The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.

Evaluation of a Student-Led Community-Based Veterinary Clinic for Disabled Low-Income Clients: A Case Study of the University of Florida PAWS Program.

People with disabilities experiencing low socioeconomic position are priority populations when considering access to veterinary care. In this population, intersectional inequities lead to adverse health outcomes for both those individuals and the companion animals they care for. Community-based veterinary clinics provide an opportunity to target these inequities from a culturally sensitive lens, intending to improve human and animal outcomes. We conducted a process evaluation of a student-led community-based clinic for this population to better understand client satisfaction, assess learning outcomes among veterinary students, and improve program delivery and services. During academic year 2020-2021, the monthly clinics had 162 appointments in total with a median 15 DVM candidates volunteering at each clinic. Clients and volunteers responded to survey questionnaires designed to elicit information about their experiences with the clinic, including open-ended questions for further elucidation of measurable indicators of client-, patient-, and student-level impact. Clients attributed enrollment in the clinic with improved quality-of-life and reduction of financial burden; the program saved clients approximately $2,050 per pet during the evaluation year. Furthermore, the clinic widely facilitated completion of the college's core Primary Care and Dentistry learning outcomes. Beyond curriculum-standard learning objectives, students also reported positive attitude changes and increased readiness to provide care to people with disabilities and people experiencing low socioeconomic position. The results of this evaluation have significant implications for both veterinary and public health pedagogy. Especially, they highlight the significance of community health practice in veterinary trainee education.

Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. METHODS: Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. RESULTS: HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. CONCLUSIONS: Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

Hepatitis C virus pathogen associated molecular pattern (PAMP) triggers production of lambda-interferons by human plasmacytoid dendritic cells.

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.

Vital Signs: HIV diagnosis, care, and treatment among persons living with HIV--United States, 2011.

In the United States, an estimated 1.2 million persons are living with human immunodeficiency virus (HIV), a serious infection that, if untreated, leads to illness and premature death. Persons living with HIV who use antiretroviral therapy (ART) and achieve very low levels of the virus (suppressed viral load) can have a nearly normal life expectancy and have very low risk for transmitting HIV to others. However, each year in the United States, nearly 50,000 persons become infected with HIV. Each step along the HIV care continuum (HIV diagnosis, prompt and sustained HIV medical care, and ART) is essential for achieving a suppressed viral load.

Development of a reference interval for urinary ammonia-to-creatinine ratio in feline patients.

BACKGROUND: Disruption of acid-base homeostasis can lead to many clinical problems. Ammonia excretion by the kidneys is critical to maintaining acid-base homeostasis through bicarbonate production. Measurement of ammonia excretion may help determine if the kidneys are properly functioning in maintaining acid-base balance. Reference intervals are essential tools for clinical decision-making but do not currently exist for urinary ammonia-to-creatinine ratio (UACR) in feline patients. OBJECTIVE: This study aimed to generate a reference interval (RI) for UACR in healthy adult cats. METHODS: The study used samples from client-owned adult healthy cats that presented to the University of Florida Primary Care and Dentistry service (n = 92). Physical examination, serum biochemistry, urinalysis, urine ammonia, and creatinine concentrations were measured. Cats were excluded if there were significant abnormalities in their urinalysis or biochemistry panel. The RI for UACR was calculated according to the recommendation of the American Society for Veterinary Clinical Pathology. The UACR was evaluated for correlation with serum bicarbonate, weight, age, and sex. RESULTS: The RI for UACR was 3.4-20.7 with 90% confidence intervals for the lower and upper limits of (3.0-3.7) and (16.0-23.7), respectively. No significant correlation with age, sex, or weight was found. There was no discernable relationship between serum bicarbonate and UACR. CONCLUSIONS: Establishing an RI for UACR in healthy adult cats will allow further studies to determine if changes in UACR are observed during specific disease states.

Race- and gender-related variation in natural killer p46 expression associated with differential anti-hepatitis C virus immunity.

