RESUMO
Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Vírus da Influenza B , Influenza Humana , Neuraminidase , Neuraminidase/imunologia , Humanos , Vírus da Influenza B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Vacinas contra Influenza/imunologia , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Virais/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Compostos Férricos , Interações entre Hospedeiro e Microrganismos , Ferro , Organelas , Animais , Camundongos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/imunologia , Clostridioides difficile/metabolismo , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Ferro/metabolismo , Organelas/metabolismo , Homeostase , Compostos Férricos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Modelos Animais de Doenças , Complexo Antígeno L1 Leucocitário/metabolismo , Viabilidade Microbiana , Inflamação/metabolismo , Inflamação/microbiologia , Intestinos/metabolismo , Intestinos/microbiologiaRESUMO
Rationale: Type 2 inflammation has been described in people with cystic fibrosis (CF). Whether loss of CFTR (cystic fibrosis transmembrane conductance regulator) function contributes directly to a type 2 inflammatory response has not been fully defined. Objectives: The potent alarmin IL-33 has emerged as a critical regulator of type 2 inflammation. We tested the hypothesis that CFTR deficiency increases IL-33 expression and/or release and deletion of IL-33 reduces allergen-induced inflammation in the CF lung. Methods: Human airway epithelial cells (AECs) grown from non-CF and CF cell lines and Cftr+/+ and Cftr-/- mice were used in this study. Pulmonary inflammation in Cftr+/+ and Cftr-/- mice with and without IL-33 or ST2 (IL-1 receptor-like 1) germline deletion was determined by histological analysis, BAL, and cytokine analysis. Measurements and Main Results: After allergen challenge, both CF human AECs and Cftr-/- mice had increased IL-33 expression compared with control AECs and Cftr+/+ mice, respectively. DUOX1 (dual oxidase 1) expression was increased in CF human AECs and Cftr-/- mouse lungs compared with control AECs and lungs from Cftr+/+ mice and was necessary for the increased IL-33 release in Cftr-/- mice compared with Cftr+/+ mice. IL-33 stimulation of Cftr-/- CD4+ T cells resulted in increased type 2 cytokine production compared with Cftr+/+ CD4+ T cells. Deletion of IL-33 or ST2 decreased both type 2 inflammation and neutrophil recruitment in Cftr-/- mice compared with Cftr+/+ mice. Conclusions: Absence of CFTR reprograms airway epithelial IL-33 release and licenses IL-33-dependent inflammation. Modulation of the IL-33/ST2 axis represents a novel therapeutic target in CF type 2-high and neutrophilic inflammation.
Assuntos
Fibrose Cística , Camundongos , Animais , Humanos , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Interleucina-33/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Alérgenos , Células Epiteliais/metabolismoRESUMO
Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.
Assuntos
Encefalomielite Autoimune Experimental , Doenças dos Roedores , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/veterinária , Fixadores , Formaldeído , Imuno-Histoquímica , Camundongos , Perfusão/veterinária , Polímeros , Fixação de Tecidos/métodos , Fixação de Tecidos/veterináriaRESUMO
BACKGROUND: /Objectives: The pathogenesis of hyperglycemia during acute pancreatitis (AP) remains unknown due to inaccessibility of human tissues and lack of animal models. We aimed to develop an animal model to study the mechanisms of hyperglycemia and impaired glucose tolerance in AP. METHODS: We injected ferrets with intraperitoneal cerulein (50 µg/kg, 9 hourly injections) or saline. Blood samples were collected for glucose (0, 4, 8, 12, 24h); TNF-α, IL-6 (6h); amylase, lipase, insulin, glucagon, pancreatic polypeptide (PP), glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) (24h). Animals underwent oral glucose tolerance test (OGTT), mixed meal tolerance test (MMTT) at 24h or 3 months, followed by harvesting pancreas for histopathology and immunostaining. RESULTS: Cerulein-injected ferrets exhibited mild pancreatic edema, neutrophil infiltration, and elevations in serum amylase, lipase, TNF-α, IL-6, consistent with AP. Plasma glucose was significantly higher in ferrets with AP at all time points. Plasma glucagon, GLP-1 and PP were significantly higher in cerulein-injected animals, while plasma insulin was significantly lower compared to controls. OGTT and MMTT showed abnormal glycemic responses with higher area under the curve. The hypoglycemic response to insulin injection was completely lost, suggestive of insulin resistance. OGTT showed low plasma insulin; MMTT confirmed low insulin and GIP; abnormal OGTT and MMTT responses returned to normal 3 months after cerulein injection. CONCLUSIONS: Acute cerulein injection causes mild acute pancreatitis in ferrets and hyperglycemia related to transient islet cell dysfunction and insulin resistance. The ferret cerulein model may contribute to the understanding of hyperglycemia in acute pancreatitis.
