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The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

Die Inzidenz von Morbus Crohn, einer chronisch-entzündlichen Darmerkrankung, steigt an. Obwohl die Therapiemöglichkeiten verbessert wurden, entwickelt ein Großteil der PatientInnen Komplikationen, die eine chirurgische Intervention benötigen. So können neben Stenosen auch Perforationen oder Fisteln entstehen, die eine dringliche chirurgische Darmresektion notwendig machen. Aufgrund des hohen Lebenszeitrisikos für die Notwendigkeit einer chirurgischen Therapie von Komplikationen, bedarf es einer optimalen Koordination von konservativer und operativer Therapie. Dies inkludiert sowohl Indikation zur Art der Chirurgie, als auch die postoperative Rezidivprophylaxe, sodass die Behandlung wegen der komplexen Interaktion in einem spezialisierten Zentrum erfolgen sollte, um den besten Operationszeitpunkt zu wählen. Um nach einer Darmresektion die Rekonvaleszenz zu beschleunigen und das Komplikationsrisiko zu minimieren, sind in den letzten Jahren einerseits die Laparoskopie als Standardzugang, andererseits spezielle chirurgische Anastomosen- und Resektionstechniken propagiert worden. Zusätzlich sollte in der Nachsorge die Einbindung der PatientInnen in ein gastroenterologisches Nachsorgeprogramm mit regelmäßigen, endoskopischen Untersuchungen erfolgen, um Rezidive zu erkennen und zu behandeln. Risikofaktoren für ein Frührezidiv nach Operationen beinhalten Rauchen, vorherige Operationen, das Fehlen von prophylaktischer medikamentöser Therapie, penetrierende/fistulierende Komplikationen bei Erstoperation, perianale Komplikationen, Granulome und myenterische Plexitis. In diesem Artikel fassen wir die gängigen Richtlinien sowie aktuelle Studien, die möglicherweise Einzug in die gängige Praxis finden, zusammen.

Guidelines for interviewers frequently include instructions to read question texts exactly as they are worded. Deviations from these guidelines on standardized interviewing might affect the comparability of survey answers and impair the quality of data. This paper contributes to the literature on interviewer behavior by analyzing how interviewers change their reading behavior during fieldwork and whether this behavioral change influences the response behavior of survey respondents. We use item-level paradata from the Survey of Health, Ageing and Retirement in Europe (SHARE) to measure interviewers’ reading times and focus our analyses on introductory questions that do not require an immediate response by the respondent. In contrast to prior research, this focus enables us to disentangle the reading times of interviewers from the response times of respondents. Based on fixed effects regression, our results show systematic changes in interviewers’ reading times of introductory items: First, reading times significantly decrease over the survey’s field period, even after controlling for relevant respondent characteristics and specific aspects of the interview situation. Second, a cross-national comparison, including fourteen European countries plus Israel, reveals that the decrease is uniform in almost all countries, suggesting its generalizability across different cultural contexts. Third, the decrease in reading times influences response behavior to varying degrees. Response behavior is affected if introductions contain relevant information for understanding or fulfilling the required task and especially if the response refers to within-survey requests. On the basis of these findings, we discuss the possible consequences for questionnaire design, interviewer training, and fieldwork monitoring.

In this first paper of a three-part series, we present the extension and validation of the high-order discontinuous Galerkin scheme in DLR’s CFD-solver trace for scale-resolving simulations of unsteady row interactions. The translational movement of rows in linear cascade experiments is represented in the numerical model by solving the equations in the relative frame of reference. To couple rows in different frames of reference, a sliding interface approach based on the mortar technique for non-conforming meshes has been developed. The verification of the approach is exemplified by three canonical test cases. First, the experimental order of convergence is verified for the isentropic vortex convection. Subsequently, the suitability of the sliding interface approach for scale-resolving simulations is tested on the Taylor–Green vortex flow and a turbulent cylinder flow. Finally, the LES solver is applied to the T106D cascade with upstream moving bars at an exit Reynolds number of 200,000 and exit Mach number of 0.4. The flow physics with and without bars is discussed in terms of the instantaneous flow field, and time- and phase-averaged quantities. The comparison with experimental data shows overall a good agreement, especially for the total pressure losses in the wake, but also reveals uncertainties related to the reproduction of an experiment in the numerical model.

