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People living with human immunodeficiency virus (PLWH) are a significant population globally. Research delineating our understanding of coinfections in PLWH is critical to care for those navigating infection with other pathogens. The recent COVID-19 pandemic underscored the urgent need for studying the effects of SARS-CoV-2 infections in therapy-controlled and uncontrolled immunodeficiency viral infections. This study established the utility of a feline model for the in vivo study of coinfections. Domestic cats are naturally infected with SARS-CoV-2 and Feline Immunodeficiency Virus, a lentivirus molecularly and pathogenically similar to HIV. In this study, comparisons are made between FIV-positive and FIV-negative cats inoculated with SARS-CoV-2 (B.1.617.2.) in an experimental setting. Of the FIV+ cats, three received Zidovudine (AZT) therapy in the weeks leading up to SARS-CoV-2 inoculation, and two did not. SARS-CoV-2 viral RNA was quantified, histopathologic comparisons of respiratory tissues were made, and T-cell populations were analyzed for immune phenotype shifts between groups. CD4+ T lymphocyte responses varied, with FIV+-untreated cats having the poorest CD4+ response to SARS-CoV-2 infection. While all cats had significant pulmonary inflammation, key histopathologic features of the disease differed between groups. Additionally, viral genomic analysis was performed, and results were analyzed for the presence of emerging, absent, amplified, or reduced mutations in SARS-CoV-2 viral RNA after passage through the feline model. Positive selection is noted, especially in FIV+ cats untreated with AZT, and mutations with potential relevance were identified; one FIV+-untreated cat had persistent, increasing SARS-CoV-2 RNA in plasma five days post-infection. These findings and others support the utility of the feline model for studying coinfection in people with HIV and highlight the importance of antiretroviral therapy in clearing SARS-CoV-2 coinfections to minimize transmission and emergence of mutations that may have deleterious effects.
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Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population.
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Anticorpos Antivirais , Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vacinas de Partículas Semelhantes a Vírus , Animais , Feminino , Humanos , Camundongos , Administração Intranasal , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Pulmão/virologia , Pulmão/patologia , Pulmão/imunologia , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/genética , Vacinação , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/genética , Redução de PesoRESUMO
Targeting the gut-bone axis with probiotics and prebiotics is considered as a promising strategy to reduce the risk of osteoporosis. Gut-derived short chain fatty acids (SCFA) mediate the effects of probiotics on bone via Tregs, but it is not known whether prebiotics act through a similar mechanism. We investigated how 2 different prebiotics, tart cherry (TC) and fructooligosaccharide (FOS), affect bone, and whether Tregs are required for this response. Eight-wk-old C57BL/6 female mice were fed with diets supplemented with 10% w/w TC, FOS, or a control diet (Con; AIN-93M) diet, and they received an isotype control or CD25 Ab to suppress Tregs. The FOS diet increased BMC, density, and trabecular bone volume in the vertebra (~40%) and proximal tibia (~30%) compared to the TC and control diets (Con), irrespective of CD25 treatment. Both prebiotics increased (P < .01) fecal SCFAs, but the response was greater with FOS. To determine how FOS affected bone cells, we examined genes involved in osteoblast and osteoclast differentiation and activity as well as genes expressed by osteocytes. The FOS increased the expression of regulators of osteoblast differentiation (bone morphogenetic protein 2 [Bmp2], Wnt family member 10b [Wnt10b] and Osterix [Osx]) and type 1 collagen). Osteoclasts regulators were unaltered. The FOS also increased the expression of genes associated with osteocytes, including (Phex), matrix extracellular phosphoglycoprotein (Mepe), and dentin matrix acidic phosphoprotein 1 (Dmp-1). However, Sost, the gene that encodes for sclerostin was also increased by FOS as the number and density of osteocytes increased. These findings demonstrate that FOS has a greater effect on the bone mass and structure in young adult female mice than TC and that its influence on osteoblasts and osteocytes is not dependent on Tregs.
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Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/ß T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.