Assuntos
Hiperglicemia , Resistência à Insulina , Pancreatite , Doença Aguda , Amilases , Animais , Glicemia , Ceruletídeo/toxicidade , Furões , Polipeptídeo Inibidor Gástrico , Glucagon , Peptídeo 1 Semelhante ao Glucagon , Humanos , Insulina , Interleucina-6 , Lipase , Pancreatite/induzido quimicamente , Pancreatite/veterinária , Fator de Necrose Tumoral alfaRESUMO
Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay's specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay's specificity and build confidence in a study's conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study's results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.
Assuntos
Anticorpos , Neoplasias , Animais , Imuno-Histoquímica , Neoplasias/veterinária , Sensibilidade e EspecificidadeRESUMO
Angiotensin II (ANG II) Agtr1a receptor (AT1A) is expressed in cells of the arcuate nucleus of the hypothalamus that express the leptin receptor (Lepr) and agouti-related peptide (Agrp). Agtr1a expression in these cells is required to stimulate resting energy expenditure in response to leptin and high-fat diets (HFDs), but the mechanism activating AT1A signaling by leptin remains unclear. To probe the role of local paracrine/autocrine ANG II generation and signaling in this mechanism, we bred mice harboring a conditional allele for angiotensinogen (Agt, encoding AGT) with mice expressing Cre-recombinase via the Lepr or Agrp promoters to cause cell-specific deletions of Agt (AgtLepr-KO and AgtAgrp-KO mice, respectively). AgtLepr-KO mice were phenotypically normal, arguing against a paracrine/autocrine AGT signaling mechanism for metabolic control. In contrast, AgtAgrp-KO mice exhibited reduced preweaning survival, and surviving adults exhibited altered renal structure and steroid flux, paralleling previous reports of animals with whole body Agt deficiency or Agt disruption in albumin (Alb)-expressing cells (thought to cause liver-specific disruption). Surprisingly, adult AgtAgrp-KO mice exhibited normal circulating AGT protein and hepatic Agt mRNA expression but reduced Agt mRNA expression in adrenal glands. Reanalysis of RNA-sequencing data sets describing transcriptomes of normal adrenal glands suggests that Agrp and Alb are both expressed in this tissue, and fluorescent reporter gene expression confirms Cre activity in adrenal gland of both Agrp-Cre and Alb-Cre mice. These findings lead to the iconoclastic conclusion that extrahepatic (i.e., adrenal) expression of Agt is critically required for normal renal development and survival.
Assuntos
Glândulas Suprarrenais/metabolismo , Proteína Relacionada com Agouti/metabolismo , Angiotensinogênio/metabolismo , Núcleo Arqueado do Hipotálamo/metabolismo , Metabolismo Energético , Rim/metabolismo , Receptores para Leptina/metabolismo , Glândulas Suprarrenais/crescimento & desenvolvimento , Proteína Relacionada com Agouti/deficiência , Proteína Relacionada com Agouti/genética , Angiotensinogênio/deficiência , Angiotensinogênio/genética , Animais , Núcleo Arqueado do Hipotálamo/crescimento & desenvolvimento , Comunicação Autócrina , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Masculino , Camundongos Knockout , Miocárdio/metabolismo , Comunicação Parácrina , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Albumina Sérica/genética , Albumina Sérica/metabolismo , Transdução de SinaisRESUMO
Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12-/- mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12-/- neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12-/- dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses.
