RESUMO
Tuberculosis is the world's most deadly infectious disease, with 10 million people falling ill and 1.5 million people dying from the disease every year. With the increasing number of drug-resistant Mycobacterium tuberculosis (MTB) strains and prevalence of coinfection of MTB with human immunodeficiency virus, many challenges remain in the prevention and treatment of tuberculosis. Therefore, the development of safe and effective tuberculosis vaccines is an urgent issue. In this study, we identified cytotoxic T lymphocyte epitopes on drug resistance-associated membrane protein efflux pumps of MTB, the ATP-binding cassette and the major facilitator superfamilies. First, three online software were used to predict HLA-A2-restricted epitopes. Then, the candidate epitopes were confirmed with the T2A2 cell binding affinity and peptide/MHC (pMHC) complex stability assays and in vitro immune activity experiments. Two drug-resistant T lymphocyte epitopes, designated Rv1218c-p24 and Rv2477c-p182, were selected, and their immunogenic activities studied in vivo in genetically engineered mice. The immune activities of these two epitopes were improved with the help of complete Freund's adjuvant (CFA). The epitopes identified here provide a foundation for the diagnosis and treatment of patients infected with drug resistant and the future development of a multiepitope vaccine.
Assuntos
Epitopos de Linfócito T/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/terapia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linhagem Celular , Mapeamento de Epitopos , Adjuvante de Freund/administração & dosagem , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Imunogenicidade da Vacina , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Cultura Primária de Células , Linfócitos T Citotóxicos/metabolismo , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Epitopos/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Animais , Epitopos/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In this study we analyzed the diversity of the humoral immune response of bFcRn Tg mice, using a length distribution analysis (spectratyping) and next generation sequencing (NGS) of the immunoglobulin heavy chain variable regions. Our analysis showed that in response to immunization with ovalbumin or transfected cells that expressed a unique membrane protein, our Tg animals developed a more diverse plasma cell repertoire than controls, which manifested in greater numbers of different clones, and clusters with fewer highly expanded large clones, as identified by the variable region (CDR3) of the immunoglobulin heavy chain. The increased antibody diversity in Tg mice after immunization was observed at both IgM and IgG levels, indicating that the increased humoral immune diversity in Tg mice is due to a higher number of both activated, antigen-specific naïve and isotype switched B cells. We thus demonstrated that the BCR repertoire of the immunized bFcRn Tg animals is more diverse compared to wild type mice, which likely makes these Tg mice a better choice for monoclonal antibody production against challenging antigens, including the extracellular regions of cell membrane proteins.
Assuntos
Linfócitos B/metabolismo , Regiões Determinantes de Complementaridade/genética , Genes de Cadeia Pesada de Imunoglobulina , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunidade Humoral , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Receptores Fc/metabolismo , Células 3T3 , Animais , Linfócitos B/imunologia , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunização , Imunoglobulina G/genética , Imunoglobulina M/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores Fc/genética , Transdução de Sinais , Regulação para CimaRESUMO
Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings.
Assuntos
Anticorpos Antibacterianos/química , Proteínas de Bactérias/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Anticorpos de Domínio Único/química , Zinco/metabolismo , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Sítios de Ligação , Camelus , Staphylococcus aureus Resistente à Meticilina/imunologia , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Zinco/químicaRESUMO
Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B4 in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB4 BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB4 and LTC4 from LTA4 are competitive, and MRP1 is the efflux pump for LTC4 Inhibition of MRP1 will increase LTB4 production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC4 and subsequently increased LTB4 levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB4 production and antimicrobial activity through LTB4/BLT1 signaling.