UNLABELLED: Major racial and gender differences have been documented in the natural history and treatment responses of chronic hepatitis C virus (HCV) infection; however, distinct mechanisms have remained enigmatic. We hypothesized that racial- and gender-related differences in natural killer (NK) cell populations may explain altered natural history and treatment responses. Our study cohort consisted of 29 African-American (AA; 55% male) and 29 Caucasian-American (CA; 48% male) healthy uninfected control subjects. Multiparameter flow cytometric analysis was used to characterize levels, phenotype with respect to 14 NK receptors, and lymphokine-activated killing (LAK) function. Gene expression was assessed by real-time reverse-transcriptase polymerase chain reaction after 6-hour in vitro stimulation with Toll-like receptor (TLR) ligands. The ability to control HCV infection was assessed in the Huh-7.5/JFH-1 coculture system. NK expression of natural cytotoxicity receptor NKp46 was strongly associated with CA race and female gender and correlated positively with LAK activity (P = 0.0054). NKp46(high) NKs were more efficient at controlling HCV than their NKp46(low) counterparts (P < 0.001). Similarly, ligation of NKp46 on isolated NK cells resulted in a significant reduction in the HCV copy number detected in Huh-7.5/JFH-1 coculture (multiplicity of infection: 0.01) at an effector:target ratio of 5:1 (P < 0.005). After TLR stimulation, genes involved in cytotoxicity, but not cytokine genes, were significantly up-regulated in NKp46(high) NKs. Cytokine stimulation (interleukin [IL]-12 and IL-15) demonstrated that NKp46(high) NK cells have significantly higher interferon-gamma production than NKp46(low) cells. TLR stimulation significantly induced degranulation as well as tumor necrosis factor alpha (TNF-α)-related apoptosis-inducing ligand, Fas, and TNF-α protein expression in NKp46(high) NKs. NKp46 ligand was induced on HCV-infected hepatocytes. CONCLUSIONS: NKp46 expression may contribute to differential HCV responses. NKp46 expression correlates with anti-HCV activity in vitro and thus may prove to be a useful therapeutic target.

Diagnostic accuracy of rapid antigen tests for COVID-19 compared to the viral genetic test in adults: a systematic review protocol.

OBJECTIVE: The objective of this diagnostic accuracy review is to evaluate the effectiveness of rapid antigen tests versus viral genetic PCR-based tests on COVID-19 diagnostic accuracy in adults 18 years and over. INTRODUCTION: Due to the rapidly changing nature of the COVID-19 pandemic, it is imperative that clinicians have access to the most relevant and effective tools and information required to combat this disease. Testing strategies are being developed continuously and need to be evaluated to ensure their appropriate implementation into clinical practice. INCLUSION CRITERIA: This systematic review will include publications that are in the English language (originally or translated) and any gray literature pertaining to the tests of interest. All races, ages over 18, and geographic locations will be considered. METHODS: MEDLINE (PubMed), Embase (Elsevier), Scopus (Elsevier), Qinsight (Quertle), and WHO COVID-19 database (World Health Organization) will be searched. Scopus, Qinsight, and WHO COVID-19 include gray literature. Studies in English published from November 2019 to the present will be considered. Animal studies and studies including pregnant women will be excluded. Retrieval of full-text studies, data extraction, and assessment of methodological quality will be performed independently by two reviewers. A custom data extraction table will be used. Findings will be graphically represented with two forest plots, one for sensitivity and the other for specificity. The strategy for meta-analysis includes producing a summary receiver operating characteristic curve and estimating the summary sensitivity/specificity for each threshold provided in the articles. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020224250.

RIG-I-like receptors direct inflammatory macrophage polarization against West Nile virus infection.

RIG-I-Like Receptors (RLRs) RIG-I, MDA5, and LGP2, are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) infections continue to grow in the US. Here, we use a systems biology approach to define the contributions of each RLR in the innate immune response to WNV. Genome-wide RNAseq and bioinformatics analyses of macrophages from mice lacking either RLR reveal that the RLRs drive distinct immune gene activation and response polarization to mediate an M1/inflammatory signature while suppressing the M2/wound healing phenotype. While LGP2 functions to modulate inflammatory signaling, RIG-I and MDA5 together are essential for M1 macrophage polarization in vivo and the control of WNV infection through potential downstream control of ATF4 and SMAD4 to regulate target gene expression for cell polarization. These analyses reveal the RLR-driven signature of macrophage polarization, innate immune protection, and immune programming against WNV infection.