Assuntos
Quimiocina CXCL1/metabolismo , Vírus da Influenza A/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Permeabilidade Capilar/genética , Quimiocina CXCL1/genética , Células Dendríticas/imunologia , Feminino , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
In cystic fibrosis (CF), there is early destruction of the exocrine pancreas, and this results in a unique form of diabetes that affects approximately half of adult CF individuals. An animal model of cystic fibrosis-related diabetes has been developed in the ferret, which progresses through phases of glycemic abnormalities because of islet remodeling during and after exocrine destruction. Herein, we quantified the pancreatic histopathological changes that occur during these phases. There was an increase in percentage ductal, fat, and islet area in CF ferrets over time compared with age-matched wild-type controls. We also quantified islet size, shape, islet cell composition, cell proliferation (Ki-67), and expression of remodeling markers (matrix metalloprotease-7, desmin, and α-smooth muscle actin). Pancreatic ducts were dilated with scattered proliferating cells and were surrounded by activated stellate cells, indicative of tissue remodeling. The timing of islet and duct proliferation, stellate cell activation, and matrix remodeling coincided with the previously published stages of glycemic crisis and inflammation. This mapping of remodeling events in the CF ferret pancreas provides insights into early changes that control glycemic intolerance and subsequent recovery during the evolution of CF pancreatic disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Furões/metabolismo , Técnicas de Inativação de Genes , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Tecido Adiposo/patologia , Envelhecimento/patologia , Animais , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Humanos , Hiperplasia , Antígeno Ki-67/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Modelos Biológicos , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Regulação para Cima/genéticaRESUMO
Melanocortin 1 receptor (MC1R) is under investigation as a target for drug delivery for metastatic melanoma therapy and imaging. The purpose of this study was to determine the potential of using BRAF inhibitors (BRAFi) and histone deacetylase inhibitors (HDACi) to enhance the delivery of MC1R-targeted radiolabeled peptide ([212Pb]DOTA-MC1L) by pharmacologically upregulating the MC1R expression in metastatic melanoma cells and tumors. MC1R expression was analyzed in de-identified melanoma biopsies by immunohistochemical staining. Upregulation of MC1R expression was determined in BRAFV600E cells (A2058) and BRAF wild-type melanoma cells (MEWO) by quantitative real-time polymerase chain reaction, flow cytometry, and receptor-ligand binding assays. The role of microphthalmia-associated transcription factor (MITF) in the upregulation of MC1R was also examined in A2058 and MEWO cells. The effectiveness of [212Pb]DOTA-MC1L α-particle radiotherapy in combination with BRAFi and/or HDACi was determined in athymic nu/nu mice bearing A2058 and MEWO human melanoma xenografts. High expression of MC1R was observed in situ in clinical melanoma biopsies. BRAFi and HDACi significantly increased the MC1R expression (up to 10-fold in mRNA and 4-fold in protein levels) via MITF-dependent pathways, and this increase led to enhanced ligand binding on the cell surface. Inhibition of MITF expression antagonized the upregulation of MC1R in both BRAFV600E and BRAFWT cells. Combining [212Pb]DOTA-MC1L with BRAFi and/or HDACi improved the tumor response by increasing the delivery of 212Pb α-particle emissions to melanoma tumors via augmented MC1R expression. These data suggest that FDA-approved HDACi and BRAFi could improve the effectiveness of MC1R-targeted therapies by enhancing drug delivery via upregulated MC1R.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Melanoma/tratamento farmacológico , Melanoma/radioterapia , Receptor Tipo 1 de Melanocortina/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/radioterapia , Regulação para Cima/efeitos dos fármacos , Partículas alfa/uso terapêutico , Animais , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imidazóis/farmacologia , Radioisótopos de Chumbo/química , Melanoma/patologia , Camundongos Nus , Fator de Transcrição Associado à Microftalmia , Oximas/farmacologia , Fenilbutiratos/farmacologia , Projetos Piloto , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Melanocortina/genética , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Infecções/microbiologia , Inflamação/microbiologia , Pneumopatias/microbiologia , Pulmão/microbiologia , Pulmão/fisiopatologia , Infecções Respiratórias/microbiologia , Animais , Modelos Animais de Doenças , Furões/microbiologia , Infecções/fisiopatologia , Inflamação/fisiopatologia , Pneumopatias/fisiopatologia , Infecções Respiratórias/fisiopatologiaRESUMO
