RESUMEN
Smallpox was the most rampant infectious disease killer of the 20th century, yet much remains unknown about the pathogenesis of the variola virus. Using archived tissue from a study conducted at the Centers for Disease Control and Prevention we characterized pathology in 18 cynomolgus macaques intravenously infected with the Harper strain of variola virus. Six macaques were placebo-treated controls, six were tecovirimat-treated beginning at 2 days post-infection, and six were tecovirimat-treated beginning at 4 days post-infection. All macaques were treated daily until day 17. Archived tissues were interrogated using immunohistochemistry, in situ hybridization, immunofluorescence, and electron microscopy. Gross lesions in three placebo-treated animals that succumbed to infection primarily consisted of cutaneous vesicles, pustules, or crusts with lymphadenopathy. The only gross lesions noted at the conclusion of the study in the three surviving placebo-treated and the Day 4 treated animals consisted of resolving cutaneous pox lesions. No gross lesions attributable to poxviral infection were present in the Day 2 treated macaques. Histologic lesions in three placebo-treated macaques that succumbed to infection consisted of proliferative and necrotizing dermatitis with intracytoplasmic inclusion bodies and lymphoid depletion. The only notable histologic lesion in the Day 4 treated macaques was resolving dermatitis; no notable lesions were seen in the Day 2 treated macaques. Variola virus was detected in all three placebo-treated animals that succumbed to infection prior to the study's conclusion by all utilized methods (IHC, ISH, IFA, EM). None of the three placebo-treated animals that survived to the end of the study nor the animals in the two tecovirimat treatment groups showed evidence of variola virus by these methods. Our findings further characterize variola lesions in the macaque model and describe new molecular methods for variola detection.
Asunto(s)
Dermatitis , Viruela , Virus de la Viruela , Animales , Benzamidas , Isoindoles , Macaca fascicularis , Viruela/tratamiento farmacológico , Viruela/patología , Estados UnidosRESUMEN
Type IV pili are involved in adhesion, twitching motility, aggregation, biofilm formation and virulence in a variety of Gram-negative bacteria. Burkholderia pseudomallei, the causative agent of melioidosis and a Tier 1 biological select agent, is a Gram-negative bacterium with eight type IV pili-associated loci (TFP1 to TFP8). Most have not been fully characterized. In this study, we investigated BPSS2185, an uncharacterized TFP8 gene that encodes a type IVB pilus protein subunit. Using genetic deletion and complementation analysis in B. pseudomallei JW270, we demonstrate that BPSS2185 plays an important role in twitching motility and adhesion to A549 human alveolar epithelial cells. Compared to JW270, the JW270 ΔBPSS2185 mutant failed to display twitching motility and did not adhere to the epithelial cells. These phenotypes were partially reversed by the complementation of BPSS2185 in the mutant strain. The study also shows that BPSS2185 is expressed only during the onset of mature biofilm formation and at the dispersal of a biofilm, suggesting that the motility characteristic is required to form a biofilm. Our study is the first to suggest that the BPSS2185 gene in TFP8 contributes to twitching motility, adhesion and biofilm formation, indicating that the gene may contribute to B. pseudomallei virulence.
Asunto(s)
Burkholderia pseudomallei , Proteínas Fimbrias , Biopelículas , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Células Epiteliales/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismoRESUMEN
BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (nâ =â 7), intravenous fluids plus levofloxacin (nâ =â 2), or a control group (nâ =â 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; Pâ =â .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.
Asunto(s)
Cuidados Críticos , Fiebre Hemorrágica Ebola , Unidades de Cuidados Intensivos , Animales , Modelos Animales de Enfermedad , Ebolavirus , Fiebre Hemorrágica Ebola/terapia , Macaca mulatta , Primates , Estudios RetrospectivosRESUMEN
BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.