Assuntos
Lesão Pulmonar Aguda , Infecções por Escherichia coli , Escherichia coli/imunologia , Vesículas Extracelulares , Células-Tronco Mesenquimais/imunologia , Pneumonia Bacteriana , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Animais , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/terapia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/patologia , Vesículas Extracelulares/transplante , Humanos , Leucotrieno B4/imunologia , Leucotrieno C4/imunologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/terapia , Propionatos/farmacologia , Quinolinas/farmacologia , Células RAW 264.7RESUMO
AIMS: ATP-binding cassette subfamily C member 3 (ABCC3) is involved in multidrug resistance and is overexpressed in some solid tumors. Recent work revealed an increase in circulating anti-ABCC3 antibodies in lung and esophageal cancers. This in vitro study was undertaken to investigate the effects of the natural IgG antibody against the ABCC3-derived peptide antigen on proliferation of oral squamous cell carcinoma (OSCC) cells and augment the development of efficient and effective treatments in patients with OSCC. SUBJECTS AND METHODS: An in-house enzyme-linked immunosorbent assay was applied to detect anti-ABCC3 IgG antibody in human plasma. Two OSCC cell lines, CAL27 and SCC15, were cultured with 20% plasma either positive or negative for anti-ABCC3 IgG. Cell proliferation was quantified by the CCK-8 method, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. The expression of the ABCC3 gene in the cell lines was analyzed by reverse transcriptase quantitative real-time polymerase chain reaction. RESULTS: The results showed that plasma anti-ABCC3 IgG significantly inhibited the proliferation of CAL27 cells but not SCC15 cells, although ABCC3 was expressed in both cell lines. The proportion of apoptotic cells was significantly higher in CAL27 cells treated with anti-ABCC3 IgG-positive plasma than in those treated with IgG-negative plasma. Cell cycle progression was arrested in CAL27 cells treated with anti-ABCC3 IgG-positive plasma. CONCLUSIONS: Our data suggest that human plasma anti-ABCC3 IgG may be a promising agent in anti-OSCC therapy, although further studies are needed to arrive at a definitive conclusion.
Assuntos
Apoptose/efeitos dos fármacos , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/imunologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Bucais/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Autoanticorpos/administração & dosagem , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Neoplasias Bucais/sangue , Neoplasias Bucais/patologia , Células Tumorais CultivadasRESUMO
In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Colite/imunologia , Doença de Crohn/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Apirase/genética , Apirase/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bilirrubina/imunologia , Bilirrubina/farmacologia , Hipóxia Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/genética , Doença de Crohn/genética , Doença de Crohn/patologia , Sulfato de Dextrana/administração & dosagem , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/imunologia , Mucosa/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Receptores de Hidrocarboneto Arílico/genética , Ritonavir/farmacologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologiaRESUMO
BACKGROUND & AIMS: Immunotherapy is a promising treatment option for cholangiocarcinoma. We compared cytotoxic T lymphocyte (CTL) responses against several tumour-associated antigen (TAA)-derived epitopes in cholangiocarcinoma patients to identify candidate epitopes for immunotherapy. METHODS: Twenty-six TAAs were selected, and the expression of TAAs in 6 cholangiocarcinoma cell lines and 9 specimens were measured using real-time polymerase chain reaction (PCR). CTL responses against 38 TAA-derived epitopes were measured using samples from 26 cholangiocarcinoma patients by interferon-γ enzyme linked immunospot (ELISPOT)-assay. RESULTS: Most TAAs were expressed in cholangiocarcinoma cell lines and specimens in PCR. Epitopes that stimulated a specific immune response were defined as those that elicited a CTL response in more than 3 patients and little response in healthy volunteers, as measured by ELISPOT-assay. Based on these criteria, there were 18 epitopes that stimulated specific immune responses: squamous cell carcinoma antigen recognized by T cells (SART)1690 , P53161 , multidrug resistance-associated protein (MRP)3503 , Survivin2B80 , melanoma-associated antigen (MAGE)-A4143 , receptor tyrosine kinase ErbB-2/neu (Her2/neu)63 , Wilms tumour (WT1)235 , WT1417 , ß-catenin29 , carcinoembryonic antigen (CEA)268 , CEA652 , epithelial cell adhesion molecule (EpCAM)173 , enhancer of zeste homolog (EZH)2291 , mucin 5AC (MUC5AC)716 , glypican-3 (GPC3)298 and kinesin family member 20A (KIF20A)66 . Furthermore, the absolute number of lymphocytes in peripheral blood was significantly correlated with the TAA-specific response. Lastly, the overall survival was significantly prolonged in patients with 2 or more TAA-specific CTL responses compared with none to one. CONCLUSIONS: These results demonstrated several TAAs may be promising for immunotherapy for cholangiocarcinoma, and patients with high lymphocyte counts may benefit more from immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Colangiocarcinoma/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/imunologia , Antígeno CTLA-4/imunologia , Estudos de Casos e Controles , Colangiocarcinoma/terapia , ELISPOT , Molécula de Adesão da Célula Epitelial/imunologia , Feminino , Humanos , Imunoterapia , Cinesinas/metabolismo , Neoplasias Hepáticas/terapia , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Proteína Supressora de Tumor p53/imunologiaRESUMO
In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1ß in PNAC. Pharmacological antagonism of IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1ß) prevents PNAC in mice. IL-1ß increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1ß, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.
