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1.
J Radiat Res ; 62(3): 483-493, 2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-33899102

RESUMO

We developed a confidence interval-(CI) assessing model in multivariable normal tissue complication probability (NTCP) modeling for predicting radiation-induced liver disease (RILD) in primary liver cancer patients using clinical and dosimetric data. Both the mean NTCP and difference in the mean NTCP (ΔNTCP) between two treatment plans of different radiotherapy modalities were further evaluated and their CIs were assessed. Clinical data were retrospectively reviewed in 322 patients with hepatocellular carcinoma (n = 215) and intrahepatic cholangiocarcinoma (n = 107) treated with photon therapy. Dose-volume histograms of normal liver were reduced to mean liver dose (MLD) based on the fraction size-adjusted equivalent uniform dose. The most predictive variables were used to build the model based on multivariable logistic regression analysis with bootstrapping. Internal validation was performed using the cross-validation leave-one-out method. Both the mean NTCP and the mean ΔNTCP with 95% CIs were calculated from computationally generated multivariate random sets of NTCP model parameters using variance-covariance matrix information. RILD occurred in 108/322 patients (33.5%). The NTCP model with three clinical and one dosimetric parameter (tumor type, Child-Pugh class, hepatitis infection status and MLD) was most predictive, with an area under the receiver operative characteristics curve (AUC) of 0.79 (95% CI 0.74-0.84). In eight clinical subgroups based on the three clinical parameters, both the mean NTCP and the mean ΔNTCP with 95% CIs were able to be estimated computationally. The multivariable NTCP model with the assessment of 95% CIs has potential to improve the reliability of the NTCP model-based approach to select the appropriate radiotherapy modality for each patient.

2.
Transplant Cell Ther ; 27(1): 92.e1-92.e5, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32961376

RESUMO

Although mesenchymal stromal cell (MSC) transfer has long drawn attention owing to its immunosuppressive potential to treat immune-mediated diseases, the role of endogenous MSCs in immune regulation in vivo has remained largely unclear. MSCs constitute the hematopoietic stem cell (HSC) niche, perhaps contributing to immune protection of HSCs, termed immune privilege. Our recent study demonstrates that immune privilege of HSCs is endowed by niche-residential regulatory T cells (Tregs), which promote allogeneic HSC engraftment. This immune privilege depends on cell surface ectoenzymes CD39 and CD73 on niche Tregs, which generate extracellular adenosine, a nucleotide known to suppress immunity and potentiate Tregs. Another niche constituent, leptin receptor-expressing (lepr+) perivascular MSCs, also highly express CD39 and CD73, prompting us to study their roles in immune privilege. This work demonstrates an unexpected negative regulation of immune privilege by MSC-derived adenosine. CD39 deletion in lepr+ cells increased and potentiated effector memory-like niche Tregs, promoting allogeneic HSC engraftment. CD39 deletion in Tregs also activated niche Tregs, while abrogating engraftment. These observations demonstrate paradoxical effects of MSC-derived adenosine to activate immunity, revealing a previously undescribed dual roles of adenosine. Adenosine from both Tregs and MSCs inhibits niche Tregs, whereas adenosine from Tregs, but not that from MSCs, acts as an effector molecule of immune privilege.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Adenosina , Medula Óssea , Privilégio Imunológico
4.
Nat Med ; 25(11): 1691-1698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31700187

RESUMO

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.


Assuntos
Pneumopatias/terapia , Pulmão/crescimento & desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Regeneração/genética , Acilação/genética , Animais , Blastocisto/metabolismo , Diferenciação Celular/genética , Metilação de DNA/genética , Modelos Animais de Doenças , Histonas/genética , Humanos , Pulmão/patologia , Pneumopatias/patologia , Camundongos , Organogênese/genética , Células-Tronco Pluripotentes/transplante , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , beta Catenina/genética
5.
Haematologica ; 104(6): 1136-1142, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30545927