Asunto(s)
Duodeno/metabolismo , Edición Génica , Mucosa Intestinal/metabolismo , Síndromes de Malabsorción/genética , Microvellosidades/patología , Mucolipidosis/genética , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Transportador 1 de Sodio-Glucosa/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Técnicas de Cocultivo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Duodeno/patología , Predisposición Genética a la Enfermedad , Humanos , Mucosa Intestinal/patología , Síndromes de Malabsorción/metabolismo , Síndromes de Malabsorción/patología , Microvellosidades/genética , Microvellosidades/metabolismo , Mucolipidosis/metabolismo , Mucolipidosis/patología , Mutación Missense , Fenotipo , Sodio/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Intercambiador 3 de Sodio-Hidrógeno/genética , Sus scrofaRESUMEN
Animal models recapitulating human COVID-19 disease, especially severe disease, are urgently needed to understand pathogenesis and to evaluate candidate vaccines and therapeutics. Here, we develop novel severe-disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than those in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and was uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2 but also play an early role in protection from acute disease.IMPORTANCE Syrian hamsters are in use as a model of disease caused by SARS-CoV-2. Pathology is pronounced in the upper and lower respiratory tract, and disease signs and endpoints include weight loss and viral RNA and/or infectious virus in swabs and organs (e.g., lungs). However, a high dose of virus is needed to produce disease, and the disease resolves rapidly. Here, we demonstrate that immunosuppressed hamsters are susceptible to low doses of virus and develop more severe and prolonged disease. We demonstrate the efficacy of a novel neutralizing monoclonal antibody using the cyclophosphamide transient suppression model. Furthermore, we demonstrate that RAG2 knockout hamsters develop severe/fatal disease when exposed to SARS-CoV-2. These immunosuppressed hamster models provide researchers with new tools for evaluating therapies and vaccines and understanding COVID-19 pathogenesis.
Asunto(s)
Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Modelos Animales de Enfermedad , Mesocricetus , Neumonía Viral/inmunología , Neumonía Viral/patología , Inmunidad Adaptativa , Animales , Animales Modificados Genéticamente , Betacoronavirus/fisiología , COVID-19 , Ciclofosfamida , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Inmunosupresores , Pandemias , SARS-CoV-2 , Índice de Severidad de la EnfermedadRESUMEN
Deficiency in diacylglycerol acyltransferase (DGAT1) is a rare cause of neonatal diarrhea, without a known mechanism or in vitro model. A patient presenting at our institution at 7 weeks of life with failure to thrive and diarrhea was found by whole-exome sequencing to have a homozygous DGAT1 truncation mutation. Duodenal biopsies showed loss of DGAT1 and deficits in apical membrane transporters and junctional proteins in enterocytes. When placed on a very low-fat diet, the patient's diarrhea resolved with normalization of brush border transporter localization in endoscopic biopsies. DGAT1 knockdown in Caco2-BBe cells modeled the deficits in apical trafficking, with loss of apical DPPIV and junctional occludin. Elevation in cellular lipid levels, including diacylglycerol (DAG) and phospholipid metabolites of DAG, was documented by lipid analysis in DGAT1 knockdown cells. Culture of the DGAT1 knockdown cells in lipid-depleted media led to re-establishment of occludin and return of apical DPPIV. DGAT1 loss appears to elicit global changes in enterocyte polarized trafficking that could account for deficits in absorption seen in the patient. The in vitro modeling of this disease should allow for investigation of possible therapeutic targets.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Diarrea Infantil/genética , Enfermedades del Sistema Digestivo/genética , Células CACO-2 , Preescolar , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Diarrea Infantil/patología , Enfermedades del Sistema Digestivo/patología , Humanos , Lactante , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Transporte de ProteínasRESUMEN
Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50â¯=â¯603â¯CFU) as compared to mice challenged with the parent (LD50â¯=â¯1â¯CFU) or complemented strain (LD50â¯=â¯1â¯CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.
Asunto(s)
Francisella tularensis/enzimología , Francisella tularensis/patogenicidad , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , Tularemia/inmunología , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas/inmunología , Línea Celular , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Francisella tularensis/genética , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Proteínas de Unión a las Penicilinas , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , Tularemia/microbiología , Vacunas Atenuadas/inmunología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Centrosoma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/genética , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Centrosoma/ultraestructura , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Humanos , Imagenología Tridimensional , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Centro Organizador de los Microtúbulos/ultraestructura , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteómica/métodos , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.
Asunto(s)
Enterocitos/ultraestructura , Microvellosidades/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Células CACO-2 , Polaridad Celular , Endosomas/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Transporte de ProteínasRESUMEN
Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.
Asunto(s)
Betacelulina , Polaridad Celular , Secuencia de Aminoácidos , Animales , Betacelulina/genética , Betacelulina/metabolismo , Perros , Familia de Proteínas EGF , Receptores ErbB/genética , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Células de Riñón Canino Madin Darby , Mutación , Señales de Clasificación de Proteína/genética , Estructura Terciaria de ProteínaRESUMEN
Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.
