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1.
PLoS Pathog ; 20(3): e1012128, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38547254

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to suppress the type I interferon (IFNs-α/ß) response during infection. PRRSV also activates the NF-κB signaling pathway, leading to the production of proinflammatory cytokines during infection. In swine farms, co-infections of PRRSV and other secondary bacterial pathogens are common and exacerbate the production of proinflammatory cytokines, contributing to the porcine respiratory disease complex (PRDC) which is clinically a severe disease. Previous studies identified the non-structural protein 1ß (nsp1ß) of PRRSV-2 as an IFN antagonist and the nucleocapsid (N) protein as the NF-κB activator. Further studies showed the leucine at position 126 (L126) of nsp1ß as the essential residue for IFN suppression and the region spanning the nuclear localization signal (NLS) of N as the NF-κB activation domain. In the present study, we generated a double-mutant PRRSV-2 that contained the L126A mutation in the nsp1ß gene and the NLS mutation (ΔNLS) in the N gene using reverse genetics. The immunological phenotype of this mutant PRRSV-2 was examined in porcine alveolar macrophages (PAMs) in vitro and in young pigs in vivo. In PAMs, the double-mutant virus did not suppress IFN-ß expression but decreased the NF-κB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV-2. Co-infection of PAMs with the mutant PRRSV-2 and Streptococcus suis (S. suis) also reduced the production of NF-κB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV-2 and S. suis. The double-mutant PRRSV-2 was clinically attenuated, and the expressions of proinflammatory cytokines and chemokines were significantly reduced in pigs after bacterial co-infection. Compared to the wild-type PRRSV-2 and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV-2 exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-κB activation functions of PRRSV-2 may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS-2 and PRDC during bacterial co-infection.


Assuntos
Coinfecção , Interferon Tipo I , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Suínos , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Citocinas/genética , Citocinas/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Macrófagos Alveolares/metabolismo , Interferon Tipo I/metabolismo , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo
2.
Hepatology ; 80(3): 566-577, 2024 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-38373106

RESUMO

BACKGROUND AND AIMS: Sustained inflammation and hepatocyte injury in chronic liver disease activate HSCs to transdifferentiate into fibrogenic, contractile myofibroblasts. We investigated the role of protocadherin 7 (PCDH7), a cadherin family member not previously characterized in the liver, whose expression is restricted to HSCs. APPROACH AND RESULTS: We created a PCDH7 fl/fl mouse line, which was crossed to lecithin retinol acyltransferase-Cre mice to generate HSC-specific PCDH7 knockout animals. HSC contraction in vivo was tested in response to the HSC-selective vasoconstrictor endothelin-1 using intravital multiphoton microscopy. To establish a PCDH7 null HSC line, cells were isolated from PCDH7 fl/fl mice and infected with adenovirus-expressing Cre. Hepatic expression of PCDH7 was strictly restricted to HSCs. Knockout of PCDH7 in vivo abrogated HSC-mediated sinusoidal contraction in response to endothelin-1. In cultured HSCs, loss of PCDH7 markedly attenuated contractility within collagen gels and led to altered gene expression in pathways governing adhesion and vasoregulation. Loss of contractility in PCDH7 knockout cells was impaired Rho-GTPase signaling, as demonstrated by altered gene expression, reduced assembly of F-actin fibers, and loss of focal adhesions. CONCLUSIONS: The stellate cell-specific cadherin, PCDH7, is a novel regulator of HSC contractility whose loss leads to cytoskeletal remodeling and sinusoidal relaxation.