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Doença das Mucosas por Vírus da Diarreia Viral Bovina , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina Tipo 2 , Vírus da Diarreia Viral Bovina , Animais , Bovinos , Deltainfluenzavirus , Imunidade , Atrofia , Anticorpos AntiviraisRESUMO
This prospective clinical study sought to determine the accuracy of cytopathologic examination and needle-core biopsy (NCB) against diagnoses obtained by excisional histopathology (EH) for canine splenic masses. Twenty-five masses were evaluated ex vivo by ultrasound-guided fine-needle aspiration (FNA) and NCB tissue sampling. Each spleen was placed in a container and artificial skin placed over its surface. Ultrasound-guided FNA using a 22-gauge needle and 2 NCB samples [14-gauge (NCB-14), 16-gauge (NCB-16)] were obtained and submitted for analysis. Results were compared to results obtained by splenic excisional histopathology (EH). There was no difference noted between FNA, NCB-14, or NCB-16 analyses. In addition, there was no difference in accuracy between FNA and NCB-14 or between FNA and NCB-14 versus NCB-16. Reported accuracy of FNA was 0.72, NCB-14 was 0.72, and NCB-16 was 0.64, respectively. Both FNA and NCB-14 displayed a sensitivity of 71% and NCB-16 a sensitivity of 53%. Both FNA and NCB-14 displayed a specificity of 75% and NCB-16 a specificity of 88%. The results demonstrated that NCB had no advantage clinically over FNA at diagnosing splenic pathology. This study further demonstrates that preoperative diagnostic evaluation of the spleen is not highly accurate and cannot be recommended prior to splenectomy.
Cette étude clinique prospective visait à déterminer la précision de l'examen cytopathologique et de la biopsie au trocart (NCB) par rapport aux diagnostics obtenus par histopathologie excisionnelle (EH) pour les masses spléniques canines. Vingt-cinq masses ont été évaluées ex vivo par aspiration à l'aiguille fine guidée par ultrasons (FNA) et prélèvement de tissu par NCB. Chaque rate a été placée dans un récipient et une peau artificielle placée sur sa surface. Une FNA guidée par ultrasons à l'aide d'une aiguille de calibre 22 et de 2 échantillons de NCB (calibre 14 (NCB-14), calibre 16 (NCB-16)) ont été obtenues et soumises pour analyse. Les résultats ont été comparés aux résultats obtenus par histopathologie excisionnelle splénique (EH). Aucune différence n'a été notée entre les analyses FNA, NCB-14 ou NCB-16. De plus, il n'y avait aucune différence de précision entre FNA et NCB-14 ou entre FNA et NCB-14 par rapport à NCB-16. La précision rapportée de FNA était de 0,72, celle de NCB-14 de 0,72 et de NCB-16 était de 0,64, respectivement. FNA et NCB-14 ont affiché une sensibilité de 71 % et NCB-16 une sensibilité de 53 %. FNA et NCB-14 ont affiché une spécificité de 75 % et NCB-16 une spécificité de 88 %. Les résultats ont démontré que la NCB n'avait aucun avantage clinique sur la FNA pour diagnostiquer la pathologie splénique. Cette étude démontre en outre que l'évaluation diagnostique préopératoire de la rate n'est pas très précise et ne peut être recommandée avant la splénectomie.(Traduit par Docteur Serge Messier).
