EGF receptor modulates HEV entry in human hepatocytes.

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

A Survey on the Choice of Spinal Cord Stimulation Parameters and Implantable Pulse Generators and on Reasons for Explantation.

Objective: Spinal Cord Stimulation (SCS) is a vital treatment for chronic intractable pain. In the last few years, the field has undergone dramatic changes in new waveform and frequency introduction as well as device miniaturization. It is important to understand contemporary practice patterns regarding these parameters. Methods: We surveyed the active membership of Spine Intervention Society (SIS), and American Society of Regional Anesthesia (ASRA) on their practices regarding various aspects of Spinal Cord Stimulation therapy. Here we report on SCS waveform usage, battery types, and causes of explant in this cohort of providers. Results: There was similar degree of usage of tonic, burst, and 10 kHz usage at 71.5%, 74.1% and 61.7% respectively. Dorsal root ganglion stimulation was used by 32.6% and other modes of stimulation by 13.5%. Rechargeable systems were often or always used by 67.2% whereas 10% never used a rechargeable system. Most common cause of explant was loss of effectiveness, reported by 53.7%. Conclusion: There has been significant adoption of new waveforms in daily practice of spinal cord stimulation therapy and there is robust mixed usage of new waveforms and frequencies. Rechargeable systems are the most commonly used but primary cell is also used in significant numbers. Loss of efficacy remains the most common cause of explant for the majority of practitioners. This survey establishes practice patterns of SCS usage regarding these important variables against which future changes can be gauged.

Biologics and atherosclerotic cardiovascular risk in rheumatoid arthritis: a review of evidence and mechanistic insights.

Introduction: Cardiovascular disease is a leading comorbidity in rheumatoid arthritis. Timely introduction of biologic therapies in a treat-to-target approach has optimized disease-related outcomes and attenuated accrual of comorbidities, including cardiovascular risk.Areas covered: A literature search in MEDLINE (via PubMed) was performed between January 2009 and November 2020. This manuscript explores recent developments in atherosclerotic cardiovascular risk in RA compared with non-RA individuals; it synopsizes differences in vascular function and inflammation, prevalence, burden, vulnerability, and progression of atherosclerotic plaque and their underlying cellular and molecular mechanisms. Finally, it reviews the recent literature on cardioprotective benefits of biologics and draws mechanistic links with inhibition of new plaque formation, stabilization of high-risk lesions and improvement in endothelial function, arterial stiffness, lipid metabolism, and traditional cardiac risk factors.Expert opinion: Increasing evidence points to a solid cardioprotective influence of earlier, longer, and ongoing use of biologic treatments in RA. Nevertheless, the precise mechanistic effects of plaque progression and remodeling, vascular stiffness, endothelial dysfunction, lipid metabolism, and traditional cardiac risk factors are less rigorously characterized.

Beta-2-glycoprotein-I IgA antibodies predict coronary plaque progression in rheumatoid arthritis.