G protein-coupled receptor signaling mechanisms are implicated in many aspects of cardiovascular control, and dysfunction of such signaling mechanisms is commonly associated with disease states. Investigators have identified a large number of regulator of G protein signaling (RGS) proteins that variously contribute to the modulation of intracellular second-messenger signaling kinetics. These many RGS proteins each interact with a specific set of second-messenger cascades and receptor types and exhibit tissue-specific expression patterns. Increasing evidence supports the contribution of RGS proteins, or their loss, in the pathogenesis of cardiovascular dysfunctions. This review summarizes the current understanding of the functional contributions of RGS proteins, particularly within the B/R4 family, in cardiovascular disorders of pregnancy including gestational hypertension, uterine artery dysfunction, and preeclampsia.
Assuntos
Fenômenos Fisiológicos Cardiovasculares/genética , Polimorfismo de Nucleotídeo Único , Proteínas RGS/genética , Transdução de Sinais/genética , Animais , Feminino , Humanos , Gravidez , Ligação Proteica , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR-/-) mice lack the ability to detect trimannose. When MR-/- mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.
Assuntos
Leishmania major , Leishmaniose Cutânea/patologia , Manose/análogos & derivados , Animais , Feminino , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Manose/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microesferas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing the lungs to chronic infection and inflammation. In young infants with CF, structural airway defects are increasingly recognized before the onset of significant lung disease, which suggests a developmental origin and a possible role in lung disease pathogenesis. The role(s) of CFTR in lung development is unclear and developmental studies in humans with CF are not feasible. Young CF pigs have structural airway changes and develop spontaneous postnatal lung disease similar to humans; therefore, we studied lung development in the pig model (non-CF and CF). CF trachea and proximal airways had structural lesions detectable as early as pseudoglandular development. At this early developmental stage, budding CF airways had smaller, hypo-distended lumens compared to non-CF airways. Non-CF lung explants exhibited airway lumen distension in response to forskolin/IBMX as well as to fibroblast growth factor (FGF)-10, consistent with CFTR-dependent anion transport/secretion, but this was lacking in CF airways. We studied primary pig airway epithelial cell cultures and found that FGF10 increased cellular proliferation (non-CF and CF) and CFTR expression/function (in non-CF only). In pseudoglandular stage lung tissue, CFTR protein was exclusively localized to the leading edges of budding airways in non-CF (but not CF) lungs. This discreet microanatomic localization of CFTR is consistent with the site, during branching morphogenesis, where airway epithelia are responsive to FGF10 regulation. In summary, our results suggest that the CF proximal airway defects originate during branching morphogenesis and that the lack of CFTR-dependent anion transport/liquid secretion likely contributes to these hypo-distended airways.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Pulmão/embriologia , Animais , Células Cultivadas , AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Feminino , Fator 10 de Crescimento de Fibroblastos/fisiologia , Humanos , Morfogênese , Suínos , Traqueia/anormalidadesRESUMO
The role of TRAF2 and TRAF5 in TNFα-induced NF-κB activation has become complicated owing to the accumulation of conflicting data. Here, we report that 7-day-old TRAF2-knockout (KO) and TRAF2 TRAF5 double KO (TRAF2/5-DKO) mice exhibit enhanced canonical IκB kinase (IKK) and caspase-8 activation in spleen and liver, and that subsequent knockout of TNFα suppresses the basal activity of caspase-8, but not of IKK. In primary TRAF2 KO and TRAF2/5-DKO cells, TNFα-induced immediate IKK activation is impaired, whereas delayed IKK activation occurs normally; as such, owing to elevated basal and TNFα-induced delayed IKK activation, TNFα stimulation leads to significantly increased induction of a subset of NF-κB-dependent genes in these cells. In line with this, both TRAF2 KO and TRAF2/5-DKO mice succumb to a sublethal dose of TNFα owing to increased expression of NF-κB target genes, diarrhea and bradypnea. Notably, depletion of IAP1 and IAP2 (also known as BIRC2 and BIRC3, respectively) also results in elevated basal IKK activation that is independent of autocrine TNFα production and that impairs TNFα-induced immediate IKK activation. These data reveal that TRAF2, IAP1 and IAP2, but not TRAF5, cooperatively regulate basal and TNFα-induced immediate IKK activation.