Assuntos
Caderinas , Células Estreladas do Fígado , Camundongos Knockout , Animais , Camundongos , Caderinas/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Protocaderinas , Endotelina-1/metabolismo , Células Cultivadas
3.
Hepatology ; 78(3): 820-834, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36988383

RESUMO

BACKGROUND AND AIMS: The scarcity of suitable donor livers highlights a continuing need for innovation to recover organs with reversible injuries in liver transplantation. APPROACH AND RESULTS: Explanted human donor livers (n = 5) declined for transplantation were supported using xenogeneic cross-circulation of whole blood between livers and xeno-support swine. Livers and swine were assessed over 24 hours of xeno-support. Livers maintained normal global appearance, uniform perfusion, and preservation of histologic and subcellular architecture. Oxygen consumption increased by 75% ( p = 0.16). Lactate clearance increased from -0.4 ± 15.5% to 31.4 ± 19.0% ( p = 0.02). Blinded histopathologic assessment demonstrated improved injury scores at 24 hours compared with 12 hours. Vascular integrity and vasoconstrictive function were preserved. Bile volume and cholangiocellular viability markers improved for all livers. Biliary structural integrity was maintained. CONCLUSIONS: Xenogeneic cross-circulation provided multisystem physiological regulation of ex vivo human livers that enabled functional rehabilitation, histopathologic recovery, and improvement of viability markers. We envision xenogeneic cross-circulation as a complementary technique to other organ-preservation technologies in the recovery of marginal donor livers or as a research tool in the development of advanced bioengineering and pharmacologic strategies for organ recovery and rehabilitation.


Assuntos
Transplante de Fígado , Fígado , Humanos , Suínos , Animais , Fígado/patologia , Transplante de Fígado/métodos , Bile , Perfusão/métodos , Preservação de Órgãos/métodos
4.
Genes Dev ; 30(7): 786-97, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-27013235

RESUMO

The Hippo signaling pathway is a major regulator of organ size. In the liver, Hippo pathway deregulation promotes hyperplasia and hepatocellular carcinoma primarily through hyperactivation of its downstream effector, YAP. The LATS2 tumor suppressor is a core member of the Hippo pathway. A screen for LATS2-interacting proteins in liver-derived cells identified the transcription factor SREBP2, master regulator of cholesterol homeostasis. LATS2 down-regulation caused SREBP activation and accumulation of excessive cholesterol. Likewise, mice harboring liver-specific Lats2 conditional knockout (Lats2-CKO) displayed constitutive SREBP activation and overexpressed SREBP target genes and developed spontaneous fatty liver disease. Interestingly, the impact of LATS2 depletion on SREBP-mediated transcription was clearly distinct from that of YAP overexpression. When challenged with excess dietary cholesterol, Lats2-CKO mice manifested more severe liver damage than wild-type mice. Surprisingly, apoptosis, inflammation, and fibrosis were actually attenuated relative to wild-type mice, in association with impaired p53 activation. Subsequently, Lats2-CKO mice failed to recover effectively from cholesterol-induced damage upon return to a normal diet. Additionally, decreased LATS2 mRNA in association with increased SREBP target gene expression was observed in a subset of human nonalcoholic fatty liver disease cases. Together, these findings further highlight the tight links between tumor suppressors and metabolic homeostasis.


Assuntos
Fígado Gorduroso/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Colesterol na Dieta/farmacologia , Fígado Gorduroso/genética , Deleção de Genes , Regulação da Expressão Gênica/genética , Células Hep G2 , Homeostase/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos Knockout , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética
5.
J Intern Med ; 291(1): 11-31, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34564899

RESUMO

Non-alcoholic fatty liver disease is comprised of either simple steatosis (non-alcoholic fatty liver) or a more advanced inflammatory and fibrogenic stage (non-alcoholic steatohepatitis [NASH]). NASH affects a growing proportion of the global adult and pediatric population, leading to rising rates of liver fibrosis and hepatocellular carcinoma. NASH is a multifactorial disease that is part of a systemic metabolic disorder. Here, we provide an overview of the metabolic underpinnings of NASH pathogenesis and established drivers of inflammation and fibrosis. Clarification of underlying fibrogenic and inflammatory mechanisms will advance the development of novel treatment strategies as there are no approved therapies at present. We discuss emerging experimental approaches and potential novel investigational strategies derived from animal models including the inflammasome, epigenetic reprogramming, Hippo signaling, Notch signaling, engineered T cells to remove fibrogenic HSCs, and HSC-specific targeting therapies. Recently completed and ongoing clinical trials and antifibrotics are discussed, illuminating the growing expectation that one or more therapies will yield clinical benefit in NASH in the coming years.