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Baço , Ultrassonografia de Intervenção , Animais , Cães , Biópsia por Agulha Fina/veterinária , Estudos Prospectivos , Ultrassonografia/veterinária , Ultrassonografia de Intervenção/veterinária , Sensibilidade e Especificidade , Estudos RetrospectivosRESUMO
Antivenom is currently the standard-of-care treatment for snakebite envenoming, but its efficacy is limited by treatment delays, availability, and in many cases, species specificity. Many of the rapidly lethal effects of envenoming are caused by venom-derived toxins, such as phospholipase A2 (sPLA2); therefore, small molecule direct toxin inhibitors targeting these toxins may have utility as initial and adjunct therapies after envenoming. Varespladib (intravenous, IV) and varespladib-methyl (oral) have been shown to potently inhibit sPLA2s from snake venoms in murine and porcine models, thus supporting their further study as potential treatments for snakebite envenoming. In this pilot study, we tested the ability of these compounds to reverse neurotoxic effects of venom from the Australian and Papuan taipan (Oxyuranus scutellatus) subspecies in juvenile pigs (Sus domesticus). The mean survival time for control animals receiving Australian taipan venom (0.03 mg/kg, n = 3) was 331 min ± 15 min; for those receiving Papuan taipan venom (0.15 mg/kg, n = 3) it was 178 ± 31 min. Thirteen pigs received Australian taipan venom and treatment with either IV or oral varespladib (or with IV to oral transition) and all 13 survived the duration of the study (≥96 h). Eight pigs received Papuan taipan venom followed by treatment: Briefly: Two animals received antivenom immediately and survived to the end of the study. Two animals received antivenom treatment delayed 45 min from envenoming and died within 4 h. Two animals received similarly delayed antivenom treatment and were rescued by varespladib. Two animals were treated with varespladib alone after a 45-min delay. Treatment with varespladib only was effective but required repeat dosing over the course of the study. Findings highlight both the importance of early treatment and, as well, a half-life for the investigational inhibitors now in Phase II clinical trials for snakebite. Varespladib rapidly reversed weakness even when administered many hours post-envenoming and, overall, our results suggest that varespladib and varespladib-methyl could be efficacious tools in the treatment of sPLA2-induced weakness from Oxyuranus envenoming. Further clinical study as initial therapy and as potential method of rescue from some types of antivenom-resistant envenomings are supported by these data.
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Fosfolipases A2 Secretórias , Mordeduras de Serpentes , Animais , Suínos , Camundongos , Antivenenos/farmacologia , Antivenenos/uso terapêutico , Mordeduras de Serpentes/tratamento farmacológico , Projetos Piloto , Austrália , Venenos Elapídicos/toxicidadeRESUMO
Background: Commensal gut bacteria, including Lactobacillus, can produce metabolites that stimulate the release of gut antimicrobial peptides (AMPs) via the signal transducer and activator of transcription (STAT)3 pathway and prevent obesity-associated leaky gut and chronic inflammation. We have previously reported that wheat germ (WG) selectively increased cecal Lactobacillus in obese mice. Objectives: This study investigated the effects of WG on gut STAT3 activation and AMPs (Reg3γ and Reg3ß) as well as the potential of WG to inhibit nuclear Nf-κB-activation and immune cell infiltration in the visceral adipose tissue (VAT) of mice fed a Western diet (i.e., high-fat and sucrose diet [HFS]). Methods: Six-wk-old male C57BL/6 mice were randomly assigned to 4 groups (n = 12/group): control (C, 10% fat and sucrose kcal) or HFS (45% fat and 26% sucrose kcal) diet with or without 10% WG (wt/wt) for 12 wk. Assessments include serum metabolic parameters jejunal AMPs genes, inflammatory markers, and phosphorylation of STAT3 as well as VAT NF-κBp65. Independent and interaction effects of HFS and WG were analyzed with a 2-factor ANOVA. Results: WG significantly improved markers of insulin resistance and upregulated jejunal Il10 and Il22 genes. The HFS + WG group had a 15-fold increase in jejunal pSTAT3 compared with the HFS group. Consequently, WG significantly upregulated jejunal mRNA expression of Reg3γ and Reg3ß. The HFS group had a significantly higher VAT NF-κBp65 phosphorylation than the C group, while the HFS + WG group suppressed this to the level of C. Moreover, VAT Il6 and Lbp genes were downregulated in the HFS + WG group compared with HFS. Genes related to macrophage infiltration in the VAT were repressed in the WG-fed mice. Conclusion: These findings show the potential of WG to influence vital regulatory pathways in the gut and adipose tissue which may reduce the chronic inflammatory burden on these tissues that are important targets in obesity and insulin resistance.