RESUMO

Various extrinsic signals tightly control hematopoietic stem cell quiescence. Our recent study showed that hematopoietic stem cells are regulated by a special FoxP3+ regulatory T-cell population with high expression of a hematopoietic stem cell marker, CD150. Extracellular adenosine generated via a cell-surface ectoenzyme CD39 on CD150high regulatory T cells maintained hematopoietic stem cell quiescence. It remains unclear how conventional T cells and the other cell-surface ectoenzyme, CD73, contribute to regulation of hematopoietic stem cells. This work shows that CD150high regulatory T cells as well as unique CD150high CD4+ conventional T cells regulate hematopoietic stem cells via CD73. Global CD73 deletion increased the numbers of hematopoietic stem cells, cycling stem cell frequencies, and levels of reactive oxygen species in hematopoietic stem cells. In vivo antioxidant treatment inhibited the increase of hematopoietic stem cells in CD73 knockout mice, suggesting that CD73 maintains stem cell quiescence by preventing oxidative stress. High levels of CD73 expression were frequently found on CD150high regulatory T cells and CD150high FoxP3-CD4+ T cells within the bone marrow. Transfer of these CD150high regulatory T cells and CD150high CD4+ conventional T cells abolished the increase of hematopoietic stem cells in CD73 knockout mice. In addition, the increase of stem cells in CD73 knockout mice was also inhibited by pharmacological activation of adenosine receptor 2A which is highly expressed by hematopoietic stem cells. Taken together, these results suggest that CD73 of CD150high regulatory T cells and CD150high CD4+ conventional T cells protects hematopoietic stem cells from oxidative stress, maintaining stem cell quiescence via adenosine receptor 2A.


Assuntos
5'-Nucleotidase/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Biomarcadores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Proteínas Ligadas por GPI/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunofenotipagem , Ativação Linfocitária , Camundongos , Camundongos Knockout , Estresse Oxidativo , Agonistas do Receptor Purinérgico P1/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia
6.
Cancer Sci ; 109(5): 1672-1681, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29575390

RESUMO

Discovery of a high-risk group for pancreatic cancer is important for prevention of pancreatic cancer. The present study was conducted as a nested case-control study including 170 pancreatic cancer cases and 340 matched controls of our population-based cohort study involving 30 239 subjects who answered a baseline questionnaire and supplied blood samples. Twelve targeted metabolites were quantitatively analyzed by gas chromatography/tandem mass spectrometry. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were calculated using conditional logistic regression models. Statistically significant P-value was defined as P < .05. Increasing 1,5-anhydro-d-glucitol (1,5-AG) levels were associated with a decreasing trend in pancreatic cancer risk (OR of quartile 4 [Q4], 0.50; 95% CI, 0.27-0.93; P = .02). Increasing methionine levels were also associated with an increasing trend of pancreatic cancer risk (OR of Q4, 1.79; 95% CI, 0.94-3.40: P = .03). Additional adjustment for potential confounders attenuated the observed associations of 1,5-AG and methionine (P for trend = .06 and .07, respectively). Comparing subjects diagnosed in the first 0-6 years, higher levels of 1,5-AG, asparagine, tyrosine and uric acid showed a decreasing trend for pancreatic cancer risk (P for trend = .04, .04, .04 and .02, respectively), even after adjustment for potential confounders. We found that the 12 target metabolites were not associated with pancreatic cancer risk. However, metabolic changes in the subjects diagnosed in the first 0-6 years showed a similar tendency to our previous reports. These results might suggest that these metabolites are useful for early detection but not for prediction of pancreatic cancer.


Assuntos
Metaboloma , Neoplasias Pancreáticas/metabolismo , Adulto , Idoso , Asparagina/análise , Estudos de Casos e Controles , Desoxiglucose/análise , Feminino , Humanos , Modelos Logísticos , Masculino , Metionina/análise , Pessoa de Meia-Idade , Neoplasias Pancreáticas/etiologia , Estudos Prospectivos , Risco
7.
Cell Stem Cell ; 22(3): 445-453.e5, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29456159

RESUMO

A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. Here, we describe a Treg subpopulation that controls hematopoietic stem cell (HSC) quiescence and engraftment. These Tregs highly expressed an HSC marker, CD150, and localized within the HSC niche in the bone marrow (BM). Specific reduction of BM Tregs achieved by conditional deletion of CXCR4 in Tregs increased HSC numbers in the BM. Adenosine generated via the CD39 cell surface ectoenzyme on niche Tregs protected HSCs from oxidative stress and maintained HSC quiescence. In transplantation settings, niche Tregs prevented allogeneic (allo-) HSC rejection through adenosine and facilitated allo-HSC engraftment. Furthermore, transfer of niche Tregs promoted allo-HSC engraftment to a much greater extent than transfer of other Tregs. These results identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, abundance, and engraftment, further highlighting their therapeutic utility.