2019 AAHA Dental Care Guidelines for Dogs and Cats.

The 2019 AAHA Dental Care Guidelines for Dogs and Cats outline a comprehensive approach to support companion animal practices in improving the oral health and often, the quality of life of their canine and feline patients. The guidelines are an update of the 2013 AAHA Dental Care Guidelines for Dogs and Cats. A photographically illustrated, 12-step protocol describes the essential steps in an oral health assessment, dental cleaning, and periodontal therapy. Recommendations are given for general anesthesia, pain management, facilities, and equipment necessary for safe and effective delivery of care. To promote the wellbeing of dogs and cats through decreasing the adverse effects and pain of periodontal disease, these guidelines emphasize the critical role of client education and effective, preventive oral healthcare.

Rapid reversal of innate immune dysregulation in blood of patients and livers of humanized mice with HCV following DAA therapy.

RESULTS: First, in patients receiving two different combinations of DAAs, we found that DAAs induced not only rapid viral clearance, but also a re-setting of antiviral immune responses in the peripheral blood. Specifically, we see a rapid decline in the expression of genes associated with chronic IFN stimulation (IFIT3, USP18, IFIT1) as well as a rapid decline in genes associated with inflammation (IL1ß, CXCL10, CXCL11) in the peripheral blood that precedes the complete removal of virus from the blood. Interestingly, this rapid reversal of innate immune activation was not seen in patients who successfully clear chronic HCV infection using IFN-based therapy. Next, using a novel humanized mouse model (Fah-/-RAG2-/-IL2rgnull-FRG), we assessed the changes that occur in the hepatic tissue following DAA treatment. DAA-mediated rapid HCV clearance resulted in blunting of the expression of proinflammatory responses while functionally restoring the RIG-I/MAVS axis in the liver of humanized mice. CONCLUSIONS: Collectively, our data demonstrate that the rapid viral clearance following treatment with DAAs results in the rebalancing of innate antiviral response in both the peripheral blood and the liver as well as enhanced antiviral signaling within previously infected hepatocytes.

Transmitted/founder hepatitis C viruses induce cell-type- and genotype-specific differences in innate signaling within the liver.

UNLABELLED: Hepatitis C virus (HCV) infection leads to persistence in the majority of cases despite triggering complex innate immune responses within the liver. Although hepatocytes are the preferred site for HCV replication, nonparenchymal cells (NPCs) can also contribute to antiviral immunity. Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference have identified full-length transmitted/founder (T/F) viruses. Here, we tested the effect of HCV T/F viral RNA (vRNA) on innate immune signaling within hepatocytes and NPCs, including the HepG2 and Huh 7.5.1 cell lines, a human liver endothelial cell line (TMNK-1), a plasmacytoid dendritic cell line (GEN2.2), and a monocytic cell line (THP-1). Transfection with hepatitis C T/F vRNA induced robust transcriptional upregulation of type I and III interferons (IFNs) within HepG2 and TMNK-1 cells. Both the THP-1 and GEN2.2 lines demonstrated higher type I and III IFN transcription with genotype 3a compared to genotype 1a or 1b. Supernatants from HCV T/F vRNA-transfected TMNK-1 cells demonstrated superior viral control. Primary human hepatocytes (PHH) transfected with genotype 3a induced canonical pathways that included chemokine and IFN genes, as well as overrepresentation of RIG-I (DDX58), STAT1, and a Toll-like receptor 3 (TLR3) network. Full-length molecular clones of HCV induce broad IFN responses within hepatocytes and NPCs, highlighting that signals imparted by the various cell types within the liver may lead to divergent outcomes of infection. In particular, the finding that HCV genotypes differentially induce antiviral responses in NPCs and PHH might account for relevant clinical-epidemiological observations (higher clearance but greater necroinflammation in persistence with genotype 3). IMPORTANCE: Hepatitis C virus (HCV) has become a major worldwide problem, and it is now the most common viral infection for which there is no vaccine. HCV infection often leads to persistence of the virus and is a leading cause of chronic hepatitis, liver cancer, and cirrhosis. There are multiple genotypes of the virus, and patients infected with different viral genotypes respond to traditional therapy differently. However, the immune response to the virus within the liver has not been fully elucidated. Here, we determined the responses to different genotypes of HCV in cell types of the liver. We found that the immune response varied according to both cell type and HCV genotype, leading to a more pronounced induction of inflammatory pathways after exposure to certain genotypes. Therefore, inflammatory pathways that are being robustly activated by certain HCV genotypes could lead to more severe damage to the liver, inducing diverse outcomes and responses to therapy.

Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT) 1 protein expression.

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or ß-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

Education of global veterinarians.