OBJECTIVES: To evaluate whether anti-Beta-2-Glycoprotein-I (anti-ß2GPI) IgA antibodies associate with progression of coronary atherosclerosis and cardiovascular disease (CVD) events in rheumatoid arthritis (RA). METHODS: One hundred-fifty patients underwent plaque evaluation (total, non-calcified, mixed and calcified) with coronary computed tomography angiography; 101 were re-imaged within 6.9±0.3 years to assess progression. The Framingham-D'Agostino score assessed cardiovascular risk. Coronary artery calcium (CAC) and segment involvement score quantified plaque burden. RESULTS: Anti-ß2GPI IgA were seen in 45 (30%) patients. Despite no link to baseline plaque burden, anti-ß2GPI IgA associated with segment involvement score increase (adjusted-RR=1.64 [95%CI 1.02-2.63]), CAC change (adjusted-ß=0.33 [95%CI 0.002-0.656]) and developing new extensive or obstructive plaque at follow-up (adjusted-OR=4.24 [95%CI 1.30-13.87]). Adding anti-ß2GPI IgA to logistic regression models with conventional risk factors predicting plaque progression outcomes increased Area under the receiver-operator curve and improved Net Reclassification and Integrated Discrimination Improvement indices (all P<0.05). In per-segment analyses, anti-ß2GPI IgA predicted mixed plaque formation (adjusted-OR=3.20 [95%CI 1.01-10.09]) and lower likelihood of transition of mixed to calcified plaque (adjusted-OR=0.19 [95%CI 0.04-0.96]). Anti-ß2GPI IgA moderated the effect of C-reactive protein on CAC change such that C-reactive protein associated with CAC change (ß=0.26 [95%CI 0.14-0.38]) and CVD risk (adjusted-HR=1.89 [95%CI 1.02-3.51]) only in anti-ß2GPI IgA positive patients. CONCLUSION: Anti-ß2GPI IgA addition to clinical risk models improved prediction accuracy of CAC, plaque progression and transition to extensive/obstructive disease. They associated with new high-risk mixed plaques and delayed healing to calcified lesions. Anti-ß2GPI IgA further modified the effect of inflammation on plaque progression and CVD events.

Toll-Like Receptors Induce Signal-Specific Reprogramming of the Macrophage Lipidome.

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

Zoonotic Vectorborne Pathogens and Ectoparasites of Dogs and Cats in Eastern and Southeast Asia.

To provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and Southeast Asia during 2017-2018. Overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. Our data cover a wide geographic distribution of several pathogens, including Leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. Because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. These results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies.

Analysis of sulfate metabolites of the doping agents oxandrolone and danazol using high performance liquid chromatography coupled to tandem mass spectrometry.

The direct detection of sulfate conjugates of anabolic androgenic steroids (AAS) can be a powerful tool in doping control analysis. By skipping the solvolysis step analysis time can be reduced, and due to long term sulfate metabolites the detection time can be significantly extended as demonstrated for some AAS. This study presents the successful identification of sulfate metabolites of the doping agents oxandrolone and danazol in excretion urines by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The sulfate conjugate of 17ß-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one could be identified as a new metabolite of oxandrolone. Sulfate conjugates of the danazol metabolites ethisterone and 2α-hydroxymethylethisterone were identified in an excretion urine for the first time. In addition, these sulfate conjugates were synthesized successfully. For a confirmation analysis, the number of analytes can be increased by additional sulfate conjugates of danazol metabolites (2-hydroxymethyl-1,2-dehydroethisterone and 6ß-hydroxy-2-hydroxymethylethisterone), which were also identified for the first time. The presented validation data underline the suitability of the identified sulfate conjugates for doping analysis with regard to the criteria given by the technical documents of the World Anti-Doping Agency (WADA).

Novel Single Puncture Approach for Simplicity 3 Sacral Plexus Radiofrequency Ablation: Technical Note.

UNLABELLED: Radiofrequency (RF) ablation of the lateral sacral plexus has been used for the treatment of sacroiliac joint pain including as an adjunct to other palliative therapies for the treatment of painful osseous metastasis. The treatment goal is targeted ablation of the dorsal lateral branches of S1-S4. Though several techniques have been described, the Simplicity III (Neurotherm, Middleton, MA) system allows for ablation to be achieved with a single RF probe by utilizing a multi-electrode curved RF probe to create a continuous ablation line across all sacral nerves. In the standard approach, there is sequential introduction of a spinal needle along the desired ablation tract for local anesthesia followed by separate placement of the ablation probe. Though fluoroscopic guidance is utilized, multiple needle passes increase the risk of complication such as bowel perforation or probe insertion through a neural foramen. It may also extend procedure time and increase radiation dose. We illustrate a technique for Simplicity III RF ablation of the dorsal sacral plexus using a modified Seldinger approach for treatment of a patient with sacroiliac joint pain due to osseous renal cell carcinoma metastasis. The desired ablation tract is initially anesthetized via a hollow micropuncture needle. The needle is then exchanged for a peelaway sheath. The RF probe is inserted through the peelaway sheath thus ensuring the probe is placed precisely along the previously anesthetized tract allowing the procedure to be completed using a single percutaneous puncture. We believe that this approach decreases the risks of bowel perforation, patient discomfort as a result of multiple percutaneous punctures, and procedure time. KEY WORDS: Simplicity 3, sacral plexus ablation, image-guided approach, modified Seldinger, chronic sacral pain, thin wall introducer needle.

