequent expression of Dkk1 was found in lymph nodes metastases and bone metastases compared with adjacent non-tumoral breast tissues and primary breast tumours. Tissue expression of β-catenin was positive in the vast majority of all tested tissue types indicating activated Wnt/β-catenin signalling. Our results suggested that Wnt/β-catenin signalling in breast tumours and their secondary lymph nodes- and bone metastases is dysregulated and this could be related to aberrant Dkk1 expression levels. Hence, Dkk1 protein might provide insights into the continued development of novel comprehensive and therapeutic strategies for breast cancer and its bone metastases.

To investigate the interaction between Wnt/β-catenin and estrogen signaling pathways in endometrial cancer (EC). 119 women were involved in this study, including 65 women with histologically confirmed EC and 54 healthy women as a control group. Serum protein levels of Dkk1 were measured using ELISA. Protein expression levels of Dkk1, β-catenin, ER-β isoforms (β1, β2, β5), and ER-α were tested in paraffin-embedded tissues using IHC. Gene expression levels of Dkk1, CTNNB, ESR1, and ESR2 were tested in fresh tumorous and normal endometrium tissues using RT-PCR. EC patients had significantly higher serum levels of Dkk1 protein compared with healthy women. Dkk1 and β-catenin showed different expression pattern in tumor cells compared to it in normal cells at the protein level but not at the gene level. Protein expression levels of ERβ2 and ERα were significantly lower in tumor cells compared with tumor-adjacent normal cells. Increased protein expression levels of ERα were associated with favorable clinicopathological features and better overall survival rate (OS). Protein expression levels of ERα were correlated with protein expression levels of Dkk1 and cytoplasmic β-catenin. The association between ERα expression levels and OS was no more significant when tested in regard to Dkk1- and cytoplasmic β-catenin expression levels. Our data demonstrated that Wnt/β-catenin and estrogen signaling systems are dysregulated in EC showing; for the first time, a potential crosstalk between certain components of these two pathways, which in turn has affected the specificity of these molecules in disease characteristics. Understanding the signaling networks in EC is crucial in designing clinical trials to evaluate the efficacy of molecular-targeted agents and providing more successful therapies in the future.

BACKGROUND Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross-reactive tropomyosins. However, the functional and clinical relevance of their cross-reactivity is still debated. OBJECTIVE To analyse the humoral and cellular cross-reactivity of recombinant Aed a 10.01, Aed a 10.02 and Der p 10. METHODS Sera from 15 Austrian house dust mite-allergic, Der p 10-sensitized individuals were tested for IgE reactivity to recombinant tropomyosins in ELISA, inhibition ELISA and basophil activation tests. BALB/c mice were immunized with Aed a 10.01 or Aed a 10.02, and their sera were assessed for reactivity to all tropomyosins. Splenocytes were stimulated with all tropomyosins and synthetic peptides representing the amino acid sequence of Aed a 10.01. RESULTS IgE antibodies of Der p 10-sensitized patients cross-reacted with both tropomyosins from A. aegypti. Aed a 10.01 was a more potent inhibitor of IgE binding to Der p 10 and a stronger activator of basophils sensitized with Der p 10-specific IgE than Aed a 10.02. Murine antibodies raised against Aed a 10.01 and Aed a 10.02 cross-reacted with Der p 10. Aed a 10.01-specific antibody showed stronger cross-reactivity with Der p 10 than Aed a 10.02-specific antibody. Splenocytes from both groups of mice proliferated similarly to all tropomyosins. Five cross-reactive T cell-activating regions were identified. CONCLUSION AND CLINICAL RELEVANCE Tropomyosins from D. pteronyssinus and A. aegypti show humoral and cellular cross-reactivity, involving 5 potential T cell-activating regions. The more pronounced cross-reactivity of Aed a 10.01 and Der p 10 matched the higher sequence similarity of both proteins.

Background Birch pollen–related apple allergy is among the most prevalent food allergies in adolescent/adult subjects and mainly results from sensitization to the major birch pollen allergen Bet v 1 and subsequent cross-reaction with the apple protein Mal d 1. However, specific immunotherapy with birch pollen has inconsistent effects on apple allergy. Objective We sought to compare the safety and efficacy of sublingual immunotherapy (SLIT) with 2 formulations containing either rMal d 1 or rBet v 1 on birch pollen–related apple allergy. Methods Sixty participants with birch pollen–related apple allergy were randomized to daily sublingual application of placebo (n = 20) or 25 μg of rMal d 1 (n = 20) or rBet v 1 (n = 20) for 16 weeks. Adverse events were regularly recorded. Sublingual challenges with standardized doses of rMal d 1, skin prick tests with recombinant allergens, and measurements of allergen-specific IgE and IgG 4 antibodies were performed before and after treatment. Results Both formulations caused comparable, mainly local adverse events. No systemic reactions occurred. Compared with the placebo and rBet v 1–treated groups, SLIT with rMal d 1 reduced rMal d 1–induced oral symptoms ( P = .001 and P = .038) accompanied by longitudinally reduced rMal d 1–specific cutaneous reactions ( P = .022) and enhanced IgG 4 /IgE ratios ( P = .012). SLIT with rBet v 1 neither improved the clinical reactivity to rMal d 1 nor enhanced rMal d 1–specific IgG 4 /IgE ratios. Participants receiving placebo showed no allergen-specific changes. Conclusion Sublingual treatment with a recombinant food allergen was safe and clinically effective, as determined by using standardized challenges. We present a promising approach for the effective treatment of birch pollen–related apple allergy.

Background: Enhancing the quality and yield of protein production in heterologous expression systems is an important issue for developing new biopharmaceuticals. It has been shown that the dynamics of protein folding is influenced by codon frequencies. As codon usage frequencies are species specific, this can affect heterologous protein expression. In this respect, “codon harmonization,” that is, the usage of synonymous codons with usage frequencies in the host resembling the usage frequencies in the native organism, is a promising strategy. As recombinant proteins are important tools in the area of allergy research, we investigated in this study the influence of codon harmonization on the production of the major birch pollen allergen Bet v 1.0101. Methods: To accomplish this task, parallel production of several batches of rBet v 1, BWT, together with a harmonized variant, BH, was applied. The expression yield of soluble and insoluble protein was assayed via densitometric analysis of ­SDS-PAGEs for every batch. The quality of purified proteins was assessed with a variety of physicochemical methods including mass spectrometry, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy, in vitro degradation, and 1-anilino-8-naphthalene sulfonate-binding assays. Patients’ IgE reactivity was tested in enzyme-linked immunosorbent assays and rat basophil mediator release experiments. Results: No significant differences in the ligand-binding capacity and secondary structure elements, as well as, in immunological assays could be found; however, the production yield was drastically increased for BH. Conclusion: We could show that codon harmonization is a powerful method to enhance protein yields in heterologous expression systems and should be considered especially for difficult-to-express proteins.

Activated allergen-specific Th2 and Th1 cells release cytokines that transform neutrophils into functional APCs characterized by the expression of HLA-DR and CD58 as well as enhanced survival and antigen uptake, irrespectively of the presence of IL-10, which reduces allergen uptake by neutrophils.

BACKGROUND Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.