Asunto(s)
Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Células Parietales Gástricas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Atrofia/metabolismo , Atrofia/patología , Quinasas Similares a Doblecortina , Mucosa Gástrica/patología , Metaplasia , Ratones , Células Parietales Gástricas/patología , Proteínas Serina-Treonina Quinasas/genética , Estómago/patologíaRESUMEN
The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , VIH-1/metabolismo , Proteínas de la Membrana/metabolismo , Ensamble de Virus , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/virología , Células Cultivadas , Infecciones por VIH/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Transporte de Proteínas , Interferencia de ARN , Replicación Viral , Proteínas de Unión al GTP rab/genéticaRESUMEN
Autophagy plays a critical role in multiple aspects of the immune system, including the development and function of T lymphocytes. In mammalian cells, the class III PI3K vacuolar protein sorting (Vps)34 is thought to play a critical role in autophagy. However, recent studies have cast doubt on the role of Vps34 in autophagy, at least in certain cell types. To study the effects of Vps34 on autophagy in T lymphocytes, we generated mice that selectively lack Vps34 in the T cell lineage. Vps34 ablation in T cells caused profound defects in autophagic flux, resulting in accumulation of cellular organelles and apoptosis. These animals exhibited normal intrathymic development of conventional T cells, but they were profoundly impaired in the intrathymic development of invariant NKT cells. In peripheral organs, T cell-specific ablation of Vps34 had a profound impact on T cell homeostasis and function. Furthermore, aged animals developed an inflammatory wasting syndrome characterized by weight loss, intestinal inflammation, and anemia. Consistent with this phenotype, Vps34 was required for the peripheral maintenance and function of CD4(+)Foxp3(+) regulatory T cells. Collectively, our study reveals a critical role for Vps34 in autophagy and for the peripheral homeostasis and function of T lymphocytes.
Asunto(s)
Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Linfocitos T Reguladores/inmunología , Síndrome Debilitante/genética , Síndrome Debilitante/inmunología , Traslado Adoptivo , Envejecimiento , Animales , Apoptosis/genética , Colitis/inmunología , Factores de Transcripción Forkhead/metabolismo , Inflamación , Interleucina-2/biosíntesis , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.
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Mpox , Orthopoxvirus , Viruela , Virus de la Viruela , Animales , Ratones , Humanos , Nucleósidos/uso terapéutico , Viruela/tratamiento farmacológico , Viruela/prevención & control , Modelos Animales de EnfermedadRESUMEN
The role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca(2+)-dependent membrane-binding protein Annexin B9 (AnxB9), apical ß(Heavy)-spectrin (ß(H)) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of ß(H) spliceoforms, and loss of AnxB9 results in an increase in basolateral ß(H) and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when ß(H)-positive endosomes are generated either by upregulation of ß(H) in pak mutants or through the expression of the dominant-negative version of ß(H). In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or ß(H). Loss of AnxB9 or ß(H) function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes ß(H)) mutants. Reduction of AnxB9 results in degradation of the apical-lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to ß(H). These results indicate that AnxB9 and ß(H) are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.
Asunto(s)
Anexinas/metabolismo , Proteínas de Drosophila/metabolismo , Espectrina/metabolismo , Animales , Anexinas/deficiencia , Anexinas/genética , Cadherinas/metabolismo , Drosophila , Proteínas de Drosophila/genética , Endosomas/genética , Endosomas/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Membranas/metabolismo , Cuerpos Multivesiculares , Transducción de Señal , Espectrina/genética , Ubiquitinas/metabolismoRESUMEN
Human monkeypox (mpox) has recently become a global public health emergency; however, assays that detect mpox infection are not widely available, largely due to cross-reactivity within the Orthopoxvirus genus. Immunoassay development was largely confined to researchers who focus on biothreats and endemic areas (Central and West Africa) until the 2022 outbreak. As was noted in the COVID-19 pandemic, antigen detection assays, integrated with molecular assays, are necessary to help curb the spread of disease. Antigen-detecting immunoassays offer the advantage of providing results ranging from within min to h and in lateral flow formats; they can be deployed for point-of-care, home, or field use. This study reports the development of an mpox-specific antigen detection immunoassay developed on a multiplexed, magnetic-bead-based platform utilizing reagents from all research sectors (commercial, academic, and governmental). Two semi-quantitative assays were developed in parallel and standardized with infectious mpox virus (MPXV) cell culture fluid and MPXV-positive non-human primate (NHP) sera samples. These assays could detect viral antigens in serum, were highly specific toward MPXV as compared to other infectious orthopoxviruses (vaccinia virus, cowpox virus, and camelpox virus), and exhibited a correlation with quantitative PCR results from an NHP study. Access to a toolbox of assays for mpox detection will be key for identifying cases and ensuring proper treatment, as MPXV is currently a global traveler.