In the second paper of this three-part series, we focus on the simulation of transonic test cases for turbomachinery applications using a high-order discontinuous Galerkin spectral element method (DGSEM). High-fidelity simulations of transonic compressors and turbines are particularly challenging, as they typically occur at high Reynolds numbers and require additional treatment to reliably capture the shock waves characterizing such flows. A recently developed finite-volume subcell shock capturing scheme tailored for the DGSEM is applied and evaluated with regard to the shock sensor. To this end, we conduct implicit large eddy simulations of a high-pressure turbine cascade from the public literature and a transonic compressor cascade measured at the German Aerospace Center, both at a high Reynolds number above 106. Based on the results, we examine modal-energy and flow-feature based shock indicator functions, compare the simulation data to experimental and numerical studies, and present an analysis of the unsteady features of the flows.

In this final paper of a three-part series, we apply the numerical test rig based on a high-order discontinuous Galerkin scheme to the MTU T161 low-pressure turbine with diverging end walls at off-design Reynolds number of 90,000, Mach number of 0.6, and inflow angle of 41 deg. The inflow end wall boundary layers are prescribed in accordance with the experiment. Validation of the setup is shown against recent numerical references and the corresponding experimental data. Additionally, we propose and conduct a purely numerical experiment with upstream bar wake generators at a Strouhal number of 1.25, which is well above what was possible in the experiment. We discuss the flow physics at midspan and in the end wall region and highlight the influence of the wakes from the upstream row on the complex secondary flow system using instantaneous flow visualization, phase averages, and modal decomposition techniques.

We investigate the unsteady behavior of an in-line jet impingement array of nine jets at a Reynolds number of 10,000 in a narrow channel subjected to a developing cross flow of up to 25% of the bulk jet velocity. To this end, we present an improved version of a previously published large eddy simulation (LES) now with resolved turbulence at the inflow boundaries. After a careful analysis of the transient behavior and statistical convergence of the LES, we discuss the time-averaged heat transfer characteristics of the configuration compared to numerical references of similar configurations. We then show how the large-scale unsteadiness increases from jet to jet. Both space-only and spectral proper orthogonal decompositions (POD and SPOD) are used to discuss the large-scale organization of single jets and multiple jets in combination. The latter shows a qualitative change in the unsteady behavior of the temperature footprint on the impingement wall with increasing cross flow.

Vladimer: Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Jaeger:Karyopharm: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; True North: Honoraria, Research Funding; Miltenyi: Consultancy, Honoraria; CDR Life AG: Consultancy, Research Funding; F. Hoffmann-La Roche: Honoraria, Research Funding; Infinity: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Krall:Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Valent:Allcyte GmbH: Research Funding; Cellgene: Honoraria, Research Funding; Pfizer: Honoraria. Wolf:Celgene: Honoraria, Research Funding. Zielinski:MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Imugene: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Merrimack: Consultancy, Honoraria, Speakers Bureau; Merck KGaA: Consultancy, Honoraria, Speakers Bureau; Fibrogen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Tesaro: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Eli Lilly: Consultancy, Honoraria, Speakers Bureau; Athenex: Consultancy, Honoraria, Speakers Bureau. Superti-Furga:Allcyte GmbH: Current equity holder in private company, Other: Founder. Snijder:Allcyte GmbH: Current equity holder in private company, Other: Founder. Staber:Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria; msd: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Background:

Für die chronisch entzündlichen Darmerkrankungen Morbus Crohn (MC) und Colitis ulcerosa (CU) stellt neben der Endoskopie die Radiologie einen wesentlichen Eckpfeiler der Diagnostik dar. Dies gilt sowohl für die Erstdiagnose als auch im Verlauf der Erkrankung. Der Radiologie kommt speziell bei der Diagnose von Komplikationen, wie Fisteln, Abszessen, Darmwandstenosen, aber auch Perforationen eine Schlüsselrolle zu, die das Management des Patienten entscheidend beeinflussen kann. Dies gilt sowohl für die internistische als auch für die chirurgische Therapie.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/zvalues have the same spatial distribution as CD3+T lymphocytes in the lymphoid follicular structures or as CD206+macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M1and anti-inflammatory M2phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/zvalues found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

In participants with Crohn disease, gallium 68 fibroblast activation protein inhibitor PET/MR enterography activity was associated with histologically assessed fibrosis in the bowel wall.

