Clinical significance of the hemodynamic gain index in patients undergoing exercise stress testing and coronary computed tomography angiography.

BACKGROUND: Many hemodynamic parameters provide limited information regarding obstructive coronary artery disease (CAD) during exercise stress testing particularly when exercise is suboptimal. Hemodynamic gain index (HGI) is a recent sensitive indicator of ischemia and has been associated with increased mortality. This study evaluated the clinical impact of HGI in patients who underwent concomitant exercise stress testing and coronary computed tomography angiography (CCTA). METHODS: A total of 284 consecutive patients from the executive health program between 2010 and 2018 were identified. Resting and peak heart rate (HR) as well as systolic blood pressure (SBP) measurements were recorded. Framingham risk score (FRS), Duke treadmill score (DTS) and HGI [Formula: see text] were calculated. The latter was divided into quartiles. CCTA was used as a reference test to detect any CAD. Multivariate analysis and artificial neural network were used to determine the independent predictors of obstructive CAD. RESULTS: Mean age was 53 ± 12 years with 83% male. Mean HGI was 1.74 ± 0.67, with cut-off value of severely blunted HGI ≤ 1.25 (Quartile 4). Patients with severely blunted HGI were older, had higher FRS, and worse DTS. Patients with obstructive CAD had lower HGI when compared to those with normal CCTA/non-obstructive CAD (1.36 ± 0.53 vs. 1.77 ± 0.67, P = 0.005), and showed a higher prevalence of severely blunted HGI (44% vs. 22%, P = 0.019). After adjusting for traditional risk factors, HGI remained an independent predictor of obstructive CAD while severely blunted HGI was associated with threefold increased odds of having obstructive CAD (P = 0.05). Using artificial intelligence analysis, severely blunted HGI independently predicted obstructive CAD with an area under the curve of 0.83 and 0.96, and normalized importance of HGI of 100% and 63%, respectively for different models. CONCLUSIONS: Among patients who underwent concomitant exercise stress testing and CCTA, severely blunted HGI independently predicted obstructive CAD after multivariate adjustment for traditional risk factors.

Role of prostaglandin D2 receptors in the pathogenesis of abdominal aortic aneurysm formation.

Prostaglandin D2 (PGD2) released from immune cells or other cell types activates its receptors, D prostanoid receptor (DP)1 and 2 (DP1 and DP2), to promote inflammatory responses in allergic and lung diseases. Prostaglandin-mediated inflammation may also contribute to vascular diseases such as abdominal aortic aneurysm (AAA). However, the role of DP receptors in the pathogenesis of AAA has not been systematically investigated. In the present study, DP1-deficient mice and pharmacological inhibitors of either DP1 or DP2 were tested in two distinct mouse models of AAA formation: angiotensin II (AngII) infusion and calcium chloride (CaCl2) application. DP1-deficient mice [both heterozygous (DP1+/-) and homozygous (DP1-/-)] were protected against CaCl2-induced AAA formation, in conjunction with decreased matrix metallopeptidase (MMP) activity and adventitial inflammatory cell infiltration. In the AngII infusion model, DP1+/- mice, but not DP1-/- mice, exhibited reduced AAA formation. Interestingly, compensatory up-regulation of the DP2 receptor was detected in DP1-/- mice in response to AngII infusion, suggesting a potential role for DP2 receptors in AAA. Treatment with selective antagonists of DP1 (laropiprant) or DP2 (fevipiprant) protected against AAA formation, in conjunction with reduced elastin degradation and aortic inflammatory responses. In conclusion, PGD2 signaling contributes to AAA formation in mice, suggesting that antagonists of DP receptors, which have been extensively tested in allergic and lung diseases, may be promising candidates to ameliorate AAA.

Perivascular adipose tissue in autoimmune rheumatic diseases.

Perivascular adipose tissue (PVAT) resides at the outermost boundary of the vascular wall, surrounding most conduit blood vessels, except for the cerebral vessels, in humans. A growing body of evidence suggests that inflammation localized within PVAT may contribute to the pathogenesis of cardiovascular disease (CVD). Patients with autoimmune rheumatic diseases (ARDs), e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriasis, etc., exhibit heightened systemic inflammation and are at increased risk for CVD. Data from clinical studies in patients with ARDs support a linkage between dysfunctional adipose tissue, and PVAT in particular, in disease pathogenesis. Here, we review the data linking PVAT to the pathogenesis of CVD in patients with ARDs, focusing on the role of novel PVAT imaging techniques in defining disease risk and responses to biological therapies.