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The objective of this study was to determine the extent that nebulized glycosylated caffeic acid phenylether ester-4-O-alpha-D-glucopyranoside (G-CAPE) attenuates particulate-induced airway inflammation in healthy horses. Our hypothesis was that nebulization with G-CAPE would result in improved respiratory scores, higher arterial oxygen partial pressure, and less inflammatory airway infiltrates in horses with induced airway inflammation, compared with untreated controls. Five healthy adult horses were housed inside a climate controlled, closed barn on straw bedding and fed ad lib moldy grass hay for 16 days to induce airway inflammation. An experimental crossover study was performed in which animals were treated with 200 mg G-CAPE dissolved in 45 mL of 10% triethanolamine (G-CAPE group) or 45 mL of 10% triethanolamine (CONTROL group), and clinical respiratory scoring, arterial blood gases, and bronchoalveolar lavages (BALs) were collected at predetermined time points up to 24 h post nebulization. While the mean neutrophil percentage decreased in treated horses compared to controls (9.3 ± 2.0 and 16.9 ± 2.4, respectively) at 6 hours post treatment (t = 6 h), the difference did not achieve statistical significance (p = 0.1154). Blood gas analysis did not differ significantly between groups. There was a significant difference in the mean respiratory scores of G-CAPE-treated horses between baseline and at 1-h post treatment (from 3.2 ± 0.7 to 1.6 ± 0.7, p = 0.0013). This study demonstrates that a single nebulized dose of G-CAPE decreased clinical respiratory scores 1 h post administration and decreased BAL percentage of neutrophils 6 h post administration in horses with particulate induced airway inflammation. This compound shows promise as an anti-inflammatory and warrants further investigation.
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Continued emergence of SARS-CoV-2 variants highlights the critical need for adaptable and translational animal models for acute COVID-19. Limitations to current animal models for SARS CoV-2 (e.g., transgenic mice, non-human primates, ferrets) include subclinical to mild lower respiratory disease, divergence from clinical COVID-19 disease course, and/or the need for host genetic modifications to permit infection. We therefore established a feline model to study COVID-19 disease progression and utilized this model to evaluate infection kinetics and immunopathology of the rapidly circulating Delta variant (B.1.617.2) of SARS-CoV-2. In this study, specific-pathogen-free domestic cats (n = 24) were inoculated intranasally and/or intratracheally with SARS CoV-2 (B.1.617.2). Infected cats developed severe clinical respiratory disease and pulmonary lesions at 4- and 12-days post-infection (dpi), even at 1/10 the dose of previously studied wild-type SARS-CoV-2. Infectious virus was isolated from nasal secretions of delta-variant infected cats in high amounts at multiple timepoints, and viral antigen was co-localized in ACE2-expressing cells of the lungs (pneumocytes, vascular endothelium, peribronchial glandular epithelium) and strongly associated with severe pulmonary inflammation and vasculitis that were more pronounced than in wild-type SARS-CoV-2 infection. RNA sequencing of infected feline lung tissues identified upregulation of multiple gene pathways associated with cytokine receptor interactions, chemokine signaling, and viral protein-cytokine interactions during acute infection with SARS-CoV-2. Weighted correlation network analysis (WGCNA) of differentially expressed genes identified several distinct clusters of dysregulated hub genes that are significantly correlated with both clinical signs and lesions during acute infection. Collectively, the results of these studies help to delineate the role of domestic cats in disease transmission and response to variant emergence, establish a flexible translational model to develop strategies to prevent the spread of SARS-CoV-2, and identify potential targets for downstream therapeutic development.
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COVID-19 , SARS-CoV-2 , Animais , Gatos , Furões , Cinética , CamundongosRESUMO
During influenza A virus (IAV) infection, it is unclear whether type I interferons (IFNs) have defensive antiviral effects or contribute to immunopathology in smokers. We treated nonsmoking (NS) and cigarette smoke (CS)-exposed mice intranasally with early (prophylactic) or late (therapeutic) IFN-ß. We compared the mortality and innate immune responses of the treated mice following challenge with IAV. In NS mice, both early and late IFN-ß administration decreased the survival rate in mice infected with IAV, with late IFN-ß administration having the greatest effect on survival. In contrast, in CS-exposed mice, early IFN-ß administration significantly increased survival during IAV infection while late IFN-ß administration did not alter mortality. With regards to inflammation, in NS mice, IFN-ß administration, especially late administration, significantly increased IAV-induced inflammation and lung injury. Early IFN-ß administration to CS-exposed mice did not increase IAV-induced inflammation and lung injury as occurred in NS mice. Our results demonstrate, although IFN-ß administration worsens the susceptibility of NS mice to influenza infection with increased immunopathology, early IFN-ß administration to CS-exposed mice, which have suppression of the intrinsic IFN response, improved outcomes during influenza infection.