The linear ubiquitin chain assembly complex (LUBAC), consisting of SHANK-associated RH-domain–interacting protein (SHARPIN), heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), and HOIL-1–interacting protein (HOIP), is a critical regulator of inflammation and immunity. This is highlighted by the fact that patients with perturbed linear ubiquitination caused by mutations in the Hoip or Hoil-1 genes, resulting in knockouts of these proteins, may simultaneously suffer from immunodeficiency and autoinflammation. TLR3 plays a crucial, albeit controversial, role in viral infection and tissue damage. We identify a pivotal role of LUBAC in TLR3 signaling and discover a functional interaction between LUBAC components and TLR3 as crucial for immunity to influenza A virus infection. On the biochemical level, we identify LUBAC components as interacting with the TLR3-signaling complex (SC), thereby enabling TLR3-mediated gene activation. Absence of LUBAC components increases formation of a previously unrecognized TLR3-induced death-inducing SC, leading to enhanced cell death. Intriguingly, excessive TLR3-mediated cell death, induced by double-stranded RNA present in the skin of SHARPIN-deficient chronic proliferative dermatitis mice (cpdm), is a major contributor to their autoinflammatory skin phenotype, as genetic coablation of Tlr3 substantially ameliorated cpdm dermatitis. Thus, LUBAC components control TLR3-mediated innate immunity, thereby preventing development of immunodeficiency and autoinflammation.

Scale-resolving simulations, such as large eddy simulations, have become affordable tools to investigate the flow in turbomachinery components. The resulting time-resolved flow field is typically analyzed using first- and second-order statistical moments. However, two sources of uncertainty are present when statistical moments from scale-resolving simulations are computed: the influence of initial transients and statistical errors due to the finite number of samples. In this paper, both are systematically analyzed for several quantities of engineering interest using time series from a long-time large eddy simulation of the low-pressure turbine cascade T106C. A set of statistical tools to either remove or quantify these sources of uncertainty is assessed. First, the Marginal Standard Error Rule is used to detect the end of the initial transient. The method is validated for integral and local quantities and guidelines on how to handle spatially varying initial transients are formulated. With the initial transient reliably removed, the statistical error is estimated based on standard error relations considering correlations in the time series. The resulting confidence intervals are carefully verified for quantities of engineering interest utilizing cumulative and simple moving averages. Furthermore, the influence of periodic content from large scale vortex shedding on the error estimation is studied. Based on the confidence intervals, the required averaging interval to reduce the statistical uncertainty to a specific level is indicated for each considered quantity.

Previously we have developed a prototype for conditionally replicating oncolytic influenza A virus which is based on deletions in the non-structural (NS1) protein. Multi-cycle replication of influenza A virus in malignant tissue is critically dependent on a protease which cleaves the viral entry protein. Here we demonstrate that the malignant colon cancer cell lines Caco-2, HT-29 and SW-620 can endogenously provide a virus-activating protease, which allows lytic multi-cycle replication of NS1 deletion viruses in those cancer cells in vitro. The oncolytic potency of an influenza NS1 deletion virus (NS1-80) was further tested in SCID mice bearing HT-29 derived tumors. The intra-tumoral injection of live, but not of heat inactivated NS1-80 virus significantly inhibited progression of established tumors. We conclude that a selected set of human cancer expressing virus activating- proteases will be a preferred target for oncolytic tumor therapy using influenza A virus mutants.

Prolonged or repeated stimulation of Toll-like receptor (TLR) 4 leads to hyporesponsiveness of monocyte-derived macrophages, which seems to be a hallmark of immunosuppression related to sepsis and cancer. Two negative regulators of TLR-4 signaling are IL-1 receptor-associated kinase M and B-cell leukemia 3. Here, we demonstrate that the expression of both proteins is inhibited when the TLR-7/TLR-8 agonist CL097 is added to monocyte cultures despite costimulation with the TLR-4 agonist LPS or hyaluronic acid. Reduction of IL-1 receptor-associated kinase M and B-cell leukemia 3 was paralleled by a significant increased cytokine induction of TNF-, IL-10, and IL-12 observed after intracellular and extracellular TLR stimulation. In ex vivostimulated whole blood of patients who have prolonged sepsis or metastatic cancer, TLR-7/TLR-8 agonists retained their ability of increased stimulation of TNF-. These data might add to the understanding of sepsis and cancer-associated immune suppression in men.

