Targeted isolation of Methanobrevibacter strains from fecal samples expands the cultivated human archaeome.

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes' adaptability in critical microbiome contexts.

T Cell Phenotyping in Individuals Hospitalized with COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become pandemic. Cytokine release syndrome occurring in a minority of SARS-CoV-2 infections is associated with severe disease and high mortality. We profiled the composition, activation, and proliferation of T cells in 20 patients with severe or critical COVID-19 and 40 matched healthy controls by flow cytometry. Unsupervised hierarchical cluster analysis based on 18 T cell subsets resulted in separation of healthy controls and COVID-19 patients. Compared to healthy controls, patients suffering from severe and critical COVID-19 had increased frequencies of activated and proliferating CD38+Ki67+ CD4+ and CD8+ T cells, suggesting active antiviral T cell defense. Frequencies of CD38+Ki67+ Th1 and CD4+ cells correlated negatively with plasma IL-6. Thus, our data suggest that patients suffering from COVID-19 have a distinct T cell composition that is potentially modulated by IL-6.

Effects of an oral synbiotic on the gastrointestinal immune system and microbiota in patients with diarrhea-predominant irritable bowel syndrome.

PURPOSE: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common functional gastrointestinal disorder. Probiotics and synbiotics have been shown to improve symptoms of IBS, although mechanisms of action are currently not understood. METHODS: We investigated the effects of a 4-week oral synbiotic treatment (OMNi-BiOTiC® Stress Repair) in ten IBS-D patients on gastrointestinal mucosal and fecal microbiota, mucosa-associated immune cells, and fecal short-chain fatty acids. The upper and lower gastrointestinal tracts were compared before and after a 4-week synbiotic treatment using endoscopic evaluation to collect mucosal specimens for FACS analysis and mucosal 16S rRNA gene analysis. In stool samples, analysis for fecal SCFAs using GC-MS, fecal zonulin using ELISA, and fecal 16S rRNA gene analysis was performed. RESULTS: Synbiotics led to an increased microbial diversity in gastric (p = 0.008) and duodenal (p = 0.025) mucosal specimens. FACS analysis of mucosal immune cells showed a treatment-induced reduction of CD4+ T cells (60 vs. 55%, p = 0.042) in the ascending colon. Short-chain fatty acids (acetate 101 vs. 202 µmol/g; p = 0.007) and butyrate (27 vs. 40 µmol/g; p = 0.037) were elevated in fecal samples after treatment. Furthermore, treatment was accompanied by a reduction of fecal zonulin concentration (67 vs. 36 ng/ml; p = 0.035) and disease severity measured by IBS-SSS (237 vs. 54; p = 0.002). CONCLUSIONS: Our findings indicate that a short-course oral synbiotic trial may influence the human gastrointestinal tract in IBS-D patients on different levels which are region specific.

The effect of disease modifying therapies on CD62L expression in multiple sclerosis.

BACKGROUND: The increasing armamentarium of disease-modifying therapies in multiple sclerosis is accompanied by potentially severe adverse effects. The cell-adhesion molecule CD62L, which facilitates leukocyte extravasation, has been proposed as a predictive marker for treatment tolerability. However, pre-analytical procedures might impact test results, thereby limiting its clinical usability. Whether the immediate analysis of CD62L expression of peripheral blood mononuclear cells can aid treatment decision making is yet unclear. OBJECTIVE: To investigate the effect of various disease-modifying therapies in multiple sclerosis on CD62L expression of CD3+CD4+ peripheral blood mononuclear cells in freshly collected blood samples. METHODS: We collected peripheral blood samples from patients with clinically isolated syndrome and multiple sclerosis (baseline/follow up n = 234/n = 98) and healthy controls (n = 51). CD62L+CD3+CD4+ expression was analysed within 1 hour by fluorescence-activated cell sorting. RESULTS: CD62L+CD3+CD4+ expression was significantly decreased in patients treated with natalizumab (n = 26) and fingolimod (n = 20) and increased with dimethyl-fumarate (n = 15) compared to patients receiving interferon/glatiramer acetate (n = 90/30) or no disease-modifying therapies (n = 53) and controls (n = 51) (p<0.001). CD62L expression showed temporal stability during unchanged disease-modifying therapy usage, but increased after natalizumab withdrawal and decreased upon fingolimod introduction. CONCLUSION: CD62L+CD3+CD4+ expression is altered in patients treated with different disease-modifying therapies when measured in freshly collected samples. The clinical meaning of CD62L changes under disease-modifying therapies warrants further investigation.