Assuntos
Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Criança , Fibrose , Via de Sinalização Hippo , Humanos , Fígado/patologia , Cirrose Hepática/terapia , Hepatopatia Gordurosa não Alcoólica/terapia
6.
Am J Pathol ; 191(2): 309-319, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33159885

RESUMO

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin ß1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin ß1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell-cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor ß (TGF-ß) and widespread fibrosis. Targeted deletion of integrin ß1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin ß1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-ß. Taken together, these findings identify integrin ß1 as a key determinant of liver architecture with a critical role as a regulator of TGF-ß secretion. These results suggest that disrupting the hepatocyte-extracellular matrix interaction is sufficient to drive fibrosis.


Assuntos
Integrina beta1/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Matriz Extracelular/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Camundongos , Camundongos Transgênicos
7.
Am J Geriatr Psychiatry ; 30(1): 46-53, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074610

RESUMO

OBJECTIVE: To investigate the effect of decreased cortical thickness or volume of medial temporal lobe structures on the risk of incident psychosis in patients with AD. DESIGN, SETTING, AND PARTICIPANTS: This hospital-based prospective longitudinal study enrolled 109 patients with AD. All patients with AD were evaluated at 3-month intervals to investigate the effect of decreased cortical thickness or volume of medial temporal lobe structures on the risk of incident psychosis in patients with AD. OUTCOME MEASURE: The main outcome measure was time-to-progression from AD to incident psychosis. The thickness or volume of medial temporal lobe structures (i.e., the hippocampus, entorhinal cortex, and parahippocampus) were measured using magnetic resonance imaging and the Freesurfer automated segmentation pipeline at baseline. RESULTS: Multivariate Cox proportional hazards regression analysis revealed that a decreased cortical thickness or volume of medial temporal region was associated with a higher risk of incident psychosis in patients with AD. The hazard ratios for decreased cortical thickness of the left entorhinal cortex and decreased cortical volume of the right hippocampus were 4.291 (95% confidence interval [CI], 1.196-15.384) and 2.680 [(CI, 1.003-1.196]), respectively. CONCLUSION: Our study revealed that decreased cortical thickness or volume of medial temporal sub-regions is a risk factor for incident psychosis in patients with AD. A careful assessment of the thickness or volume of the medial temporal lobe structures in AD may improve early detection and intervention of psychosis in AD.


Assuntos
Doença de Alzheimer , Transtornos Psicóticos , Lobo Temporal , Doença de Alzheimer/complicações , Humanos , Incidência , Estudos Longitudinais , Imageamento por Ressonância Magnética , Tamanho do Órgão , Estudos Prospectivos , Transtornos Psicóticos/epidemiologia , Fatores de Risco , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/patologia
8.
J Hepatol ; 75(4): 865-878, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33992698

RESUMO

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.


Assuntos
Carcinoma Hepatocelular/genética , Biologia Molecular/estatística & dados numéricos , Hepatopatia Gordurosa não Alcoólica/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/etiologia , Feminino , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Biologia Molecular/métodos , Hepatopatia Gordurosa não Alcoólica/complicações , Fatores de Risco
9.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31748396

RESUMO

Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 PFU of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs.IMPORTANCE JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.


Assuntos
Vírus Junin/imunologia , Vírus Junin/patogenicidade , Receptores da Transferrina/metabolismo , Animais , Arenavirus/imunologia , Arenavirus/patogenicidade , Células CHO , Chlorocebus aethiops , Cricetulus , Modelos Animais de Doenças , Feminino , Glicoproteínas/metabolismo , Cobaias/imunologia , Cobaias/metabolismo , Células HEK293 , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Febres Hemorrágicas Virais/imunologia , Febres Hemorrágicas Virais/virologia , Humanos , Vírus Junin/metabolismo , Macrófagos/virologia , Masculino , Receptores da Transferrina/imunologia , Células Vero , Internalização do Vírus , Replicação Viral
10.
Hepatology ; 70(6): 2107-2122, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31016744