ABSTRACTBILR 355 is a second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) under clinical development for the treatment of human immunodeficiency virus infection, particularly in those who harbor virus resistant to the currently available NNRTIs. Two single-center, double-blinded, placebo-controlled, parallel dose-escalation studies were conducted to evaluate the pharmacokinetics and safety of oral BILR 355 administration alone and after coadministration with ritonavir (RTV) at 100 mg. Following a single dose of BILR 355 in oral solution, the mean half life (t1/2) was 2 to 4 h, with peak concentrations occurring at 0.5 to 1 h postadministration. The mean apparent clearance (CL/F) ranged from 79.2 to 246 liters/h for administered doses of 12.5 mg to 100 mg. This observed nonlinearity in CL/Fresulted from the increased bioavailability attributed to a saturated absorption and/or elimination process at higher doses. In contrast, after the coadministration of single doses of 5 mg to 87.5 mg of BILR 355 with RTV, the mean CL/Franged from 5.88 to 8.47 liters/h. Over the dose range (5 to 87.5 mg) studied, systemic BILR 355 exposures were approximately proportional to the doses administered when they were coadministered with RTV. With RTV coadministration, the mean t1/2increased to 10 to 16 h, and the mean time of the maximum concentration in plasma lengthened to 1.5 to 5 h. Compared to the values for BILR 355 given alone, the mean area under the concentration-time curve from time zero to infinity, the maximum concentration in plasma, and the t1/2of BILR 355 achieved after coadministration with RTV increased 15- to 30-fold, 2- to 5-fold, and 3- to 5-fold, respectively. In both studies, BILR 355 appeared to be safe and well tolerated in healthy volunteers when the outcomes in the treated volunteers were compared with those in the placebo group.

AbstractThe use of sorbents has been proposed to remove volatile organic compounds (VOCs) present in ambient air at concentrations in the parts-per-billion (ppb) range, which is typical of indoor air quality applications. Sorbent materials, such as granular activated carbon and molecular sieves, are used to remove VOCs from gas streams in industrial applications, where VOC concentrations are typically in the parts-per-million range. A method for evaluating the VOC removal performance of sorbent materials using toluene concentrations in the ppb range is described. Breakthrough times for toluene at concentrations from 2 to 7500 ppb are presented for a hydrophobic molecular sieve at 25% relative humidity. By increasing the ratio of challenge gas flow rate to the mass of the sorbent bed and decreasing both the mass of sorbent in the bed and the sorbent particle size, this method reduces the required experimental times by a factor of up to several hundred compared with the proposed American Society of Heating, Refrigerating, and Air-Conditioning Engineers method, ASHRAE 145P, making sorbent performance evaluation for ppb-range VOC removal more convenient. The method can be applied to screen sorbent materials for application in the removal of VOCs from indoor air.

Catecholamines play a central role in the treatment of sepsis-associated hypotension. However, these hormones have also been shown to modulate the lipopolysaccharide (LPS)-induced induction of cytokines such as tumor necrosis factor alpha, interleukin (IL)-10, and IL-6 in vitroand in human endotoxemia. We hypothesized that catecholamines applied therapeutically in septic shock also influence cytokine patterns. We studied the cytokine response in tissues of the splanchnic compartment in a porcine endotoxin shock model up to 4 h. Shock was induced by a short infusion of LPS, and animals were treated either with fluid resuscitation alone or in combination with continuous epinephrine or norepinephrine. Animals, receiving epinephrine therapy, showed a significantly prolonged upregulation of IL-6 mRNA expression at 4 h after LPS application in liver (P0.0014), spleen (P< 0.0001), and mesenteric lymph nodes (P0.0078) as compared with animals treated with norepinephrine or fluid resuscitation. Serum IL-6 increased over time in all groups. The total concentration of the cytokine (area under the curve) was significantly higher in the epinephrine group as compared with the norepinephrine and fluid resuscitation groups (P0.017). The peak of serum tumor necrosis factor alpha at 1 h after LPS application was already significantly reduced by epinephrine, which was only administered at a mean of less than 0.05 g/kg/min at this time point (P< 0.01). None of the catecholamines had a significant effect on IL-10 serum levels when compared with animals receiving fluid resuscitation alone. Our data suggest that the therapeutic application of epinephrine but not of norepinephrine is associated with a profound effect on the IL-6 response of splanchnic reticuloendothelial tissues.