Mucosal biopsy shows immunologic changes of the colon in patients with early MS.

OBJECTIVE: To investigate immune cells of the colonic mucosa and fecal short-chain fatty acids (SCFAs) in treatment-naive patients with a clinically isolated syndrome (CIS) or early relapsing MS. METHODS: In this cross-sectional proof-of-concept study, we obtained mucosal specimens during ileocolonoscopy from 15 untreated patients with CIS/MS and 10 controls. Mucosal immune cells were analyzed by FACS, and gas chromatography-mass spectrometry measurements of stool samples served to determine SCFA. RESULTS: The number of total dendritic cells (DCs), CD103+ tolerogenic DCs, and CD4+25+127-regulatory T cells (Tregs) was significantly reduced in the distal colon of patients with CIS/MS compared with controls, whereas we found no differences in the proximal colon. The patients' fecal samples also showed a substantially lower content of SCFA and especially lower levels of butyrate and acetate. CONCLUSIONS: Our findings indicate a disturbed homeostasis of colonic DCs and Tregs in patients with MS which could be associated with colonic SCFA depletion. Although not implying causality, these findings confirm parallel abnormalities of the gut in MS and warrant further research if modulation of the colonic SCFA profile or the colonic Treg pool can serve to modify the course of MS.

Quantifying low-value services by using routine data from Austrian primary care.

BACKGROUND: Open debates about the reduction of low-value services, unnecessary diagnostic tests and ineffective therapeutic procedures and initiatives like "Choosing Wisely "in the USA and Canada are still absent in Austria. The objectives of this study are: (i) to establish a list of ineffective or low-value services possibly provided in Austrian primary care, (ii) to explore how many of these services are quantifiable using routine data and (iii) to estimate the number of affected beneficiaries and avoidable costs arising from the provision of these services. METHODS: In May 2014, we identified low-value care services relevant for primary care in Austria. For our analysis we used routine data sets from the Austrian health insurance. All analysis refer to the insured population of the Lower Austrian Sickness Fund (n = 1 168 433) in the year 2013. RESULTS: (i) We found 453 low-value services possibly offered in Austrian primary care. (ii) Only 34 (7.5%) services were quantifiable using routine data. (iii) In the year 2013, these 34 services were provided to at least 246 131 beneficiaries and the estimated avoidable costs arising were at least 11.38 million Euros. This accounts for 1.2% of overall spending of the Lower Austrian Sickness Fund for drugs and services provided by primary care doctors in the year 2013. CONCLUSION: The absence of a homogeneous, transparent and accessible coding system for diagnosis in Austrian primary care restrained our assessment. However, our study findings illustrate the potential utility and limitations of using claims-based measures to identify low-value care.

Design of a wearable perturbator for human knee impedance estimation during gait.

Mechanical impedance modulation is the key to natural, stable and efficient human locomotion. An improved understanding of this mechanism is necessary for the development of the next generation of intelligent prosthetic and orthotic devices. This paper documents the design methodologies that were employed to realize a knee perturbator that can experimentally estimate human knee impedance during gait through the application of angular velocity perturbations. The proposed experiment requires a light, transparent, wearable, and remotely actuated device that closely follows the movement of the biological joint. A genetic algorithm was used to design a polycentric hinge whose instantaneous center of rotation is optimized to be kinematically compatible with the human knee. A wafer disc clutch was designed to switch between a high transparency passive mode and a high impedance actuated mode. A remote actuation and transmission scheme was designed to enable high power output perturbations while minimizing the device's mass. Position and torque sensors were designed for device control and to provide data for post-processing and joint impedance estimation. Pending the fabrication and mechanical testing of the device, we expect this knee perturbator to be a valuable tool for experimental investigation of locomotive joint impedance modulation.

Increasing arterial oxygen partial pressure during cardiopulmonary resuscitation is associated with improved rates of hospital admission.

