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ZusammenfassungDie Standardmethode zur Messung der pulmonalen Hämodynamik ist der Rechtsherzkatheter (RHK) unter Ruhebedingungen, welcher international standardisiert ist. In den letzten Jahren wurden Bemühungen unternommen, auch die methodischen Aspekte des RHK unter Belastung zu vereinheitlichen und die somit gewonnenen Daten hinsichtlich ihrer prognostischen Aussagekraft zu definieren. Die Spiroergometrie als eine nichtinvasive Methode kann zusätzliche Aspekte der Pathophysiologie unter Belastung darstellen, und beide Methoden werden zunehmend kombiniert (als sog. invasive Spiroergometrie, iCPET) und ermöglichen eine sehr differenzierte Analyse der Belastungsreaktion.In dieser Übersicht werden die bisherigen Erfahrungen mit der invasiven Spiroergometrie dargestellt und insbesondere methodische Details erläutert.Die bisherigen Daten zur invasiven Spiroergometrie zeigen, dass diese komplexe Untersuchung besonders bei der Differenzierung der zugrundeliegenden Ursachen einer unklaren Dyspnoe hilfreich ist. Es zeichnet sich ab, dass über die Kombination aus hämodynamischen sowie ventilatorischen und gasanalytischen Daten schon frühe Formen einer kardialen bzw. pulmonalvaskulären Störung zu detektieren sind. Durch die zunehmende Validierung gewonnener Parameter aus der iCPET sind zukünftig Grenzwerte zur Unterscheidung normaler vs. pathologischer Belastungsreaktionen zu erwarten, die genauer als durch die alleinige Anwendung der jeweiligen Methoden möglich werden.

ZusammenfassungAktuell werden in deutschen Weaning-Zentren ungeachtet des enormen personellen und materiellen Ressourceneinsatzes etwa 20 % der Patienten nicht erfolgreich von der mechanischen Beatmung entwöhnt und in die außerklinische Beatmung überführt.Wir haben im Rahmen eines Pilotprojektes 33 Patienten (48 % weiblich; mittleres Alter 68 ± 11 Jahre bei Aufnahme) zwischen 03/2015 bis 11/2019 zur Re-Evaluation einer möglichen Entwöhnung von der invasiven Beatmung stationär aufgenommen. Zu diesem Zeitpunkt waren die Patienten seit Entlassung aus der Akutklinik bereits 475 ± 775 Tage invasiv beatmet. Die mittlere Liegedauer der Patienten auf der Weaning-Station betrug 26 ± 19 Tage, wobei wir 24/33 (73,53 %) erfolgreich entwöhnen konnten. Von diesen erfolgreich entwöhnten Patienten wurden 11/24 (44 %) mit einer nicht-invasiven Beatmung (NIV) entlassen. Die komplett entwöhnten Patienten zeigten ein 3- bzw. 12-Monats-Überleben von 92 % bzw. 77 %. Bei den Patienten mit einer NIV lagen die Daten bei 91 % bzw. 81 % und bei den invasiv beatmeten Patienten bei 86 % bzw. 71 %.Die vorgelegten Ergebnisse zeigen die Möglichkeit einer erfolgreichen Entwöhnung auch nach langer invasiver außerklinischer Beatmung. Somit erscheinen aus unserer Sicht regelhafte Re-Evaluationen zur Frage einer Entwöhnung der Patienten mit einer bestehenden Beatmungsnotwendigkeit im ambulanten Bereich sinnvoll.

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of Spike and lost neutralization efficacy against continuously emerging SARS-CoV-2 variants, which especially mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralizing antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both, NGS and epitope mapping of a monoclonal antibody with known binding site. The most prominent epitope captured represented parts of Spike´s fusion peptide (FP). It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell and the serine protease TMPRSS2 cleaves Spike within this sequence. Blocking of this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence were rather rare among SARS-CoV-2 variants so far, this may be an advantage in the fight against future virus variants.