Analysis of tolvaptan and its metabolites in sports drug testing by high-performance liquid chromatography coupled to tandem mass spectrometry.

Tolvaptan is prohibited by the World Anti-doping Agency (WADA) under class S5 - Diuretics and masking agents. Less than 1% of the administrated dose is excreted by humans in urine. Knowledge concerning the metabolism in humans, and especially the excretion of metabolites in human urine, is limited. An analysis method based on the dilute-and-shoot approach using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for detection was developed and validated. Ion transitions, which are part of this method, can easily be included in already existing screening methods used in routine doping analysis for the detection of diuretics. After administration of a single dose of tolvaptan to one male subject, low concentrations of the drug itself could be detected in urine samples over a time period of 24 h. In addition, hydroxyl metabolites of tolvaptan and one carboxyl metabolite with a cleaved benzazepine ring system were identified. These metabolites showed detection times of up to 150 h. An inclusion of these metabolites in the methods used in doping control analysis seems therefore to be of value. Copyright © 2016 John Wiley & Sons, Ltd.

Stochastic model for estimating the risk of transfusion-transmitted hepatitis B in Vietnam.

BACKGROUND AND OBJECTIVE: Transfusion-transmitted hepatitis B virus (HBV) infection may originate from hepatitis B surface antigen (HBsAg) false-negative blood donors, HBsAg negative and anti-HBc positive blood donors and blood donors with both tests negative. HBV DNA may be present in all these cases and blood may be infectious. The aim of the study was to estimate the risk of transfusion-transmitted HBV in Vietnam using a stochastic Monte Carlo model. METHODS: A cross-sectional study of HBV prevalence in 1200 potential blood donors in rural Vietnam is used as basis for the Monte Carlo model together with expert panel estimates of occult hepatitis B infection (OBI) prevalence in blood donors. RESULTS: With 1 000 000 blood donors running in the model, the potential OBI ranged from 658 to 747 blood units per million at 5 percentile and from 1342 to 2507 blood units per million at 95 percentile resulting in the risk of post-transfusion hepatitis ranging from 66 to 250 blood units per million assuming that risk of post-transfusion from potential OBI is 10%. Using the manufacturer's HBsAg sensitivity, the mean rate of blood units per million donations having false-negative HBsAg results was 298 (5-95 percentile: 14-893). When the test sensitivity was set lower, false-negative tests was observed at a mean of 1087 per million (5-95 percentile: 762-3220). The fraction of potential OBI donors increased with the increasing age in both genders. CONCLUSION: Current HBsAg screening in Vietnam is insufficient in eliminating the risk of transfusion-transmitted HBV infection. The major risk factors are HBsAg false-negative results and OBI. Increased test sensitivity and locally validated HBsAg assays are recommended.

Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt(+) innate lymphocytes.

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.

RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells.

The mucosal immune system of the intestine is separated from a vast array of microbes by a single layer of epithelial cells. Cues from the commensal microflora are needed to maintain epithelial homeostasis, but the molecular and cellular identities of these cues are unclear. Here we provide evidence that signals from the commensal microflora contribute to the differentiation of a lymphocyte population coexpressing stimulatory natural killer cell receptors and the transcription factor RORgammat that produced interleukin 22 (IL-22). The emergence of these IL-22-producing RORgammathiNKp46+NK1.1(int) cells depended on RORgammat expression, which indicated that these cells may have been derived from lymphoid tissue-inducer cells. IL-22 released by these cells promoted the production of antimicrobial molecules important in the maintenance of mucosal homeostasis.