Assuntos
Colestase/genética , Interleucina-1beta/genética , Fígado/imunologia , Macrófagos/imunologia , NF-kappa B/genética , Receptores CCR2/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/imunologia , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Caspase 1/genética , Caspase 1/imunologia , Caspases/genética , Caspases/imunologia , Caspases Iniciadoras , Colestase/etiologia , Colestase/imunologia , Colestase/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Recém-Nascido , Interleucina-1beta/imunologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Fígado/patologia , Receptores X do Fígado/genética , Receptores X do Fígado/imunologia , Macrófagos/patologia , Masculino , Camundongos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , NF-kappa B/imunologia , Nutrição Parenteral/efeitos adversos , Receptores CCR2/deficiência , Receptores CCR2/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/imunologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Transdução de SinaisRESUMO
Whole parasite immunization strategies employing genetically attenuated parasites (GAP), which arrest during liver-stage development, have been applied successfully for induction of sterile malaria protection in rodents. Recently, we generated a Plasmodium berghei GAP-lacking expression of multidrug resistance-associated protein (MRP2) (PbΔmrp2) that was capable of partial schizogony in hepatocytes but showed complete growth arrest. Here, we investigated the protective efficacy after intravenous (IV) immunization of BALB/c and C57BL/6J mice with PbΔmrp2 sporozoites. Low-dose immunization using 400 PbΔmrp2 sporozoites induced 100% sterile protection in BALB/c mice after IV challenge with 10,000 wild-type sporozoites. In addition, almost full protection (90%) was obtained after three immunizations with 10,000 sporozoites in C57BL/6J mice. Parasite liver loads in nonprotected PbΔmrp2-challenged C57BL/6J mice were reduced by 86% ± 5% on average compared with naive control mice. The mid-to-late arresting PbΔmrp2 GAP was equipotent in induction of protective immunity to the early arresting PbΔb9Δslarp GAP. The combined data support a clear basis for further exploration of Plasmodium falciparum parasites lacking mrp2 as a suitable GAP vaccine candidate.
Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Parasitemia/prevenção & controle , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Relação Dose-Resposta Imunológica , Feminino , Imunização , Fígado/imunologia , Fígado/parasitologia , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Organismos Geneticamente Modificados/imunologia , Organismos Geneticamente Modificados/metabolismo , Parasitemia/imunologia , Parasitemia/parasitologia , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Esporozoítos/metabolismo , Vacinas AtenuadasRESUMO
Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes.
Assuntos
Células Endoteliais/imunologia , Hepatócitos/imunologia , Fígado/imunologia , Modelos Biológicos , Monócitos/imunologia , Organoides/imunologia , Albuminas/genética , Albuminas/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Apolipoproteínas B/genética , Apolipoproteínas B/imunologia , Caderinas/genética , Caderinas/imunologia , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/imunologia , Comunicação Celular/imunologia , Técnicas de Cocultura , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Inflamação , Dispositivos Lab-On-A-Chip , Lipopolissacarídeos/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Organoides/citologia , Organoides/efeitos dos fármacos , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaAssuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Policitemia Vera/genética , Mielofibrose Primária/genética , Pirazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células/efeitos dos fármacos , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Mutação , Proteínas de Neoplasias/imunologia , Nitrilas , Fito-Hemaglutininas/farmacologia , Policitemia Vera/tratamento farmacológico , Policitemia Vera/imunologia , Policitemia Vera/patologia , Cultura Primária de Células , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/imunologia , Mielofibrose Primária/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas , Transdução de Sinais , Resultado do TratamentoRESUMO
Multidrug resistance-associated protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters. In this study, we investigated the safety and immunogenicity of a MRP3-derived peptide (MRP3765) as a vaccine and characterized the MRP3-specific T cell responses induced. Twelve hepatocellular carcinoma (HCC) patients treated with hepatic arterial infusion chemotherapy (HAIC) were enrolled. The MRP3-derived peptide was emulsified in incomplete Freund's adjuvant and administered via subcutaneous immunization three times weekly. No serious adverse drug reactions to the peptide vaccine were observed, and the vaccination was well tolerated. The vaccination induced MRP3-specific immunity in 72.7% of the patients. In a phenotypic analysis, the largest post-vaccinated increase in MRP3-specific T cells was due to an increase in cells with the effector memory phenotype. Among the 12 patients, one patient showed a partial response, nine showed a stable disease, and two showed a progressive disease. The median overall survival time was 14.0 months. In conclusion, the safety, effects of immune boosting, and possible prolongation of overall survival by the MRP3-derived peptide demonstrate the potential of the peptide to provide clinical benefit in HCC patients.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Fragmentos de Peptídeos/imunologia , Idoso , Citotoxicidade Imunológica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , VacinaçãoRESUMO
Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.