RESUMO

The growing burden of liver fibrosis and lack of effective antifibrotic therapies highlight the need for identification of pathways and complementary model systems of hepatic fibrosis. A rare, monogenic disorder in which children with mutations in mannose phosphate isomerase (MPI) develop liver fibrosis led us to explore the function of MPI and mannose metabolism in liver development and adult liver diseases. Herein, analyses of transcriptomic data from three human liver cohorts demonstrate that MPI gene expression is down-regulated proportionate to fibrosis in chronic liver diseases, including nonalcoholic fatty liver disease and hepatitis B virus. Depletion of MPI in zebrafish liver in vivo and in human hepatic stellate cell (HSC) lines in culture activates fibrotic responses, indicating that loss of MPI promotes HSC activation. We further demonstrate that mannose supplementation can attenuate HSC activation, leading to reduced fibrogenic activation in zebrafish, culture-activated HSCs, and in ethanol-activated HSCs. Conclusion: These data indicate the prospect that modulation of mannose metabolism pathways could reduce HSC activation and improve hepatic fibrosis.


Assuntos
Células Estreladas do Fígado/fisiologia , Cirrose Hepática/etiologia , Manose-6-Fosfato Isomerase/fisiologia , Manose/farmacologia , Animais , Células Cultivadas , Glicosilação , Humanos , Masculino , Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra
11.
Hepatology ; 69(5): 2196-2213, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30520052

RESUMO

Autophagy is important for hepatic homeostasis, nutrient regeneration, and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy because of the lack of autophagy-related gene 7 or autophagy-related gene 5, key autophagy-related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of tauromuricholic acid/taurocholic acid in the bile, increased hepatic bile acid load, abnormal bile canaliculi, and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and up-regulated expression of farnesoid X receptor (Fxr) in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly bile salt export pump, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy-deficient livers. In addition, codeletion of nuclear factor erythroid 2-related factor 2 (Nrf2) in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy-deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion. Conclusion: Together with earlier reports that FXR can suppress autophagy, our findings indicate that autophagy and FXR form a regulatory loop and deficiency of autophagy causes abnormal FXR functionality, leading to the development of intracellular cholestasis and liver injury.


Assuntos
Autofagia , Colestase Intra-Hepática/etiologia , Fator 2 Relacionado a NF-E2/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Colestase Intra-Hepática/metabolismo , Feminino , Privação de Alimentos , Fígado/ultraestrutura , Masculino , Camundongos Transgênicos
12.
J Biomed Sci ; 27(1): 21, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906951

RESUMO

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.


Assuntos
Toxinas Bacterianas/metabolismo , Metilação de DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Melatonina/farmacologia , Mucina-2/biossíntese , Receptor MT2 de Melatonina/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Células HT29 , Humanos , Camundongos , Vibrioses/patologia
13.
PLoS Biol ; 14(5): e1002467, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27213272

RESUMO

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.


Assuntos
Sistema Nervoso Central/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular/genética , Cromatina/metabolismo , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteínas Proto-Oncogênicas/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , alfa Carioferinas/metabolismo
14.
J Hepatol ; 69(2): 385-395, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29572095