Complete postsurgical left ventricular-aortic discontinuity and pseudoaneurysm formation.

BACKGROUND: Left ventricular outflow tract pseudoaneurysm is a rare but potentially fatal complication of aortic valve replacement, infective endocarditis (IE), and suture dehiscence. Left ventricular-aortic discontinuity is a severe and uncommon manifestation of IE. For patients who have a long-standing history of endocarditis, periannular lesions in the aortic valve may rupture, leading to the rare occurrence of complete, or total, left ventricular-aortic discontinuity. METHODS: We present a case of complete postoperative left ventricular-aortic discontinuity and massive circumferential left ventricular outflow tract pseudoaneurysm discovered during a 3-month follow-up visit. Appropriate consent was obtained from all parties before submission of this case report. RESULTS: Postoperative cardiac computed tomography of a patient demonstrated dehiscence of a recently placed surgical aortic valve from the left ventricular outflow tract, with massive circumferential pseudoaneurysm formation. Only a small remnant of the membranous interventricular septum connected the aortic root to the heart, informing the diagnosis of complete left ventricular-aortic discontinuity. CONCLUSION: The clinical presentation of a left ventricular outflow tract pseudoaneurysm with concomitant left ventricular-aortic discontinuity is commonly nonspecific or clinically silent; thus, it requires a high index of suspicion and use of multimodality imaging for diagnosis and management.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BACKGROUND: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential. RESULTS: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived. CONCLUSION: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Perivascular Adipose Tissue and Vascular Perturbation/Atherosclerosis.

Atherosclerosis is orchestrated by complex interactions between vascular and inflammatory cells. Traditionally, it has been considered to be an intimal inflammatory disease, characterized by endothelial dysfunction, inflammatory cell recruitment, lipid oxidation, and foam cell formation. This inside-out signaling paradigm has been accepted as dogma for many years, despite the fact that inflammatory cells are far more prevalent in the adventitia compared with the intima. For decades, the origin of adventitial inflammation in atherosclerosis was unknown. The fact that these inflammatory cells were observed to cluster at the margin of perivascular adipose tissues-a unique and highly inflammatory adipose depot that surrounds most atherosclerosis-prone blood vessels-has stimulated interest in perivascular adipose tissue-mediated outside-in signaling in vascular pathophysiology, including atherosclerosis. The phenotype of perivascular adipocytes underlies the functional characteristics of this depot, including its role in adventitial inflammatory cell recruitment, trafficking to the intima via the vasa vasorum, and atherosclerosis perturbation. This review is focused on emerging concepts pertaining to outside-in signaling in atherosclerosis driven by dysfunctional perivascular adipose tissues during diet-induced obesity and recent strategies for atherosclerosis prediction and prognostication based upon this hypothesis.

Endoscopic vacuum assisted closure (E-VAC) of upper gastrointestinal leakages.

OBJECTIVES: Endoscopic vacuum-assisted closure (E-VAC) of leaks of the upper gastrointestinal tract is an increasingly applied endoscopic technique. Data on indication, clinical success, complications and prognostic factors are still sparse. METHODS: Patients treated with E-VAC between 2012 and 2019 at a tertiary referral center have been retrospectively analyzed. RESULTS: Overall, 116 patients treated with E-VAC were identified. Indication for E-VAC placement was postoperative leakage in 94/116 (81%), iatrogenic perforations 7/116 (6%) and others 15/116 (13%). In 92/116 (79%) of the patients E-VAC therapy showed successful wound closure. The first E-VAC after detection of insufficiency was significantly more often placed intracavitary in patients with E-VAC failure (p = .031). There was a trend for longer intensive care unit treatment for patients with E-VAC failure (p = .069). Complications occurred significantly more often in patients with E-VAC failure (p = .009). Platelet count was significantly higher in patients with E-VAC success at day of insufficiency detection (257/Thsd/µL (interquartile range [IQR], 185-362) vs. 195 (IQR, 117-309); p = .039). Platelet count (375 Thsd/µL (IQR, 256-484) vs. 190 (IQR, 129-292)), hemoglobin (9.5 g/dL (IQR, 8.8-10.1) vs. 8.7 g/dL (IQR, 8.15-9.35)) and C-reactive protein level (79 mg/L (IQR, 39.7-121.9) vs. 152 mg/L (IQR, 73.7-231)) at day 14 differed significantly. The 30 days mortality rate was 33.3% (8/24) in E-VAC failure compared with 2.2% in patients with E-VAC success (p = .001). CONCLUSIONS: E-VAC is an emerging highly effective interventional endoscopic technique for gastrointestinal wound closure even in highly selected patients.