Purpose Weaning from mechanical ventilation is a key component of intensive care treatment; however, this process may be prolonged as some patients require care at specialised centres. Current data indicate that weaning from invasive mechanical ventilation is successful in approximately 65% of patients; however, data on long-term survival after discharge from a weaning centre are limited. Materials and methods We analysed predictors of survival among 597 patients (392 men, mean age 68 ± 11) post-discharge from a specialised German weaning centre. Results Complete weaning from mechanical ventilation was achieved in 407 (57.8%) patients, and 106 patients (15.1%) were discharged with non-invasive ventilation; thus, prolonged weaning was successful in 72.9% of the patients. The one-year and five-year survival rates post-discharge were 66.5% and 37.1%, respectively. Age, duration of mechanical ventilation, certain clusters of comorbidities, and discharged with mechanical ventilation significantly influenced survival (p Conclusions The identified predictors of survival after prolonged weaning could support therapeutic strategies during patients' intensive care unit stay. Patients should be closely monitored after discharge from a weaning centre.

The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and classified as a variant of concern (VOC). Omicron shows substantially more mutations in the spike protein than any previous variant, mostly in the receptor binding domain (RBD). We analyzed the binding of the Omicron RBD to the human ACE2 receptor (hACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta or Delta RBDs variants.All RBDs were produced in insect cells. RBD binding to hACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 58 fully vaccinated individuals and 16 booster recipients were titrated by ELISA on the fixed RBDs from the original Wuhan strain, Beta, Delta and Omicron VOC.Surprisingly, the Omicron RBD showed a weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. However, a difference of 2.5 times was observed in RBD binding while in other studies the neutralization of Omicron SARS-CoV-2 was reduced by a magnitude of 10x and more. These results indicate an immune escape focused on neutralizing antibodies.The reduced binding of sera to Omicron RBD adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

Right heart catheterization (RHC) is the internationally standardized reference method for measuring pulmonary hemodynamics under resting conditions. In recent years, increasing efforts have been made to establish the reliable assessment of exercise hemodynamics as well, in order to obtain additional diagnostic and prognostic data. Furthermore, cardiopulmonary exercise testing (CPET), as the most comprehensive non-invasive exercise test, is increasingly performed in combination with RHC providing detailed pathophysiological insights into the exercise response, so-called invasive cardiopulmonary exercise testing (iCPET).In this review, the accumulated experience with iCPET is presented and methodological details are discussed. This complex examination is especially helpful in differentiating the underlying causes of unexplained dyspnea. In particular, early forms of cardiac or pulmonary vascular dysfunction can be detected by integrated analysis of hemodynamic as well as ventilatory and gas exchange data. It is expected that with increasing validation of iCPET parameters, a more reliable differentiation of normal from pathological stress reactions will be possible.Die Standardmethode zur Messung der pulmonalen Hämodynamik ist der Rechtsherzkatheter (RHK) unter Ruhebedingungen, welcher international standardisiert ist. In den letzten Jahren wurden Bemühungen unternommen, auch die methodischen Aspekte des RHK unter Belastung zu vereinheitlichen und die somit gewonnenen Daten hinsichtlich ihrer prognostischen Aussagekraft zu definieren. Die Spiroergometrie als eine nichtinvasive Methode kann zusätzliche Aspekte der Pathophysiologie unter Belastung darstellen, und beide Methoden werden zunehmend kombiniert (als sog. invasive Spiroergometrie, iCPET) und ermöglichen eine sehr differenzierte Analyse der Belastungsreaktion.In dieser Übersicht werden die bisherigen Erfahrungen mit der invasiven Spiroergometrie dargestellt und insbesondere methodische Details erläutert.Die bisherigen Daten zur invasiven Spiroergometrie zeigen, dass diese komplexe Untersuchung besonders bei der Differenzierung der zugrundeliegenden Ursachen einer unklaren Dyspnoe hilfreich ist. Es zeichnet sich ab, dass über die Kombination aus hämodynamischen sowie ventilatorischen und gasanalytischen Daten schon frühe Formen einer kardialen bzw. pulmonalvaskulären Störung zu detektieren sind. Durch die zunehmende Validierung gewonnener Parameter aus der iCPET sind zukünftig Grenzwerte zur Unterscheidung normaler vs. pathologischer Belastungsreaktionen zu erwarten, die genauer als durch die alleinige Anwendung der jeweiligen Methoden möglich werden.

