RESUMO
Inspired by the significant ï¡-glucosidase inhibitory activities of (+)- and (-)-pericosine E, we herein designed and synthesized 16 analogs of these marine natural products bearing a methoxy group instead of a chlorine atom at C6. Four of these compounds exhibited moderate ï¡-glucosidase inhibitory activities, which were weaker than those of the corresponding chlorine-containing species. The four compounds could be prepared by coupling reactions utilizing the (-)-pericosine B moiety. An additional in silico docking simulation suggested that the reason of reduced activity of the C6-methoxylated analogs might be an absence of hydrogen bonding between a methoxy group with the surrounding amino acid residues in the active site in ï¡-glucosidase.
Assuntos
Inibidores de Glicosídeo Hidrolases/análise , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/síntese química , Ácido Chiquímico/análogos & derivados , Simulação por Computador , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Ácido Chiquímico/química , Relação Estrutura-Atividade , alfa-GlucosidasesRESUMO
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/uso terapêutico , Imunização Passiva/métodos , Virus da Influenza A Subtipo H5N1/imunologia , Infecções por Orthomyxoviridae/terapia , Ácidos Carbocíclicos , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/uso terapêutico , Linhagem Celular , Ciclopentanos/uso terapêutico , Cães , Quimioterapia Combinada , Feminino , Guanidinas/uso terapêutico , Hospedeiro Imunocomprometido/imunologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Interleucina-6/sangue , Pulmão/patologia , Pulmão/virologia , Macaca fascicularis , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Neuraminidase/antagonistas & inibidores , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Carga Viral/imunologiaRESUMO
The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of µm, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug.
Assuntos
Antineoplásicos/administração & dosagem , Esqueleto da Parede Celular/administração & dosagem , Mycobacterium bovis , Nanopartículas/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adulto , Animais , Butilidroxibutilnitrosamina , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Ratos , Ratos Endogâmicos F344 , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Neoplasias da Bexiga Urinária/induzido quimicamente , Adulto JovemRESUMO
Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Animais , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Especificidade de Anticorpos , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Modelos Animais de Doenças , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Ebolavirus/fisiologia , Epitopos/imunologia , Humanos , Imunização Passiva , Macaca mulatta , Masculino , Camundongos , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Células Vero , Carga Viral/imunologiaRESUMO
Several lines of evidence suggest the involvement of the raphe-serotonergic neurons in addiction to psychostimulants and some recreational drugs. In this study, we established rat organotypic mesencephalic slice cultures containing the raphe nuclei and examined the effects of sustained exposure to 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH). Immunostaining for tryptophan hydroxylase (TPH) studies revealed that serotonergic neurons were abundant in the slice cultures. Sustained exposure to MDMA and METH (1-1000 microM) for 4 days had little effect on the serotonin tissue content, [(3)H]citalopram binding, or expression/phosphorylation of TPH. Treatment with MDMA or METH for 30 min increased serotonin release in a concentration-dependent manner. Slice cultures were exposed to MDMA for 4 days following a 1-day withdrawal period and then challenged with MDMA (10 microM). Sustained MDMA exposure augmented MDMA-induced serotonin release in a concentration-dependent manner, indicating serotonergic sensitization. Similar serotonergic sensitization was observed for METH. The development of MDMA-induced serotonergic sensitization was attenuated by the NMDA receptor antagonist, MK-801 (10 microM). These results suggest that in mesencephalic slice cultures sustained MDMA or METH exposure induces serotonergic sensitization through activation of NMDA receptors without serotonergic neurotoxicity. The in vitro model system could help to elucidate the mechanisms underlying drug addiction.
Assuntos
Mesencéfalo/efeitos dos fármacos , Metanfetamina/farmacologia , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Neurônios/efeitos dos fármacos , Núcleos da Rafe/efeitos dos fármacos , Serotonina/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Mesencéfalo/metabolismo , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Núcleos da Rafe/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappaB reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pam3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappaB in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappaB. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events.
Assuntos
Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , Peptidoglicano/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Galinhas , Humanos , Lipoproteínas/metabolismo , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Filogenia , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Inativação Gênica , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interferência de RNA , Proteínas Repressoras/genética , Proteínas de Bactérias/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli , Regulação da Expressão Gênica , Vetores Genéticos , Repressores Lac , RNA Polimerase III/farmacologia , Proteínas Repressoras/farmacologiaRESUMO
The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Helicobacter pylori/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Células COS , Linhagem Celular , DNA Complementar/metabolismo , Ativação Enzimática , Vetores Genéticos , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia de Vídeo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Fosforilação , Testes de Precipitina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Estômago/microbiologia , Estômago/patologia , Fatores de Tempo , Transfecção , Tirosina/química , Tirosina/metabolismo , Proteínas ras/metabolismoRESUMO
Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.