AIM: As recent clinical data suggest a harmful effect of arterial hyperoxia on patients after resuscitation from cardiac arrest (CA), we aimed to investigate this association during cardiopulmonary resuscitation (CPR), the earliest and one of the most crucial phases of recirculation. METHODS: We analysed 1015 patients who from 2003 to 2010 underwent out-of-hospital CPR administered by emergency medical services serving 300,000 inhabitants. Inclusion criteria for further analysis were nontraumatic background of CA and patients >18 years of age. One hundred and forty-five arterial blood gas analyses including oxygen partial pressure (paO2) measurement were obtained during CPR. RESULTS: We observed a highly significant increase in hospital admission rates associated with increases in paO2 in steps of 100 mmHg (13.3 kPa). Subsequently, data were clustered according to previously described cutoffs (≤ 60 mmHg [8 kPa]], 61-300 mmHg [8.1-40 kPa], >300 mmHg [>40 kPa]). Baseline variables (age, sex, initial rhythm, rate of bystander CPR and collapse-to-CPR time) of the three compared groups did not differ significantly. Rates of hospital admission after CA were 18.8%, 50.6% and 83.3%, respectively. In a multivariate analysis, logistic regression revealed significant prognostic value for paO2 and the duration of CPR. CONCLUSION: This study presents novel human data on the arterial paO2 during CPR in conjunction with the rate of hospital admission. We describe a significantly increased rate of hospital admission associated with increasing paO2. We found that the previously described potentially harmful effects of hyperoxia after return of spontaneous circulation were not reproduced for paO2 measured during CPR. CLINICAL TRIAL REGISTRATION: n/a.

Spontaneous tension pneumothorax and CO2 narcosis in a near fatal episode of chronic obstructive pulmonary disease exacerbation.

Exacerbation of chronic obstructive pulmonary disease (COPD) is a disease pattern frequently seen in emergency medical services and intensive care units. Usually, exacerbations of COPD are of infectious origin, and an acute vital threat may take several days to develop. Tension pneumothorax in patients with COPD is a rare and often unexpected cause of acute vital threat. To the best of our knowledge, this is a unique case of CO2 narcosis after spontaneous tension pneumothorax in a patient with COPD. We describe the rapid development of respiratory insufficiency and near fatal pulmonary failure in a 65-year-old female patient with COPD due to spontaneous tension pneumothorax. The patient was in respiratory failure and comatose upon arrival of the emergency service. Before mechanical ventilation, coma could be confirmed to be due to CO2 narcosis caused by exorbitant arterial hypercapnia (PCO2, 193 mm Hg). Pneumothorax was diagnosed in the hospital by chest x-ray and resolved after pleural drainage. The patient could be extubated early and discharged without sequelae. In conclusion, we want to report the occurrence of a tension pneumothorax as an important and potentially overseen condition in patients with COPD with acute respiratory failure.

Immunoplasticity--triggers of regulatory function.

Regulatory T cells (Tregs) as key players of the immune system are exposed to numerous triggers including exogenous and endogenous factors. Autonomous nerve activity, melatonin, hormones such as vitamin D and glucocorticoids as well as the exposure to sunlight and microorganisms shape our immunological profile. The complexity of this system is highlighted by the power of each single trigger but more impressive by influencing each others function and potentials directly and indirectly. However, while monocausal correlations of single triggers on Tregs have been studied at length, there is much less known about the impact of numerous coexistent triggers on the dynamics of Treg activity. It can be hypothesized that the dynamics of Treg activity plays a crucial role for the control of our immune system. Therefore it is of the utmost importance that a new translational, multi- and interdisciplinary approach finds its way into future research efforts, which should lead to a more comprehensive and holistic view on the complex immunoregulatory mechanisms and to act in the sense of public health.

Students' and general practitioners' perceptions of a recently introduced clerkship in general practice. A survey from Austria.

BACKGROUND: In autumn 2007, the Medical University of Graz (MUG) upgraded the status of general practice in medical training by integrating a compulsory five-week clerkship in general practice surgeries in the sixth and last year of the curriculum. In cooperation with the Styrian Academy of General Practice (STAFAM), more than 200 general practitioners (GPs) had been accredited to introduce medical students to the specific tasks, problems and decision-making process in general practice. Between October 2007 and June 2009, more than 300 students completed the clerkship. OBJECTIVE: To explore the perceptions of students and GPs towards this new approach to undergraduate teaching in Austrian general practice. METHODS: Between March and June 2009, we conducted a cross-sectional survey by using a self-administered questionnaire with 14 items for students as well as GPs. To limit recall bias, we gave the questionnaire to all students (n = 146) and GPs (n = 146) immediately after the clerkship. The response rates were 146/146 (100%) and 114/146 (78%) for students and GPs, respectively. RESULTS: The study results show high satisfaction rates among students as well as GPs. Most of the students and GPs perceive the compulsory clerkship in general practice as an essential part of medical education. The organization of the clerkship had the least positive outcome. CONCLUSION: Our survey shows that the clerkship is well accepted among Austrian medical students and GPs.