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Fumar Cigarros , Doenças Transmissíveis , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Lesão Pulmonar , Infecções por Orthomyxoviridae , Pneumonia , Animais , Doenças Transmissíveis/patologia , Humanos , Inflamação/patologia , Interferon beta , Pulmão/patologia , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/patologia , Pneumonia/prevenção & controle , NicotianaRESUMO
Acute infection of the ocular, oral, or nasal cavity by bovine herpesvirus 1 (BoHV-1) culminates in lifelong latency in sensory neurons within trigeminal ganglia. The BoHV-1 latency reactivation cycle, including calves latently infected with commercially available modified live vaccines, can lead to reproductive complications, including abortions. Recent studies demonstrated progesterone stimulated BoHV-1 productive infection and sporadically induced reactivation from latency in male rabbits. The progesterone receptor (PR) and progesterone transactivate the immediate early transcription unit 1 (IEtu1) promoter and the infected cell protein 0 (bICP0) early promoter. These viral promoters drive expression of two viral transcriptional regulatory proteins (bICP0 and bICP4) that are crucial for productive infection. Based on these observations, we hypothesize that progesterone induces reactivation in a subset of calves latently infected with BoHV-1. These studies demonstrated progesterone was less efficient than dexamethasone at initiating reactivation from latency in female calves. Notably, heat stress correlated with enhancing the ability of progesterone to induce reactivation from latency. Previous studies demonstrated that heat stress activates the glucocorticoid receptor (GR), which suggested GR activation augments progesterone-mediated reactivation from latency. Additional studies revealed GR and PR cooperatively stimulated productive infection and synergistically transactivated the IEtu1 promoter when cultures were treated with dexamethasone. Mutating one or both GR binding sites in the IEtu1 promoter blocked transactivation. Collectively, these studies indicated that progesterone intermittently triggered reactivation from latency, and heat stress augmented reactivation from reactivation. Finally, these studies suggest progesterone enhances virus spread in tissues and cells where PR is abundantly expressed. IMPORTANCE Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. The glucocorticoid receptor (GR) and dexamethasone stimulate key viral regulatory promoters and productive infection, in part because the viral genome contains numerous consensus GR-responsive elements (GREs). The progesterone receptor (PR) and GR belong to the type I nuclear hormone receptor family. PR and progesterone specifically bind to and transactivate viral promoters that contain GREs and stimulate BoHV-1 productive infection. Although progesterone did not induce reactivation from latency in female calves as efficiently as dexamethasone, heat stress enhanced progesterone-mediated reactivation from latency. Consequently, we predict that low levels of stressful stimuli can cooperate with progesterone to induce reactivation from latency or promote virus spread.
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Infecções por Herpesviridae , Herpesvirus Bovino 1 , Progesterona , Animais , Bovinos , Dexametasona/farmacologia , Feminino , Resposta ao Choque Térmico , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/fisiologia , Masculino , Progesterona/farmacologia , Coelhos , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.
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COVID-19 , Gatos , Modelos Animais de Doenças , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/patologia , COVID-19/fisiopatologia , COVID-19/virologia , Feminino , Genoma Viral , Humanos , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Linfonodos/virologia , Masculino , RNA Viral/análise , SARS-CoV-2/genética , Organismos Livres de Patógenos Específicos , Traqueia/enzimologia , Traqueia/virologia , Conchas Nasais/enzimologia , Conchas Nasais/virologiaRESUMO
Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.