RESUMEN
Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61-68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.
Asunto(s)
Virus de la Encefalitis Equina del Este , Encefalomielitis Equina , Aerosoles , Animales , Encéfalo , Modelos Animales de Enfermedad , Encefalomielitis Equina/patología , Caballos , Macaca fascicularis , ARN Viral , Médula Espinal/patologíaRESUMEN
The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health emergency. While most human disease is mild to moderate, some infections lead to a severe disease characterized by acute respiratory distress, hypoxia, anosmia, ageusia, and, in some instances, neurological involvement. Small-animal models reproducing severe disease, including neurological sequela, are needed to characterize the pathophysiological mechanism(s) of disease and to identify medical countermeasures. Transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the K18 promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge when high viral doses are used. Here, we report on SARS-CoV-2 infection of hamsters engineered to express the hACE2 receptor under the control of the K18 promoter. K18-hACE2 hamsters infected with a relatively low dose of 100 or 1,000 PFU of SARS-CoV-2 developed a severe and lethal disease, with most animals succumbing by day 5 postinfection. Hamsters developed severe lesions and inflammation within the upper and lower respiratory system, including infection of the nasal cavities causing marked destruction of the olfactory epithelium as well as severe bronchopneumonia that extended deep into the alveoli. Additionally, SARS-CoV-2 infection spread to the central nervous system (CNS), including the brain stem and spinal cord. Wild-type (WT) hamsters naturally support SARS-CoV-2 infection, with the primary lesions present in the respiratory tract and nasal cavity. Overall, infection in the K18-hACE2 hamsters is more extensive than that in WT hamsters, with more CNS involvement and a lethal outcome. These findings demonstrate the K18-hACE2 hamster model will be valuable for studying SARS-CoV-2. IMPORTANCE The rapid emergence of SARS-CoV-2 has created a global health emergency. While most human SARS-CoV-2 disease is mild, some people develop severe, life-threatening disease. Small-animal models mimicking the severe aspects of human disease are needed to more clearly understand the pathophysiological processes driving this progression. Here, we studied SARS-CoV-2 infection in hamsters engineered to express the human angiotensin-converting enzyme 2 viral receptor under the control of the K18 promoter. SARS-CoV-2 produces a severe and lethal infection in transgenic hamsters that mirrors the most severe aspects of COVID-19 in humans, including respiratory and neurological injury. In contrast to other animal systems, hamsters manifest disease with levels of input virus more consistent with natural human infection. This system will be useful for the study of SARS-CoV-2 disease and the development of drugs targeting this virus.
Asunto(s)
COVID-19 , SARS-CoV-2 , Ratones , Animales , Cricetinae , Humanos , COVID-19/patología , Enzima Convertidora de Angiotensina 2 , Peptidil-Dipeptidasa A , Pulmón/patología , Ratones Transgénicos , Modelos Animales de EnfermedadRESUMEN
Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.
Asunto(s)
Nacimiento Prematuro , Streptococcus agalactiae , Animales , Citocinas , Femenino , Humanos , Recién Nacido , Recuento de Leucocitos , Macrófagos/microbiología , Metales , Ratones , Embarazo , Nacimiento Prematuro/microbiología , Streptococcus agalactiae/genéticaRESUMEN
Effective therapeutics have been developed against acute Ebola virus disease (EVD) in both humans and experimentally infected nonhuman primates. However, the risk of viral persistence and associated disease recrudescence in survivors receiving these therapeutics remains unclear. In contrast to rhesus macaques that survived Ebola virus (EBOV) exposure in the absence of treatment, we discovered that EBOV, despite being cleared from all other organs, persisted in the brain ventricular system of rhesus macaque survivors that had received monoclonal antibody (mAb) treatment. In mAb-treated macaque survivors, EBOV persisted in macrophages infiltrating the brain ventricular system, including the choroid plexuses. This macrophage infiltration was accompanied by severe tissue damage, including ventriculitis, choroid plexitis, and meningoencephalitis. Specifically, choroid plexus endothelium-derived EBOV infection led to viral persistence in the macaque brain ventricular system. This resulted in apoptosis of ependymal cells, which constitute the blood-cerebrospinal fluid barrier of the choroid plexuses. Fatal brain-confined recrudescence of EBOV infection manifested as severe inflammation, local pathology, and widespread infection of the ventricular system and adjacent neuropil in some of the mAb-treated macaque survivors. This study highlights organ-specific EBOV persistence and fatal recrudescent disease in rhesus macaque survivors after therapeutic treatment and has implications for the long-term follow-up of human survivors of EVD.