Introduction: Chronic obstructive pulmonary disease (COPD) is one of the most common chronic diseases associated with high mortality. Previous studies suggested a prognostic role for peak oxygen uptake (VO2peak) assessed during cardiopulmonary exercise testing (CPET) in patients with COPD. However, most of these studies had small sample sizes or short follow-up periods, and despite their relevance, CPET parameters are not included in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) tool for assessment of severity. Objectives: We therefore aimed to assess the prognostic value of CPET parameters in a large cohort of outpatients with COPD. Methods: In this retrospective, multicentre cohort study, medical records of patients with COPD who underwent CPET during 2004–2017 were reviewed and demographics, smoking habits, GOLD grade and category, exacerbation frequency, dyspnoea score, lung function measurements, and CPET parameters were documented. Relationships with survival were evaluated using Kaplan-Meier analysis, Cox regression, and receiver operating characteristic (ROC) curves. Results: Of a total of 347 patients, 312 patients were included. Five-year and 10-year survival probability was 75% and 57%, respectively. VO2peak significantly predicted survival (hazard ratio: 0.886 [95% confidence interval: 0.830; 0.946]). The optimal VO2peak threshold for discrimination of 5-year survival was 14.6 mL/kg/min (area under ROC curve: 0.713). Five-year survival in patients with VO2peak Conclusions: We confirm that VO2peak is a highly significant predictor of survival in COPD patients and recommend the incorporation of VO2peak into the assessment of COPD severity.

Background Among the various factors identified as relevant for primary care career choice, financial considerations have been consistently shown to have an impact. In Germany, reliable and easily understandable information on physicians’ earning opportunities in self-employed settings is difficult to obtain for medical students, leading to substantial misperceptions that may negatively affect respective career considerations. This study investigated medical students’ evaluation of a 45-min evidence-based workshop on earning opportunities, workload and job satisfaction in different specialties and settings to examine its effect on the perceived attractiveness of working self-employed and working in general practice. Methods The workshop was implemented as part of a mandatory general practice clerkship in the fourth study year (of six). Post-hoc evaluations of all participants between October 2017 and September 2018 (one cohort) were analysed cross-sectionally including descriptive statistics, subgroup comparisons and qualitative analysis of free-text answers regarding students’ main insights. Results Response rate was 98.1% (307/313). Participants were on average 25.0 years old, and 68.3% were women. Based on a ten-point scale ranging from 1 = ’no influence’ to 10 = ’very big influence’, 91.9% confirmed at least some (> = 2) and 57.3% a rather high (> = 5) influence of earning expectations on their career choice process. Regarding the workshop, 86.1% were overall satisfied, and 89.5% indicated they had gained new insights, primarily regarding earning opportunities in different specialties and work settings, and frequently regarding job satisfaction, workload and the structure of revenues and expenditures in a doctor’s office (according to qualitative analysis). In the opinion of 89.8% of students, the provided learning content should be part of the undergraduate curriculum. More than half of participants reported an increase regarding the attractiveness of working self-employed and working as a general practitioner, most frequently regarding earning opportunities, but also in general and in respect to job satisfaction, cost–benefit ratio and workload. This increase was significantly higher among students favouring or at least considering a general practice career. Conclusions The workshop and its content were appreciated by the students and showed clear potential to usefully complement undergraduate curricula aiming at increasing or reinforcing students’ interest in working self-employed and working in general practice.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with FcγR silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