Simultaneous heart-kidney transplantation results in respectable long-term outcome but a high rate of early kidney graft loss in high-risk recipients - a European single center analysis.

BACKGROUND: In spite of renal graft shortage and increasing waiting times for transplant candidates, simultaneous heart and kidney transplantation (HKTx) is an increasingly performed procedure established for patients with combined end-stage cardiac and renal failure. Although data on renal graft outcome in this setting is limited, reports on reduced graft survival in comparison to solitary kidney transplantation (KTx) have led to an ongoing discussion of adequate organ utilization. METHODS: This retrospective study was conducted to evaluate prognostic factors and outcomes of 27 patients undergoing HKTx in comparison to a matched cohort of 27 patients undergoing solitary KTx between September 1987 and October 2019 in one of Europe's largest transplant centers. RESULTS: Median follow-up was 100.33 (0.46-362.09) months. Despite lower five-year kidney graft survival (62.6% versus 92.1%; 111.73 versus 183.08 months; p = 0.189), graft function and patient survival (138.90 versus 192.71 months; p = 0.128) were not significantly inferior after HKTx in general. However, in case of prior cardiac surgery requiring sternotomy we observed significantly reduced early graft and patient survival (57.00 and 94.09 months, respectively) when compared to patients undergoing solitary KTx (183.08 and 192.71 months; p < 0.001, respectively) or HKTx without prior cardiac surgery (203.22 and 203.22 months; p = 0.016 and p = 0.019, respectively), most probably explained by the significantly increased rate of primary nonfunction (33.3%) and in-hospital mortality (25.0%). CONCLUSIONS: Our data demonstrates the increased rate of early kidney graft loss and thus significantly inferior graft survival in high-risk patients undergoing HKTx. Thus, we advocate for a "kidney-after-heart" program in such patients to ensure responsible and reasonable utilization of scarce resources in times of ongoing organ shortage crisis.

Functional investigation of the coronary artery disease gene SVEP1.

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE-/-Svep1+/-) compared to Svep1 wild-type mice (ApoE-/-Svep1+/+) and ApoE-/-Svep1+/- mice displayed elevated plaque neutrophil, Ly6Chigh monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE-/-Svep1+/- mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE-/-Svep1+/- mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency.

Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection.

Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.

H2 influenza A virus is not pathogenic in Tmprss2 knock-out mice.

The host cell protease TMPRSS2 cleaves the influenza A virus (IAV) hemagglutinin (HA). Several reports have described resistance of Tmprss2-/- knock-out (KO) mice to IAV infection but IAV of the H2 subtype have not been examined yet. Here, we demonstrate that TMPRSS2 is able to cleave H2-HA in cell culture and that Tmprss2-/- mice are resistant to infection with a re-assorted PR8_HA(H2) virus. Infection of KO mice did not cause major body weight loss or death. Furthermore, no significant increase in lung weights and no virus replication were observed in Tmprss2-/- mice. Finally, only minor tissue damage and infiltration of immune cells were detected and no virus-positive cells were found in histological sections of Tmprss2-/- mice. In summary, our studies indicate that TMPRSS2 is required for H2 IAV spread and pathogenesis in mice. These findings extend previous results pointing towards a central role of TMPRSS2 in IAV infection and validate host proteases as a potential target for antiviral therapy.

Expression of human thrombomodulin on porcine endothelial cells can reduce platelet aggregation but did not reduce activation of complement or endothelium - an experimental study.