Assuntos
Adenosina/metabolismo , Células da Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Nicho de Células-Tronco
8.
Cell Mol Gastroenterol Hepatol ; 3(2): 272-283, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28275693

RESUMO

BACKGROUND & AIMS: An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection. METHODS: We investigated the EV-mediated transmission of HBV infection by using a HBV infectious culture system that uses primary human hepatocytes derived from humanized chimeric mice (PXB-cells). Purified EVs were isolated by ultracentrifugation. To analyze the EVs and virions, we used stimulated emission depletion microscopy. RESULTS: Purified EVs from HBV-infected PXB-cells were shown to contain HBV DNA and to be capable of transmitting HBV DNA to naive PXB-cells. These HBV-DNA-transmitting EVs were shown to be generated through a ceramide-triggered EV production pathway. Furthermore, we showed that these HBV-DNA-transmitting EVs were resistant to antibody neutralization; stimulated emission depletion microscopy showed that EVs lacked hepatitis B surface antigen, the target of neutralizing antibodies. CONCLUSIONS: These findings suggest that EVs harbor a DNA cargo capable of transmitting viral DNA into hepatocytes during HBV infection, representing an additional antibody-neutralization-resistant route of HBV infection.

9.
Clin Chim Acta ; 468: 98-104, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28215548

RESUMO

BACKGROUND: To improve prognosis of pancreatic cancer (PC) patients, the discovery of more reliable biomarkers for the early detection is desired. METHODS: Blood samples were collected by 2 independent groups. The 1st set was included 55 early PC and 58 healthy volunteers (HV), and the 2nd set was included 16 PC and 16HV. The 16 targeted metabolites were quantitatively analyzed by gas chromatography/tandem mass spectrometry together with their corresponding stable isotopes. In the 1st set, the levels of these metabolites were evaluated, and diagnostic models were constructed via multivariate logistic regression analysis, leading to validation using the 2nd set. RESULTS: In the 1st set, model X consisting of 4 candidates based on our previous report possessed higher sensitivity (74.1%) than carbohydrate antigen 19-9 (CA19-9). Model Y, consisting of 2 metabolites newly selected from 16 metabolites via stepwise method possessed higher sensitivity (70.4%) than CA19-9. Furthermore, combining model Y with CA19-9 increased its sensitivity (90.7%) and specificity (89.5%). In the 2nd set, combining model Y with CA19-9 displayed high sensitivity (81.3%) and specificity (93.8%). In particular, it displayed very high sensitivity (100%) for resectable PC. CONCLUSIONS: Quantitative analysis confirmed that metabolomics-based diagnostic methods are useful for detecting PC early.


Assuntos
Biomarcadores Tumorais/sangue , Detecção Precoce de Câncer/métodos , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Espectrometria de Massas em Tandem , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo
10.
Biomark Med ; 10(6): 577-86, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27171159

RESUMO

AIM: To examine a novel screening method for pancreatic cancer involving gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry-based metabolomics analysis. MATERIALS & METHODS: Sera from pancreatic cancer patients (n = 59) and healthy volunteers (n = 59) were allocated to the training set or validation set. Serum metabolome analysis was carried out using our multiplatform metabolomics system. A diagnostic model was constructed using a two-phase screening method that was newly advocated. RESULTS: When the training set was used, the constructed diagnostic model exhibited high sensitivity (100%) and specificity (80%) for pancreatic cancer. When the validation set was used, the model displayed high sensitivity (84.1%) and specificity (84.1%). CONCLUSION: We successfully developed a diagnostic model for pancreatic cancer using a multiplatform serum metabolomics system.


Assuntos
Biomarcadores Tumorais/sangue , Metabolômica , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/patologia , Curva ROC , Sensibilidade e Especificidade
11.
J Hepatol ; 64(3): 547-55, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26505121

RESUMO

BACKGROUND & AIMS: Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. METHODS: We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. RESULTS: Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo. CONCLUSION: We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors.


Assuntos
Técnicas de Transferência de Genes , Hepatite B Crônica/terapia , RNA Interferente Pequeno/administração & dosagem , Animais , DNA Viral/análise , Antígenos de Superfície da Hepatite B/análise , Antígenos E da Hepatite B/análise , Vírus da Hepatite B/genética , Humanos , Concentração de Íons de Hidrogênio , Lipossomos , Camundongos
12.
PLoS One ; 10(11): e0141785, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26544200

RESUMO

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.


Assuntos
Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Animais , Diferenciação Celular , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Células-Tronco Hematopoéticas/citologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Imagem Molecular , Transplante Homólogo/efeitos adversos , Transplante Isogênico/efeitos adversos
13.
Nihon Shokakibyo Gakkai Zasshi ; 112(10): 1858-67, 2015 Oct.
Artigo em Japonês | MEDLINE | ID: mdl-26440689

RESUMO

In 2010, a 39-year-old woman presented with a cystic lesion, 16 mm in diameter, in the tail of the pancreas. Regular follow-ups were conducted to monitor this lesion; its diameter was found to increase to 45 mm in 2013. Thus, the patient was admitted to our hospital for further examination and treatment. Abdominal US, abdominal contrast-enhanced CT, and MRI showed a cystic lesion of 45 mm in diameter in the tail of the pancreas, which had internal septae and mural nodules inside. EUS revealed a cyst-in-cyst-like structure, with a thickened cystic wall along the entire circumference. Thus, distal pancreatectomy and splenectomy were performed on the basis of a diagnosis of mucinous cystic neoplasm. Histopathological examination of a resected specimen showed that the lesion comprised a substantial component of red-brown tone, with adjacent cystic components. The final diagnosis was an epidermoid cyst in an intrapancreatic accessory spleen.