ABSTRACTThe availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

Abstract: Development of donor-specific proliferative hyporeactivity has been evaluated in many studies for its usefulness in identifying transplant recipients at low risk of immunological complications. These studies often result in controversial conclusions, however. The authors claim that the discrepancy in the predictive value of mixed lymphocyte culture- (MLC) reactivity might partly be due to differences in presentation and interpretation of results. The purpose of this study is to investigate the usefulness of a normalized evaluation of antigen-specific donor-reactivity in a small number of kidney transplant recipients. This could then serve as a basis for an extended clinical study. Ten cadaveric kidney recipients were tested for proliferative reactivity to donor- and third-party antigens up to 20 months posttransplantation. Expressing donor-specific reactivity as a relation between the percentage of pretransplant responses towards donor splenocytes and the percentage of pretransplant responses towards third-party donor cells should minimize influences of e. g. uremia, current immunosuppression or infections on the evaluation of specific reactivity and thus should allow an evaluation of the donor-specificity of T-cell alloresponses independently of fluctuations in global responsiveness. Four of ten recipients acquired a state of donor-specific hyporeactivity ( < 75 % relative specific reactivity) at 20 months posttransplantation (61 � 12 %, mean � SD). Six patients were classified non-hyporeactive (98 � 10 % mean relative specific reactivity). Relative specific reactivity did not correlate with the levels of general reactivity. Three of the four hyporeactive and four of the six non-hyporeactive patients developed acute rejection. Stable graft function at 20 months posttransplantation (serum creatinine ≤ 2 mg/dl) was not closely related to the reactivity status, as five of eight patients with well-functioning grafts did not develop relative specific hyporeactivity. One recipient with chronic rejection was classified hyporeactive. One non-hyporeactive patient lost his graft due to non-immunological causes. Our data suggest that posttransplant relative specific reactivity does not predict acute rejection. Downregulation of donor-specific reactivity might not be a prerequisite for stable graft function but could help identifying recipients who require less immunosuppression. This, however, remains to be established in a prospective immunosuppression-weaning study.

AbstractDevelopment of donorspecific proliferative hyporeactivity has been evaluated in many studies for its usefulness in identifying transplant recipients at low risk of immunological complications. These studies often result in controversial conclusions, however. The authors claim that the discrepancy in the predictive value of mixed lymphocyte culture‐ (MLC) reactivity might partly be due to differences in presentation and interpretation of results. The purpose of this study is to investigate the usefulness of a normalized evaluation of antigenspecific donor‐reactivity in a small number of kidney transplant recipients. This could then serve as a basis for an extended clinical study. Ten cadaveric kidney recipients were tested for proliferative reactivity to donor‐ and third‐party antigens up to 20 months posttransplantation. Expressing donor‐specific reactivity as a relation between the percentage of pretransplant responses towards donor splenocytes and the percentage of pretransplant responses towards third‐party donor cells should minimize influences of e. g. uremia, current immunosuppression or infections on the evaluation of specific reactivity and thus should allow an evaluation of the donor‐specificity of T‐cell alloresponses independently of fluctuations in global responsiveness. Four of ten recipients acquired a state of donor‐specific hyporeactivity (<75% relative specific reactivity) at 20 months posttransplantation (61 ± 12%, mean ± SD). Six patients were classified non‐hyporeactive (98 ± 10% mean relative specific reactivity). Relative specific reactivity did not correlate with the levels of general reactivity. Three of the four hyporeactive and four of the six non‐hyporeactive patients developed acute rejection. Stable graft function at 20 months posttransplantation (serum creatinine ≤ 2 mg/dl) was not closely related to the reactivity status, as five of eight patients with well‐functioning grafts did not develop relative specific hyporeactivity. One recipient with chronic rejection was classified hyporeactive. One non‐hyporeactive patient lost his graft due to non‐immunological causes. Our data suggest that post‐transplant relative specific reactivity does not predict acute rejection. Downregulation of donor‐specific reactivity might not be a prerequisite for stable graft function but could help identifying recipients who require less immunosuppression. This, however, remains to be established in a prospective immunosuppression‐weaning study.