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Proteínas Relacionadas à Autofagia/genética , Resfriado Comum/prevenção & controle , Coronavirus Humano OC43/fisiologia , Proteína DEAD-box 58/genética , Infecções por Rhabdoviridae/prevenção & controle , Vírus da Estomatite Vesicular Indiana/fisiologia , Células A549 , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Chlorocebus aethiops , Resfriado Comum/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/prevenção & controle , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Células RAW 264.7 , Infecções por Rhabdoviridae/metabolismo , Células Vero , Estomatite Vesicular/metabolismo , Estomatite Vesicular/prevenção & controleRESUMO
The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. This study validates a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with severe COVID-19 in humans. Intra-tracheal inoculation of concentrated SARS-CoV-2 caused infected cats to develop clinical disease consistent with that observed in the early exudative phase of COVID-19. A novel clinical scoring system for feline respiratory disease was developed and utilized, documenting a significant degree of lethargy, fever, dyspnea, and dry cough in infected cats. In addition, histopathologic pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were observed due to SARS-CoV-2 infection, imitating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation exists between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were quantified in nasal turbinates, distal trachea, lung, and various other organs. Natural ACE2 expression, paired with clinicopathologic correlates between this feline model and human COVID-19, encourage use of this model for future translational studies.
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The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. In total, 630 samples were used in this study. Individual positive samples with cycle threshold (CT ) values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5), and individual positive samples with CT values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.
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COVID-19 , Humanos , Nasofaringe , RNA Viral/genética , Transcrição Reversa , SARS-CoV-2 , Manejo de EspécimesRESUMO
Low-grade inflammation is a critical pathological factor contributing to the development of metabolic disorders. ß-carotene oxygenase 2 (BCO2) was initially identified as an enzyme catalyzing carotenoids in the inner mitochondrial membrane. Mutations in BCO2 are associated with inflammation and metabolic disorders in humans, yet the underlying mechanisms remain unknown. Here, we used loss-of-function approaches in mice and cell culture models to investigate the role of BCO2 in inflammation and metabolic dysfunction. We demonstrated decreases in BCO2 mRNA and protein levels and suppression of mitochondrial respiratory complex I proteins and mitochondrial superoxide dismutase levels in the liver of type 2 diabetic human subjects. Deficiency of BCO2 caused disruption of assembly of the mitochondrial respiratory supercomplexes, such as supercomplex III2+IV in mice, and overproduction of superoxide radicals in primary mouse embryonic fibroblasts. Further, deficiency of BCO2 increased protein carbonylation and populations of natural killer cells and M1 macrophages, and decreased populations of T cells, including CD4+ and/or CD8+ in the bone marrow and white adipose tissues. Elevation of plasma inflammatory cytokines and adipose tissue hypertrophy and inflammation were also characterized in BCO2 deficient mice. Moreover, BCO2 deficient mice were more susceptible to high-fat diet-induced obesity and hyperglycemia. Double knockout of BCO2 and leptin receptor genes caused a significantly greater elevation of the fasting blood glucose level in mice at 4 weeks of age, compared to the age- and sex-matched leptin receptor knockout. Finally, administration of Mito-TEMPO, a mitochondrial specific antioxidant attenuated systemic low-grade inflammation induced by BCO2 deficiency. Collectively, these findings suggest that BCO2 is essential for mitochondrial respiration and metabolic homeostasis in mammals. Loss or decreased expression of BCO2 leads to mitochondrial oxidative stress, low-grade inflammation, and the subsequent development of metabolic disorders.