This prospective study compared exercise test and intravenous fluid challenge in a single right heart catheter procedure to detect latent diastolic heart failure in patients with echocardiographic heart failure with preserved ejection function. We included 49 patients (73% female) with heart failure with preserved ejection function and pulmonary artery wedge pressure ≤15 mmHg. A subgroup of 26 patients had precapillary pulmonary hypertension. Invasive haemodynamic and gas exchange parameters were measured at rest, 45° upright position, during exercise, after complete haemodynamic and respiratory recovery in lying position, and after rapid infusion of 500 mL isotonic solution. Most haemodynamic parameters increased at both exercise and intravenous fluid challenge, with the higher increase at exercise. Pulmonary vascular resistance decreased by –0.21 wood units at exercise and –0.56 wood units at intravenous fluid challenge ( p = 0.3); 20% (10 of 49) of patients had an increase in pulmonary artery wedge pressure above the upper limit of 20 mmHg at exercise, and 20% above the respective limit of 18 mmHg after intravenous fluid challenge. However, only three patients exceeded the upper limit of pulmonary artery wedge pressure in both tests, i.e. seven patients only at exercise and seven other patients only after intravenous fluid challenge. In the subgroup of pulmonary hypertension patients, only two patients exceeded pulmonary artery wedge pressure limits in both tests, further five patients at exercise and four patients after intravenous fluid challenge. A sequential protocol in the same patient showed a significantly higher increase in haemodynamic parameters at exercise compared to intravenous fluid challenge. Both methods can unmask diastolic dysfunction at right heart catheter procedure, but in different patient groups.

Implantable infusion pumps might improve the convenience and safety of intravenous treprostinil for pulmonary arterial hypertension. The LENUS Pro (R) pump (approved in Europe) has a fixed flow rate. Based on 126 pumps and 2853 refills, we retrospectively analyzed the actual flow rate from 09/2010 to 09/2018. A relevant flow rate variance is evident after three years; therefore, flow rate monitoring and dose adjustment are mandatory.

Background The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed. Results Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

BACKGROUND We examined safety and long-term outcomes of intravenous treprostinil administered via the implantable LENUS Pro pump in patients with severe pulmonary hypertension (PH). METHODS Patients with PH undergoing pump implantation between December 2009 and October 2016 in German referral centers were retrospectively analyzed (end of follow-up: May 2017). The primary objective was to determine long-term safety of the implantable pump. Secondary end points were 3-year survival and prognostic relevance of pre-implantation hemodynamics. RESULTS We monitored 129 patients (120 with pulmonary arterial hypertension, 1 with PH due to lung diseases, and 8 with inoperable chronic thromboembolic PH) for 260 patient-years (median follow-up, 19 months; interquartile range, 11–34 months). There were 82 complications/peri-procedural events in 60 patients; of these, 57 were serious adverse events (0.60 per 1,000 treatment-days), including 2 periprocedural deaths due to right heart failure. The incidence of complications related to the pump, catheter, infection, and pump pocket per 1,000 treatment-days was 0.074, 0.264, 0.032 (3 local infections; no bloodstream infections), and 0.380, respectively. Three-year overall and transplant-free survival were 66.5% and 55.7%, respectively (39 patients died; 16 underwent lung transplantation). Baseline cardiac index independently predicted transplant-free survival (multivariate hazard ratio, 1.90; 95% confidence interval, 1.11–3.28; p = 0.019; n = 95). CONCLUSIONS Our data suggest that intravenous treprostinil via the LENUS Pro pump in advanced PH is associated with a very low risk of bloodstream infections, but other serious adverse events may occur. Therefore, this therapy needs standardization and should be offered in specialized PH centers only. Further technical advances of the pump system and prospective studies are needed.