RESUMO

BACKGROUND AND AIMS: Although the majority of patients with non-alcoholic fatty liver disease (NAFLD) have only steatosis without progression, a sizeable fraction develop non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocellular carcinoma (HCC). Many established diet-induced mouse models for NASH require 24-52 weeks, which makes testing for drug response costly and time consuming. METHODS: We have sought to establish a murine NASH model with rapid progression of extensive fibrosis and HCC by using a western diet (WD), which is high-fat, high-fructose and high-cholesterol, combined with low weekly dose of intraperitoneal carbon tetrachloride (CCl4), which serves as an accelerator. RESULTS: C57BL/6J mice were fed a normal chow diet ±â€¯CCl4 or WD ±â€¯CCl4 for 12 and 24 weeks. Addition of CCl4 exacerbated histological features of NASH, fibrosis, and tumor development induced by WD, which resulted in stage 3 fibrosis at 12 weeks and HCC development at 24 weeks. Furthermore, whole liver transcriptomic analysis indicated that dysregulated molecular pathways in WD/CCl4 mice and immunologic features were similar to those of human NASH. CONCLUSIONS: Our mouse NASH model exhibits rapid progression of advanced fibrosis and HCC, and mimics histological, immunological and transcriptomic features of human NASH, suggesting that it will be a useful experimental tool for preclinical drug testing. LAY SUMMARY: A carefully characterized model has been developed in mice that recapitulates the progressive stages of human fatty liver disease, from simple steatosis, to inflammation, fibrosis and cancer. The functional pathways of gene expression and immune abnormalities in this model closely resemble human disease. The ease and reproducibility of this model make it ideal to study disease pathogenesis and test new treatments.


Assuntos
Dieta Ocidental , Fígado Gorduroso , Cirrose Hepática , Neoplasias Hepáticas , Camundongos Endogâmicos C57BL , Animais , Tetracloreto de Carbono/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/etiologia , Fígado Gorduroso/imunologia , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica/métodos , Inflamação/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Reprodutibilidade dos Testes
15.
J Biol Chem ; 291(28): 14620-7, 2016 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-27226621

RESUMO

Although it has been established that diabetes increases susceptibility to infections, the role of insulin (INS) in the immune response is unknown. Here, we investigated the immunological function of INS. Proinsulin dimer (pINSd) was a potent immune stimulus that induced inflammatory cytokines, but mature INS was unable to induce an immune response. An affinity-purified rabbit polyclonal antibody raised against mature IL-1α recognized IL-1α and pINS but failed to detect mature INS and IL-1ß. Analysis of the pINS sequence revealed the existence of an INS/IL-1α motif in the C-peptide of pINS. Surprisingly, the INS/IL-1α motif was recognized by monoclonal antibody raised against IL-1α. Deleting the INS/IL-1α motif in pINSd and IL-1α changed their activities. To investigate the pINSd receptor, the reconstitution of IL-1 receptor 1 (IL-1R1) in Wish cells restored pINSd activity that was reversed by an IL-1R antagonist. These data suggested that pINSd needs IL-1R1 for inflammatory cytokine induction. Mouse embryo fibroblast cells of IL-1R1-deficient mice further confirmed that pINSd promotes immune responses through IL-1R1.


Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/metabolismo , Proinsulina/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Células Cultivadas , Interleucina-1alfa/química , Camundongos , Proinsulina/química
16.
J Arthroplasty ; 32(9): 2717-2724, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28487091

RESUMO

BACKGROUND: Few studies have examined flexion contracture at the time of primary total knee arthroplasty (TKA) or how flexion contracture changes over time. The purpose of this study was to assess the ideal degree of extension immediately after TKA and to document postoperative changes in extension and clinical outcomes over 5-year follow-up. METHODS: This retrospective cohort study included 215 cases of primary TKA. Radiographic evaluations were performed on sagittal radiographs with the patient in the supine position and the knee in gravity and in passive extension using a stress device. Clinical outcomes were also measured. Four groups were defined on the basis of the extension angle during radiological evaluation: group 1, -10° to 0°; group 2, >0° to +5°; group 3, >+5° to +10°; group 4, >+10° in gravity. RESULTS: There were statistically significant differences in passive extension and gravity extension angles in groups 1, 3, and 4 with time-dependent and time*group (passive vs gravity) analyses, but not in group 2. The flexion contracture angles over 10° in gravity were decreased, although over 5° of flexion contracture remained at the final follow-up. Clinical outcomes were worse in groups 1 and 4 at the final follow-up. CONCLUSION: An extension angle between 0° and 5° in the passive extension position immediately after TKA can be considered ideal up to 5 years of follow-up. Patients with flexion contracture greater than 5° in passive extension and patients with hyperextension should be followed up to assess whether the condition will worsen.