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate–polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate–polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.

Pancreatic amyloid is found in more than 95 % of type II diabetes patients. Pancreatic amyloid is formed by the aggregation of islet amyloid polypeptide (hIAPP or amylin), which is a 37-residue peptide. Because pancreatic amyloid is cytotoxic, it is believed that its formation is directly associated with the development of the disease. We recently showed that hIAPP amyloid formation follows the nucleation-dependent polymerization mechanism and proceeds via a conformational transition of soluble hIAPP into aggregated β-sheets. Here, we report that the penta- and hexapeptide sequences, hIAPP(23–27) (FGAIL) and hIAPP(22–27) (NFGAIL) of hIAPP are sufficient for the formation of β-sheet-containing amyloid fibrils. Although these two peptides differ by only one amino acid residue, they aggregate into completely different fibrillar assemblies. hIAPP(23–27) (FGAIL) fibrils self-assemble laterally into unusually broad ribbons, whereas hIAPP(22–27) (NFGAIL) fibrils coil around each other in a typical amyloid fibril morphology. hIAPP(20–27) (SNNFGAIL) also aggregates into β-sheet-containing fibrils, whereas no amyloidogenicity is found for hIAPP(24–27) (GAIL), indicating that hIAPP(23–27) (FGAIL) is the shortest fibrillogenic sequence of hIAPP. Insoluble amyloid formation by the partial hIAPP sequences followed kinetics that were consistent with a nucleation-dependent polymerization mechanism. hIAPP(22–27) (NFGAIL), hIAPP(20–27) (SNNFGAIL), and also the known fibrillogenic sequence, hIAPP(20–29) (SNNFGAILSS) exhibited significantly lower kinetic and thermodynamic solubilities than the pentapeptide hIAPP(23–27) (FGAIL). Fibrils formed by all short peptide sequences and also by hIAPP(20–29) were cytotoxic towards the pancreatic cell line RIN5fm, whereas no cytotoxicity was observed for the soluble form of the peptides, a notion that is consistent with hIAPP cytotoxicity. Our results suggest that a penta- and hexapeptide sequence of an appropriate amino acid composition can be sufficient for β-sheet and amyloid fibril formation and cytotoxicity and may assist in the rational design of inhibitors of pancreatic amyloid formation or other amyloidosis-related diseases.

Studies performed on healthy volunteers have revealed that catecholamines downregulate the lipopolysaccharide LPSinduced production of tumor necrosis factor TNF, interleukin IL6, and IL1. We extended this observation and show that this effect is based on changes in the mRNA concentration of these cytokines. Catecholamines are increased in severe sepsis due to endogenous production and have to be administered exogenously when the disease has proceeded to the state of prolonged hypotension. We here investigated whether the immunomodulating effect of catecholamines could also be demonstrated in the blood of patients with prolonged severe sepsis and of those in prolonged septic shock. Blood was stimulated ex vivowith LPS in the presence and absence of epinephrine and the cytokine protein concentration was determined. In blood of healthy volunteers, epinephrine reduced the LPSstimulated synthesis of TNF by 62.5 P< 0.0001, of IL6 by 39 P< 0.0001, and of IL1 by 40 P0.015, and increased the LPSstimulated IL10 production by 77.8 P< 0.0001. Correspondingly, in blood of patients with prolonged severe sepsis, TNF was reduced by 67.2 P< 0.0001 and IL6 was reduced by 32.9 P< 0.0001 IL1 and IL10 were not modulated by catecholamines in these patients. In blood samples of patients in prolonged septic shock, epinephrine did not modulate cytokine levels of IL6 and IL10, and decreased TNF only by 36.4 P< 0.0001. Interestingly, epinephrine suppressed the IL1 production by 73 P< 0.0001 in blood of patients in prolonged septic shock, which was twice as much as in blood samples of healthy volunteers. The altered response of septic blood to catecholamines might be due to an altered reactivity of leukocytes in the prolonged disease although an additional role of preexisting catecholamines cannot be completely excluded.