Clinical xenotransplantation will only be feasible when present limitations can be controlled sufficiently. Activation of endothelium and complement as well as coagulopathy and thrombotic microangiopathy (TMA) is important barriers. Transgenic expression of hTBM on porcine endothelial cells is a reasonable approach to reduce activation of haemostasis. Endothelial cells from wild-type pigs as well from pigs expressing hTBM alone or in combination with hCD46 and knockout of the alpha-1,3,-galactosyltransferase (GTKO) were perfused with platelet-rich plasma in a microfluidic flow chamber. Platelet aggregation and activation, coagulation, complement and endothelial cell activation were assessed. Perfusion of wild-type porcine aortic endothelial cells (PAEC) resulted in distinct platelet aggregation. Expression of hTBM in either mono-transgenic or triple-transgenic (GTKO/hCD46/hTBM) PAEC showed significantly reduced or absent platelet aggregation. Flow cytometric analysis of platelets showed an increased CD62P expression in wild-type PAEC and significantly reduced expression in mono- or triple-transgenic PAEC. Activation of coagulation measured by TAT occured in WT PAEC and was clearly reduced in hTBM and GTKO/hCD46/hTBM PAEC. Activation of complement and endothelial cells was only reduced in GTKO/hCD46/hTBM but not in PAEC expressing hTBM alone. Expression of hTBM was able to prevent activation of coagulation and platelet aggregation in mono- and triple-transgenic PAEC, while activation of complement and endothelial cells was not reduced in mono-transgenic PAEC.

Multivisceral resection for adenocarcinoma of the pancreatic body and tail-a retrospective single-center analysis.

BACKGROUND: Adenocarcinoma of the pancreatic body and tail is associated with a dismal prognosis. As patients frequently present themselves with locally advanced tumors, extended surgery including multivisceral resection is often necessary in order to achieve tumor-free resection margins. The aim of this study was to identify prognostic factors for postoperative morbidity and mortality and to evaluate the influence of multivisceral resections on patient outcome. METHODS: This is a retrospective analysis of 94 patients undergoing resection of adenocarcinoma located in the pancreatic body and/or tail between April 1995 and December 2016 at our institution. Uni- and multivariable Cox regression analysis was conducted to identify independent prognostic factors for postoperative survival. RESULTS: Multivisceral resections, including partial resections of the liver, the large and small intestines, the stomach, the left kidney and adrenal gland, and major vessels, were carried out in 47 patients (50.0%). The median postoperative follow-up time was 12.90 (0.16-220.92) months. Median Kaplan-Meier survival after resection was 12.78 months with 1-, 3-, and 5-year survival rates of 53.2%, 15.8%, and 9.0%. Multivariable Cox regression identified coeliac trunk resection (p = 0.027), portal vein resection (p = 0.010), intraoperative blood transfusions (p = 0.005), and lymph node ratio in percentage (p = 0.001) as independent risk factors for survival. Although postoperative complications requiring surgical revision were observed more frequently after multivisceral resections (14.9 versus 2.1%; p = 0.029), postoperative survival was not significantly inferior when compared to patients undergoing standard distal or subtotal pancreatectomy (12.35 versus 13.87 months; p = 0.377). CONCLUSIONS: Our data indicates that multivisceral resection in cases of locally advanced pancreatic carcinoma of the body and/or tail is justified, as it is not associated with increased mortality and can even facilitate long-term survival, albeit with an increase in postoperative morbidity. Simultaneous resections of major vessels, however, should be considered carefully, as they are associated with inferior survival.

TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.

The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization.

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

A GXXXA Motif in the Transmembrane Domain of the Ebola Virus Glycoprotein Is Required for Tetherin Antagonism.