Assuntos
Artroplastia do Joelho/reabilitação , Contratura/fisiopatologia , Articulação do Joelho/fisiopatologia , Articulação do Joelho/cirurgia , Amplitude de Movimento Articular , Idoso , Idoso de 80 Anos ou mais , Artroplastia do Joelho/efeitos adversos , Contratura/etiologia , Feminino , Humanos , Artropatias , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Estudos Retrospectivos
17.
Semin Liver Dis ; 36(1): 87-98, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26870935

RESUMO

Fibrosis is the wound-healing response of tissues to injury. Extensive characterization of organ fibrosis mechanisms has identified common core pathways in renal, pulmonary, skin, and liver fibrosis that offer novel antifibrotic approaches across tissues, in addition to organ-specific and/or disease-specific pathways. A growing number of small molecules and biologics have been identified that are reaching clinical trials for one or more fibrotic diseases, making new antifibrotic options for liver fibrosis an emerging reality. The accelerating pace of drug development, which will also include drug repurposing or combination therapies, heightens the need for novel methods for noninvasive fibrosis assessment without liver biopsy, which is critical to establishing surrogate endpoints for patients in clinical trials who have a low risk of hepatic decompensation. In this article the authors review mechanisms of liver fibrosis and outline potential therapeutic targets and antifibrotic therapies in preclinical studies and clinical trials.


Assuntos
Cirrose Hepática/tratamento farmacológico , Fígado/efeitos dos fármacos , Terapia de Alvo Molecular , Animais , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/metabolismo , Terapia de Alvo Molecular/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
18.
J Hepatol ; 65(2): 344-353, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27154062

RESUMO

BACKGROUND & AIMS: Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis. METHODS: Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription. RESULTS: IL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-ß) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression. CONCLUSIONS: IL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs. LAY SUMMARY: We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells. TRANSCRIPT PROFILING ACCESSION NUMBER: GSE45612, GSE 68001 and GSE 25097.


Assuntos
Células Estreladas do Fígado , Animais , Interleucina-15 , Cirrose Hepática , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-15
20.
J Gen Virol ; 97(10): 2575-2591, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27498826

RESUMO

Japanese encephalitis virus (JEV) is the main cause of acute viral encephalitis, primarily affecting children and young adults in the Asia-Pacific region. JEV is a vaccine-preventable pathogen, with four types of JE vaccine licensed in different regions of the world. To date, the most common JEV strain used in vaccine development and production is SA14-14-2, an attenuated strain derived from its wild-type parental strain SA14. In this study, we directly compared the phenotypic and genotypic characteristics of SA14 and SA14-14-2 to determine the biological and genetic properties associated with their differential virulence. In susceptible BHK-21 cells, SA14-14-2 grew slightly more slowly and formed smaller plaques than SA14, but unlike SA14, it showed almost no expression of the viral protein NS1', the product of a conserved predicted RNA pseudoknot-mediated ribosomal frameshift. In weanling ICR mice, SA14-14-2 was highly attenuated in terms of both neuroinvasiveness and neurovirulence, with its median lethal doses invariably over five logs higher than those of SA14 when inoculated intramuscularly and intracerebrally. Interestingly, the neurovirulence of SA14-14-2 was dependent on mouse age, with the 1- to 7-day-old mice being highly susceptible and the 14- to 21-day-old mice becoming resistant to intracerebral inoculation. At the genome level, SA14-14-2 differed from SA14 by 57 nucleotides, including one silent G-to-A substitution at position 3599 within the predicted RNA pseudoknot for NS1' synthesis; of the 57 differences, 25 resulted in amino acid substitutions. Our data pave the way for the development of new genetically modified JE vaccines.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Vacinas contra Encefalite Japonesa/imunologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Vírus da Encefalite Japonesa (Espécie)/química , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/virologia , Feminino , Humanos , Vacinas contra Encefalite Japonesa/administração & dosagem , Vacinas contra Encefalite Japonesa/química , Vacinas contra Encefalite Japonesa/genética , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/química , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Virulência
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