Assuntos
Caspase 8/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/genética , Proteína 3 com Repetições IAP de Baculovírus , Células Cultivadas , Quimiocina CCL5/metabolismo , Ativação Enzimática/genética , Quinase I-kappa B/genética , Proteínas Inibidoras de Apoptose/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/genética , Fator de Necrose Tumoral alfa/genética , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
Hypertensive disease of pregnancy (HDP) with placental insufficiency is the most common cause of fetal growth restriction (FGR) in the developed world. Despite the known negative consequences of HDP both to the mother and fetus, little is known about the longitudinal placental changes that occur as HDP progresses in pregnancy. This is because longitudinal sampling of human placentae during each gestation is impossible. Therefore, using a mouse model of thromboxane A2-analog infusion to mimic human HDP in the last trimester, we calculated placental efficiencies based on fetal and placental weights; quantified spongiotrophoblast and labyrinth thicknesses and vascular density within these layers; examined whether hypoxia signaling pathway involving vascular endothelial growth factor A (VEGFA) and its receptors (VEGFR1, VEGFR2) and matrix metalloproteinases (MMPs) contributed to vascular change; and examined nutrient transporter abundance including glucose transporters 1 and 3 (GLUT1, GLUT3), neutral amino acid transporters 1, 2, and 4 (SNAT1, SNAT2, and SNAT4), fatty acid transporters 2 and 4 (FATP2, FATP4), and fatty acid translocase (CD36) from embryonic day 15.5 to 19 in a 20-day C57Bl/6J mouse gestation. We conclude that early-to-mid gestation hypertensive placentae show compensatory mechanisms to preserve fetal growth by increasing placental efficiencies and maintaining abundance of important nutrient transporters. As placental vascular network diminishes over late hypertension, placental efficiency diminishes and fetal growth fails. Neither hypoxia signaling pathway nor MMPs mediated the vascular diminution in this model. Hypertensive placentae surprisingly exhibit a sex-differential expression of nutrient transporters in late gestation despite showing fetal growth failure in both sexes.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Transporte de Ácido Graxo/metabolismo , Retardo do Crescimento Fetal/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Placenta/efeitos dos fármacos , Placentação/efeitos dos fármacos , Tromboxano A2/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Metaloproteinases da Matriz/metabolismo , Camundongos , Placenta/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiotensin II (ANG) stimulates the release of arginine vasopressin (AVP) from the neurohypophysis through activation of the AT1 receptor within the brain, although it remains unclear whether AT1 receptors expressed on AVP-expressing neurons directly mediate this control. We explored the hypothesis that ANG acts through AT1A receptors expressed directly on AVP-producing cells to regulate AVP secretion. In situ hybridization and transgenic mice demonstrated localization of AVP and AT1A mRNA in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN), but coexpression of both AVP and AT1A mRNA was only observed in the SON. Mice harboring a conditional allele for the gene encoding the AT1A receptor (AT1Aflox) were then crossed with AVP-Cre mice to generate mice that lack AT1A in all cells that express the AVP gene (AT1AAVP-KO). AT1AAVP-KO mice exhibited spontaneously increased plasma and serum osmolality but no changes in fluid or salt-intake behaviors, hematocrit, or total body water. AT1AAVP-KO mice exhibited reduced AVP secretion (estimated by measurement of copeptin) in response to osmotic stimuli such as acute hypertonic saline loading and in response to chronic intracerebroventricular ANG infusion. However, the effects of these receptors on AVP release were masked by complex stimuli such as overnight dehydration and DOCA-salt treatment, which simultaneously induce osmotic, volemic, and pressor stresses. Collectively, these data support the expression of AT1A in AVP-producing cells of the SON but not the PVN, and a role for AT1A receptors in these cells in the osmotic regulation of AVP secretion.