ZusammenfassungSeit der Etablierung der klinischen Beatmungsmedizin gewinnt die Entwöhnung langzeitbeatmeter Patienten vom Respirator (Weaning) zunehmend an Bedeutung. Vor diesem Hintergrund wurden ab den 1980iger Jahren Zentren geschaffen, die speziell auf das Weaning von Patienten von der maschinellen Beatmung ausgerichtet waren.In dem Artikel werden die strukturelle Entwicklung sowie die Ergebnisse aus den letzten zehn Jahren (2006 – 2015) des Weaningzentrums der Universitätsmedizin Greifswald dargestellt. Dazu wurden die Daten von 616 Patienten ausgewertet. Über die Jahre zeigt sich eine Verschiebung der zur Beatmung führenden Grunderkrankung, wobei der Anteil kardiochirurgischer Patienten rückläufig war und die Patienten mit Pneumonie/Sepsis zugenommen haben. Im Zeitverlauf haben die Tage mit Beatmung vor Aufnahme (p = 0,004) als auch die Beatmungstage im Weaningzentrum (p = 0,02) signifikant abgenommen. Ungeachtet des hohen APACHE-II-Scores bei stationärer Aufnahme konnte der Prozentsatz erfolgreich entwöhnter Patienten von 62,7 % (2006 – 10) auf 77,3 % (2011 – 2015) signifikant gesteigert werden (p Die Ergebnisse bei der Betreuung der Patienten im Weaningzentrum Greifswald bewegen sich auf vergleichbarem Niveau anderer deutscher Weaningzentren.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed an IC50 of 0.56 nM in a plaque-based live SARS-CoV-2 neutralization assay. The crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD was solved at 2.0 A resolution showing that the antibody binds at the same region as ACE2 to RBD. In contrast to other published anti-SARS-CoV-2 antibodies, the binding of STE90-C11 is not blocked by known RBD mutations, endowing our antibody with higher intrinsic resistance to those possible escape mutants. Funding: We kindly acknowledge the financial support of MWK Niedersachsen (14-76103-184CORONA-2/20) for antibody generation. We also acknowledge support of the European Union for the ATAC (“antibody therapy against corona”, Horizon 2020 number 101003650) consortium. Conflict of Interest: The authors declare a conflict of interest. F.B., D.M.,N.L., S.S., P.A.H., R.B., M.R., K.T.S.,K.D.R.R., S.Z.-E., M.B., V.F., S.T.,M.S. and M.H. are inventors on a patent application on blocking antibodies against SARS-CoV-2. A.H., A.F. and T.S. are officers of CORAT Therapeutics GmbH, a company founded by YUMAB GmbH for the clinical and regulatory development of STE90-C11 (COR-101). A.F., T.S., S.D. and M.H. are shareholders of YUMAB GmbH. Ethical Approval: Approval number, FV-2020-02. Approved by the TU Braunschweig Institute for Psychology.

While right heart catheterization (RHC) at rest is the gold standard to assess pulmonary hemodynamics in patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH), the invasive measurement of exercise hemodynamics is less well established in this group. Since exercise hemodynamics are increasingly recognized as important clinical information in patients with PH, our goal was to review the literature in this field to provide a basis for clinical use, further studies, and future recommendations. We identified 69 studies (published since 1968) reporting RHC data in 2819 patients with COPD, of whom 2561 underwent exercise testing. Few studies simultaneously measured gas exchange during exercise. Overall, these studies showed large variations in the patient populations and research questions studied and the methods and definitions employed. Despite these limitations, the data consistently demonstrated the presence of precapillary PH at rest in up to 38% of patients with COPD. With exercise, a relevant proportion of patients developed an abnormal hemodynamic response, depending on the definition used. Furthermore, some studies assessed right ventricular function during exercise and showed a blunted increase in right ventricular ejection fraction. Drug effects and the impact of interventional procedures were also studied. Again, due to large variations in the patients studied and the methods used, firm conclusions are difficult to derive. Despite the limitations of this dataset, several recommendations with respect to technical aspects (body position, exercise protocol, and data acquisition) can be inferred for this challenging patient population and may be helpful for further studies or recommendations.