Assuntos
Diagnóstico Diferencial , Cisto Epidérmico/diagnóstico , Neoplasias Císticas, Mucinosas e Serosas/diagnóstico , Esplenopatias/diagnóstico , Neoplasias Esplênicas/diagnóstico , Adulto , Feminino , Humanos , Imagem Multimodal
14.
Sci Rep ; 4: 4750, 2014 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-24756133

RESUMO

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5' untranslated region are capable of inducing strong RNAi activity. By dissecting the 5'-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.


Assuntos
Hepacivirus/genética , Hepatite C/metabolismo , Hepatite C/virologia , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Regiões 5' não Traduzidas , Animais , Linhagem Celular , Modelos Animais de Doenças , Inativação Gênica , Genoma Viral , Hepatite C/terapia , Hepatócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Transgênicos , Interferência de RNA , RNA Viral , Replicação Viral
15.
PLoS One ; 8(12): e82527, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358200

RESUMO

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.


Assuntos
Genes Virais , Genótipo , Hepacivirus/isolamento & purificação , Replicação Viral/genética , Animais , Hepacivirus/genética , Humanos , Camundongos , Mutação , RNA Viral/genética
16.
Gastroenterology ; 145(4): 865-73, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23791700

RESUMO

BACKGROUND & AIMS: Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. METHODS: We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. RESULTS: NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. CONCLUSIONS: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatócitos/virologia , Serina C-Palmitoiltransferase/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Hepacivirus/classificação , Hepacivirus/genética , Humanos , Camundongos , RNA Viral/análise
17.
PLoS One ; 8(3): e59611, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555725

RESUMO

BACKGROUND & AIMS: The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-ß in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.


Assuntos
Quimera/imunologia , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Interferons/genética , Fígado/imunologia , Fígado/virologia , Ativação Transcricional , Animais , Apoptose/imunologia , Linhagem Celular , Humanos , Imunidade Inata/genética , Interleucinas/genética , Fígado/citologia , Fígado/metabolismo , Camundongos , Polimorfismo de Nucleotídeo Único , RNA de Cadeia Dupla/genética , Especificidade da Espécie
18.
Carbohydr Polym ; 91(1): 434-43, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23044154

RESUMO

Continuous effort in research and development of nanofibers for apparel usage has been focused within their functional properties only. We investigated esthetic properties by producing colored cationic-cellulose nanofibers for the very first time for the potential application of apparel use. The cellulose acetate nanofibers were electrospun followed by deacetylation and cationization to produce functional cationic-cellulose nanofibers and then dyed with anionic reactive dyes. The spectrophotometric measurement of dyed samples was carried out to determine color coordinates and color yield values. The cationic-cellulose nanofibers showed enhanced color yield and dye fixation without addition of an electrolyte in comparison to cellulose nanofibers. The cationization of cellulose nanofibers significantly enhanced the color yield values of around 76% at dye concentrations of 5%. Excellent color fastness results demonstrate that these new colored and breathable materials can potentially be considered as future apparel for casual or fashion.


Assuntos
Celulose/química , Vestuário , Corantes/química , Nanofibras/química , Cor , Propriedades de Superfície , Temperatura , Fatores de Tempo
19.
PLoS Pathog ; 8(8): e1002860, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916015

RESUMO

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.


Assuntos
Hepacivirus/fisiologia , Hepatite C/metabolismo , Esfingolipídeos/biossíntese , Replicação Viral/fisiologia , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatite C/genética , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/genética , Transferases (Outros Grupos de Fosfato Substituídos)/biossíntese , Replicação Viral/efeitos dos fármacos
20.
Sci Rep ; 2: 259, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22355771

RESUMO

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.


Assuntos
Hepacivirus/efeitos dos fármacos , Interferon Tipo I/farmacologia , Replicação Viral/efeitos dos fármacos , Administração Oral , Animais , Western Blotting , Hepacivirus/fisiologia , Interferon Tipo I/administração & dosagem , Camundongos , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Ressonância de Plasmônio de Superfície
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