Assuntos
Receptor Tipo 1 de Angiotensina/fisiologia , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/fisiologia , Vasopressinas/biossíntese , Vasopressinas/fisiologia , Angiotensina II/administração & dosagem , Angiotensina II/farmacologia , Animais , Água Corporal , Comportamento Alimentar , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osmose , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/genética , Sódio na Dieta , Vasoconstritores/administração & dosagem , Vasoconstritores/farmacologiaRESUMO
The pathogenesis of preeclampsia (PreE), a hypertensive disorder of pregnancy, involves imbalanced T helper (TH) cell populations and resultant changes in pro- and anti-inflammatory cytokine release. Elevated copeptin (an inert biomarker of arginine vasopressin (AVP)), secretion precedes the development of symptoms in PreE in humans, and infusion of AVP proximal to and throughout gestation is sufficient to initiate cardiovascular and renal phenotypes of PreE in wild-type C57BL/6J mice. We hypothesize that AVP infusion in wild-type mice is sufficient to induce the immune changes observed in human PreE. AVP infusion throughout gestation in mice resulted in increased pro-inflammatory interferon γ (IFNg) (TH1) in the maternal plasma. The TH17-associated cytokine interleukin (IL)-17 was elevated in the maternal plasma, amniotic fluid, and placenta following AVP infusion. Conversely, the TH2-associated anti-inflammatory cytokine IL-4 was decreased in the maternal and fetal kidneys from AVP-infused dams, while IL-10 was decreased in the maternal kidney and all fetal tissues. Collectively, these results demonstrate the sufficiency of AVP to induce the immune changes typical of PreE. We investigated if T cells can respond directly to AVP by evaluating the expression of AVP receptors (AVPRs) on mouse and human CD4+ T cells. Mouse and human T cells expressed AVPR1a, AVPR1b, and AVPR2. The expression of AVPR1a was decreased in CD4+ T cells obtained from PreE-affected women. In total, our data are consistent with a potential initiating role for AVP in the immune dysfunction typical of PreE and identifies putative signaling mechanism(s) for future investigation.
Assuntos
Arginina Vasopressina/metabolismo , Pré-Eclâmpsia/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Arginina Vasopressina/farmacologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neurofisinas/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Pré-Eclâmpsia/induzido quimicamente , Gravidez , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismoRESUMO
Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.
Assuntos
Estudos Observacionais como Assunto/veterinária , Patologia Veterinária/métodos , Animais , Viés , Imuno-Histoquímica/métodos , Imuno-Histoquímica/normas , Imuno-Histoquímica/veterinária , Microscopia/veterinária , Estudos Observacionais como Assunto/métodos , Estudos Observacionais como Assunto/normas , Patologia Veterinária/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/veterinária , Reprodutibilidade dos TestesRESUMO
Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.