BACKGROUND In patients with pulmonary arterial hypertension who have an insufficient response to oral or inhaled therapies, current guidelines recommend the use of parenteral prostacyclin analogues, although the efficacy of this approach is unknown. METHODS This retrospective multicenter study evaluated patients with pulmonary arterial hypertension who received intravenous treprostinil as an add-on therapy. The risk at baseline and follow-up (6–12 months after the initiation of treprostinil) was classified as low, intermediate, or high according to current recommendations. The outcome was measured as transplant-free survival after the initiation of treprostinil therapy. RESULTS A total of 126 patients were analyzed, almost all of them pre-treated with combinations of other pulmonary arterial hypertension medications. Before the initiation of intravenous treprostinil, 2 (2%) patients had a low-risk profile; 100 (79%), an intermediate-risk profile; and 24 (19%), a high-risk profile. At follow-up, 24 (19%) patients were classified as low-risk. These patients had a 5-year transplant-free survival rate >90%. In contrast, patients who remained at intermediate or high risk had transplant-free survival rates of 76%, 43%, and 28% at 1, 3, and 5 years, respectively. Failure to reach a low risk at follow-up was an independent predictor of transplant-free survival (hazard ratio, 9.25; 95% confidence interval, 1.20–71.60; p = 0.033 1). CONCLUSIONS Risk assessment at 6–12 months after the initiation of add-on intravenous treprostinil in patients with an insufficient response to nonparenteral treatments allows the prediction of transplant-free survival over the ensuing years. Achieving a low-risk profile is associated with excellent outcomes, whereas mortality is high in patients who remain at intermediate or high risk.

Antibodies are widely used in a large variety of research applications, for diagnostics and therapy of numerous diseases, primarily cancer and autoimmune diseases. Antibodies are binding specifically to target structures (antigens). The antigen-binding properties are not only dependent on the antibody sequence, but also on the discrete antigen region recognized by the antibody (epitope). Knowing the epitope is valuable information for the improvement of diagnostic assays or therapeutic antibodies, as well as to understand the immune response of a vaccine. While huge progress has been made in the pipelines for the generation and functional characterization of antibodies, the available technologies for epitope mapping are still lacking effectiveness in terms of time and effort. Also, no technique available offers the absolute guarantee of succeeding. Thus, research to develop and improve epitope mapping techniques is still an active field. Phage display from random peptide libraries or single-gene libraries are currently among the most exploited methods for epitope mapping. The first is based on the generation of mimotopes and it is fastened to the need of high-throughput sequencing and complex bioinformatic analysis. The second provides original epitope sequences without requiring complex analysis or expensive techniques, but depends on further investigation to define the functional amino acids within the epitope. In this book chapter, we describe how to perform epitope mapping by antigen fragment phage display from single-gene antigen libraries and how to construct these types of libraries. Thus, we also provide figures and analysis to demonstrate the actual potential of this technique and to prove the necessity of certain procedural steps.

Clostridioides difficile is the causative bacterium in 15–20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity.

The amount of processing elements in chip-multiprocessors is increasing continuous. Parallel programs use locks or mutexes to access shared resources. However, coarse-grained-locks are easy to write but limit the parallelization of the code, while fine-grained locks allow more parallel execution but often lead to deadlocks or other bugs, due to a more difficult program development. Hardware Transactional Memory allows an easement in parallel program development by providing a simplified concurrency management. The basic idea of transactional memory are atomic transactions, which offer a method for synchronization of processes without having the problems of synchronization methods like locks. In this paper a concept for adopting the TCC transactional memory model to Xilinx FPGAs using MicroBlaze soft-core processor is presented and first implementation details are given.

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical “Corona” aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

Cell reports 36(4), 109433 (2021). doi:10.1016/j.celrep.2021.109433, The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC$_{50}$ in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 �� resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with Fc��R-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19., Published by Elsevier, [New York, NY]