In 2003, the University of Florida (UF) College of Veterinary Medicine (CVM) created an Office of International Programs (OIP) in response to one of ten initiatives of the UF Strategic Plan: internationalization of the curriculum. The OIP has developed coursework that provides students with an opportunity for international exposure during the veterinary curriculum at three levels. In Level 1 (on campus) students can participate in a seminar series in global health: www.ufglobalhealth.org. This is an elective course offered to professional students at the UF Health Science Center (Dentistry, Medicine, Pharmacy, Public Health, and Veterinary Medicine). In Level 2 (abroad), students can participate in structured study abroad programs under the supervision of UF faculty and international scholars from collaborative institutions abroad. In Level 3 (on campus and abroad), students can participate in a certificate program in international veterinary medicine. This is a 15-credit program, parallel to the veterinary curriculum. By offering courses on campus and abroad, we want to empower the curriculum with a global perspective of the veterinary profession, as well as with a humanist education that can help students recognize the importance of respect for cultural differences and the reasons for different degrees of development and growth in the world. In addition, this paper presents the need for veterinary medicine and other disciplines in the health sciences to communicate with other disciplines in the social sciences and natural sciences to create development practitioners equipped with cross-disciplinary knowledge and skills needed to formulate, implement and evaluate solutions aimed at breaking the cycle of poverty and disease in low income societies. Finally, this paper makes a call to the American Veterinary Medical Association Council on Education to assess the need to recognize the importance of internationalization of the veterinary curriculum as a key standard for accreditation of colleges or schools of veterinary medicine.

Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector.

Live, attenuated Salmonella strains can serve as vectors for the delivery of recombinant vaccine antigens for development of oral mucosal vaccines. Various vaccine parameters can affect the immune responses elicited by Salmonella vectors, including the expression level, location and timing of expressed antigens. We have previously established immunogenic Salmonella enterica serovar Typhimurium strains which cytoplasmically express hemagglutinin B (HagB) of Porphyromonas gingivalis, a putative periodontal pathogen. In this study, we sought to determine whether the 39 kDa HagB protein could be stably expressed on the surface of an avirulent Salmonella vaccine strain. The hagB gene was cloned into an expression plasmid as a C-terminal fusion with Lpp-OmpA, a hybrid surface display system. High expression of Lpp-OmpA-HagB proved to be toxic to the vaccine strain, and it was necessary to introduce attenuating mutations in the trc promoter. Stable expression was obtained in transformants with promoter mutations that resulted in low levels of expression. The expression of Lpp-OmpA-HagB was confirmed by ELISA and Western blot. Localization to the outer membrane/periplasm was confirmed by transmission electron microscopy using immunogold labeling, surface labeling of whole mounts using electron microscopy, flow cytometry, and by quantitation of HagB in cytoplasmic, as well as inner and outer cell membrane fractions. When delivered orally in mice, the surface-expressing strain induced higher serum IgG and IgA responses to HagB than a cytoplasmic expressing strain, while responses in secretions were comparable. These results suggest that surface localization may differentially enhance the immunogenicity of antigens expressed by live, avirulent Salmonella vaccine vectors.

IL-12 reduces the severity of Sendai virus-induced bronchiolar inflammation and remodeling.

The goal of this research was to determine whether differential pulmonary IL-12 gene expression controls susceptibility to Sendai virus-induced chronic airway inflammation and fibrosis in inbred rat strains. Sendai virus-resistant F344 rats and susceptible BN rats were studied from 1 to 14 days following virus inoculation. F344 rats had 3.4-fold higher IL-12 mRNA levels detected by real-time PCR in lung than BN rats as early as two days following inoculation. This increase in mRNA was associated at two days with increased total IL-12 protein and with a 2-fold increase in numbers of bronchiolar, OX-6-positive dendritic cells and an increased number of IL-12 p40-positive, bronchiolar macrophages and dendritic cells (p<0.05). Virus-susceptible BN rats treated with 3 mug of recombinant, mouse IL-12 intraperitoneally at the time of virus inoculation had a 22.1% decrease in severity of chronic bronchiolar inflammation and a 23.8% decrease in fibrosis compared to virus-inoculated BN rats treated with saline. IL-12 treatment induced increased IFN-gamma mRNA and protein expression after virus inoculation (p<0.05). The results demonstrate that there is differential pulmonary IL-12 gene expression between virus-susceptible and resistant rat strains and that IL-12 treatment can provide significant protection from virus-induced chronic airway inflammation and remodeling during early life.