The interferon-induced antiviral host cell protein tetherin can inhibit the release of several enveloped viruses from infected cells. The Ebola virus (EBOV) glycoprotein (GP) antagonizes tetherin, but the domains and amino acids in GP that are required for tetherin antagonism have not been fully defined. A GXXXA motif within the transmembrane domain (TMD) of EBOV-GP was previously shown to be important for GP-mediated cellular detachment. Here, we investigated whether this motif also contributes to tetherin antagonism. Mutation of the GXXXA motif did not impact GP expression or particle incorporation and only modestly reduced EBOV-GP-driven entry. In contrast, the GXXXA motif was required for tetherin antagonism in transfected cells. Moreover, alteration of the GXXXA motif increased tetherin sensitivity of a replication-competent vesicular stomatitis virus (VSV) chimera encoding EBOV-GP. Although these results await confirmation with authentic EBOV, they indicate that a GXXXA motif in the TMD of EBOV-GP is important for tetherin antagonism. Moreover, they provide the first evidence that GP can antagonize tetherin in the context of an infectious EBOV surrogate.IMPORTANCE The glycoprotein (GP) of Ebola virus (EBOV) inhibits the antiviral host cell protein tetherin and may promote viral spread in tetherin-positive cells. However, tetherin antagonism by GP has so far been demonstrated only with virus-like particles, and it is unknown whether GP can block tetherin in infected cells. Moreover, a mutation in GP that selectively abrogates tetherin antagonism is unknown. Here, we show that a GXXXA motif in the transmembrane domain of EBOV-GP, which was previously reported to be required for GP-mediated cell rounding, is also important for tetherin counteraction. Moreover, analysis of this mutation in the context of vesicular stomatitis virus chimeras encoding EBOV-GP revealed that GP-mediated tetherin counteraction is operative in infected cells. To our knowledge, these findings demonstrate for the first time that GP can antagonize tetherin in infected cells and provide a tool to study the impact of GP-dependent tetherin counteraction on EBOV spread.

Investigation of the influence of xenoreactive antibodies on activation of complement and coagulation in an ex vivo perfusion animal study using porcine kidneys.

During pig-to-primate xenotransplantation or perfusion of porcine organs with human blood, a xenogeneic coagulopathy with consecutive development of thrombotic microangiopathy (TMA) can be observed. The aim of this study was to elucidate the influence of the reduction of xenoreactive natural antibodies on the coagulopathy using an ex vivo perfusion system. Thirteen perfusion experiments using landrace wild-type porcine kidneys were performed in three different experimental groups: autologous, xenogeneic, and immunoadsorption. During and after perfusion, blood and tissue samples were collected to assess markers of coagulation, complement, inflammation, and endothelial activation. Immunoadsorption prior to perfusion did not prolong perfusion time (174 min ±28) compared to xenogeneic (182 min ±22) experiments, whereas autologous perfusion was possible for maximum of 240 min in all experiments. Activation of coagulation was similar comparing perfusions after immunoadsorption (D-Dimer 24 186 µg/l ±5813; TAT 566 µg/l ±34) to xenogeneic (D-Dimer 22 175 µg/l ±7826, TAT 600 µg/l ±0) experiments. But antibody-mediated complement activation was reduced in the immunoadsorption group. TNF-alpha and markers of endothelial cell activation were lower in the immunoadsorption group compared to the xenogeneic experiments. In this ex vivo perfusion model, we observed that marked removal of xenogeneic antibodies can reduce complement activation via the classical pathway as well as endothelial cell activation and inflammation. Immunoadsorption cannot prevent the activation of the terminal complement cascade and coagulation.

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

The interferon-induced transmembrane proteins 1-3 (IFITM1-3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1-3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein-protein interactions. Coexpression of IFITM1-3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

Virion Background and Efficiency of Virion Incorporation Determine Susceptibility of Simian Immunodeficiency Virus Env-Driven Viral Entry to Inhibition by IFITM Proteins.

Interferon-induced transmembrane proteins (IFITMs) can inhibit the cellular entry of several enveloped viruses, including simian immunodeficiency virus (SIV). The blockade of SIV by IFITMs is isolate specific, raising the question of which parameters impact sensitivity to IFITM. We show that the virion context in which SIV-Env is presented and the efficiency of virion incorporation determine Env susceptibility to inhibition by IFITMs. Thus, determinants other than the nature of the envelope protein can impact the IFITM sensitivity of viral entry. IMPORTANCE: The host cell-encoded IFITM proteins can block viral entry and are an important component of the innate defenses against viral infection. However, the determinants controlling whether a virus is susceptible to blockade by IFITM proteins are incompletely understood. Our study shows that the amount of envelope proteins incorporated into virions as well as the nature of the virion particle itself can impact the sensitivity of viral entry to IFITMs. These results show for the first time that determinants other than the viral envelope protein can impact sensitivity to IFITM and have implications for the interpretation of previously published data on inhibition of viruses by IFITM proteins. Moreover, our findings might help to define the mechanism underlying the antiviral activity of IFITM proteins.

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-ß-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins.IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.