Assuntos
Dioxigenases , beta Caroteno , Animais , Dioxigenases/metabolismo , Fibroblastos/metabolismo , Inflamação/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse OxidativoRESUMO
The onset of type 2 diabetes in obesity is associated with gut dysbiosis and a failure to confine commensal bacteria and toxins to the gut lumen while prebiotics may prevent these effects. This study evaluated the effects of pinto beans (PB) supplementation on cecal bacteria, short-chain fatty acids (SCFAs), distal ileal antigen presentation marker (major histocompatibility complex [MHC] II) and antimicrobial peptide genes during short-term high-fat, high sucrose (HFS) feeding. Six-week-old, male C57BL/6J mice were randomly assigned to four groups (n=12/group), and fed a control (C) or HFS diet with or without cooked PB (10%, wt/wt) for 30 days. Supplemental PB in both the C and HFS diets decreased the abundance of Tenericutes and the sulfate-reducing bacteria Bilophila. In contrast, PB raised the abundance of taxa within the SCFAs-producing family, Lachnospiraceae, compared to groups without PB. Consequently, fecal butyric acid was significantly higher in PB-supplemented groups compared to C and HFS groups. PB reversed the HFS-induced ablation of the distal ileal STAT3 phosphorylation, and up-regulated antimicrobial peptide genes (Reg3γ and Reg3ß). Furthermore, the expression of MHC II protein was elevated in the PB supplemented groups compared to C and HFS. Tenericutes and Bilophilia negatively correlated with activated STAT3 and MHC II proteins. Finally, supplemental PB improved fasting blood glucose, glucose tolerance and suppressed TNFα and inducible nitric oxide synthase mRNA in the visceral adipose tissue. Put together, the beneficial impact of PB supplementation on the gut may be central to its potential to protect against diet-induced inflammation and impaired glucose tolerance.
Assuntos
Disbiose/dietoterapia , Microbioma Gastrointestinal , Genes MHC da Classe II , Phaseolus , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Ceco/metabolismo , Dieta Ocidental , Suplementos Nutricionais , Disbiose/metabolismo , Ácidos Graxos Voláteis/metabolismo , Fezes/microbiologia , Expressão Gênica , Humanos , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMO
Rapid clearance of thrombolytics from blood following intravenous injection is a major clinical challenge in cardiovascular medicine. To overcome this barrier, nanoparticle (NP) based drug delivery systems have been reported. Although superior than conventional therapy, a large proportion of the injected NP is still cleared by the reticuloendothelial system. Previously, we and others showed that ex vivo attachment of bioscavengers, thrombolytics, and nanoparticles (NPs) to glycophorin A receptors on red blood cells (RBCs) improved the blood half-life. This is promising, but ex-vivo approaches are cumbersome and challenging to translate clinically. Here, we developed a novel Ter119-polymeric NP containing tissue plasminogen activator for on-demand targeting of GPA receptors in vivo. Upon intravenous injection, the Ter119-NPs achieved remarkable RBC labeling efficiencies (>95%), resulting in marked enhancement of blood residence time of tPA from minutes to several days without any morphological, hematological, and histological complications. Our approach of RBC labeling with the NPs also prevented reticuloendothelial detections and the activations of innate and adaptive immune system. Data suggest that real-time targeting of therapeutics to RBC with NPs can potentially improve outcomes and reduce complications against a variety chronic disease.
Assuntos
Nanopartículas , Ativador de Plasminogênio Tecidual , Sistemas de Liberação de Medicamentos , Eritrócitos , FibrinolíticosRESUMO
Diabetes has been identified as an independent risk factor for atrial fibrillation (AF), the most common chronic cardiac arrhythmia. Whether or not glucose and insulin disturbances observed during diabetes enhance arrhythmogenicity of the atria, potentially leading to AF, is not well-known. We hypothesized that insulin deficiency and impaired glucose transport provide a metabolic substrate for the development and maintenance of AF during diabetes. Transesophageal atrial pacing was used to induce AF in healthy, streptozotocin-induced insulin-deficient type 1 diabetic, and insulin-treated diabetic mice. Translocation of insulin-sensitive glucose transporters (GLUTs) to the atrial cell surface was measured using a biotinylated photolabeling assay in the perfused heart. Fibrosis and glycogen accumulation in the atrium were measured using histological analysis. Diabetic mice displayed mild hyperglycemia, increased duration and frequency of AF episodes vs. age-matched controls (e.g., AF duration: 19.7 ± 6.8 s vs. 1.8 ± 1.1 s, respectively, p = 0.032), whereas insulin-treated diabetic animals did not. The translocation of insulin-sensitive GLUT-4 and -8 to the atrial cell surface was significantly downregulated in the diabetic mice (by 67 and 79%, respectively; p ≤ 0.001), and rescued by insulin treatment. We did not observe fibrosis or glycogen accumulation in the atria of diabetic mice. Therefore, these data suggest that insulin and glucose disturbances were sufficient to induce AF susceptibility during mild diabetes.