According to Fowler's method, anatomical dead space (VD) can be determined graphically or computer-aided by iteration procedures by which phase III of a fraction–volume expirogramF(V) is back-extrapolated by a straight lineR(V). Whereas Fowler visually partitioned phase II into two equal areas bordered byF(V),R(V), andVD, in the present paper the area betweenF(V) andR(V) is set equal to the area of a trapezoid, one side of which is the unknownVDto be determined. We obtained two algebraic equations for both possible conditions, nonsloping and sloping alveolar plateau, and, as the main result, an even more general third equation that includes both Bohr's and Fowler's solution. The formulas exactly represent Fowler's graphical method and can be applied to all gases which are applicable in dead space determination. The derived equations were tested in experimental situations, showing equality between values of dead space determined by using the algebraic solution and the graphical method. Their major advantage is facilitating and speeding up computer-aided on-line determinations ofVD.

The effects of ethanol (0.5 to 5 Vol.-%) on identified neurons ofAplysia californica have been studied with the techniques of intracellular voltage recording and voltage and current clamping. In the visceral ganglion ethanol hyperpolarizes one group of identified cells and depolarizes a second group. The depolarizations may be up to the level of complete inactivation and are blocked in sodium-free sea water. While ethanol causes no significant effect on membrane resistance, it reduces spike amplitude, especially the overshoot. This effect on the spike potential correlates in voltage clamp with reductions of both sodium and calcium components of the inward currents by up to 50%, while the delayed potassium outward current is unaffected. In contrast a fast outward potassium-dependent current observable in some cells is slowed and reduced by up to 20%. The relatively smaller reduction of this current in contrast to the strong effect on the inward currents produces shifts in a depolarizing direction of the apparent steady state inactivation curves of the inward current. Ethanol did not alter the rate of the development of inactivation but it doubled the recovery from the inactivation following a conditioning depolarization. All of the ethanol effects are dose-dependent over the concentration range of 0.5 to 5 Vol.-% and are completely reversible.

Despite various attempts to rectify matters, the internalism-externalism (I-E) debate in epistemology remains mired in serious confusion. I present a new account of this debate, one which fits well with entrenched views on the I-E distinction and illuminates the fundamental disagreements at the heart of the debate. Roughly speaking, the I-E debate is over whether or not certain of the necessary conditions of positive epistemic status are internal. But what is the sense of ‘internal’ here? And of which conditions of which positive epistemic status are we speaking? I argue that an adequate answer to these questions requires reference to what I call the no-defeater condition which is satisfied by a subject’s belief B just in case she does not believe that B is defeated. I close by stating succinctly the main positions taken in the I-E debate, identifying the basic points of disagreement and suggesting fruitful courses for future discussion.

This research note makes a modest attempt to view the valuation of common shares, in part, in terms of a simple feedback mechanism. In so doing, the authors hope to shed light upon one of the underlying dynamics of the often mentioned tendency of the market to 'over-react' to changes in its environment.

ABSTRACTT cells in colorectal cancer (CRC) are associated with improved survival. However, checkpoint immunotherapies antagonizing the suppression of these cells are ineffective in the great majority of patients. To better understand the immune cell regulation in CRC, we compared tumor-associated T lymphocytes and macrophages to the immune cell infiltrate of normal mucosa. Human colorectal tumor specimen and tumor-distant normal mucosa tissues of the same patients were collected. Phenotypes and functionality of tissue-derived T cells and macrophages were characterized using immunohistochemistry, RNA in situ hybridization, and multiparameter flow cytometry. CRC contained significantly higher numbers of potentially immunosuppressive CD39 and Helios-expressing regulatory T cells in comparison to normal mucosa. Surprisingly, we found a concomitant increase of pro-inflammatory IFNγ -producing T cells. PD-L1+stromal cells were decreased in the tumor tissue. Macrophages in the tumor compared to tumor-distant normal tissue appear to have an altered phenotype, identified by HLA-DR, CD14, CX3CR1, and CD64, and tolerogenic CD206+macrophages are quantitatively reduced. The prognostic effect of these observed differences between distant mucosa and tumor tissue on the overall survival was examined using gene expression data of 298 CRC patients. The combined gene expression of increased FOXP3, IFNγ, CD14, and decreased CD206 correlated with a poor prognosis in CRC patients. These data reveal that the CRC microenvironment promotes the coexistence of seemingly antagonistic suppressive and pro-inflammatory immune responses and might provide an explanation why a blockade of the PD1/PD-L1 axis is ineffective in CRC. This should be taken into account when designing novel treatment strategies.