Assuntos
Carcinoma Hepatocelular/genética , Interleucina-18/imunologia , Neoplasias Hepáticas/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , NF-kappa B/genética , Receptores Citoplasmáticos e Nucleares/genética , Fator de Transcrição YY1/genética , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , NF-kappa B/imunologia , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/imunologia , Transdução de Sinais , Fator de Transcrição YY1/imunologiaRESUMO
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
Assuntos
Anticorpos/imunologia , Imunização , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Plasmídeos , Vacinas de DNA , Animais , Linhagem Celular , DNA Complementar/imunologia , DNA Complementar/farmacologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmídeos/imunologia , Plasmídeos/farmacologia , Estrutura Secundária de Proteína , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
Single-chain Fv fragments (scFvs) consist of the variable heavy-chain (VH) and variable light-chain (VL) domains, which are the smallest immunoglobulin fragments containing the whole antigen-binding site. Human soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proves to acquire a potent pro-apoptotic activity only after selective binding to a predefined tumor cell surface antigen and has no off-target effects towards normal cells. Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor and overexpresses human multidrug resistance protein 3 (MRP3). In this study, we designed a novel fusion protein, termed scFvM58-sTRAIL, in which the MRP3-specific scFv antibody M58 was genetically fused to the N-terminus of human soluble TRAIL (sTRAIL). The recombinant scFvM58-sTRAIL fusion protein, expressed in Escherichia coli, was purified by chromatography and tested for cytotoxicity. scFvM58-sTRAIL showed a significant apoptosis-inducing activity towards MRP3-positive GBM cells in vitro. The pro-apoptotic activity of scFvM58-sTRAIL towards GBM cells was strongly inhibited in the presence of the parental scFvM58 antibody, suggesting that cytotoxic activity is MRP3-restricted. In a control experiment with MRP3-negative Jurkat cells, scFvM58-sTRAIL did not induce apparent apoptosis. In addition, through target antigen-restricted binding, scFvM58-sTRAIL was capable of activating not only TRAIL-R1 but also TRAIL-R2. In conclusion, our results suggest that fusion protein scFvM58-sTRAIL with specificity for MRP3 is a highly selective therapeutic agent and may provide an alternative therapy for human GBM.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Proteínas Recombinantes de Fusão/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Apoptose/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Células Jurkat , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/uso terapêutico , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêuticoRESUMO
BaeR is the response regulator of the two-component system, BaeSR, found in Escherichia coli (E. coli) and Salmonella. Several biological functions of BaeR, related to multidrug efflux and bacterial virulence, have been described. Herein, we report a putative function of BaeR during inflammatory response of the host by using BaeR protein of Salmonella enterica Paratyphi A (S. Paratyphi A) origin overexpressed in E. coli, and RAW 264.7 and THP-1 cells as in vitro models. BaeR (3 µg/ml) upregulated iNOS mRNA expression in both cell lines, and induced significant production of NO. Greater than ten-fold (TNF-α), 24-fold (IL-1ß) and 156-fold (IL-6) increases in mRNA expression levels were observed in THP-1 cells treated with BaeR, compared to untreated controls. Furthermore, an eight-fold (IL-1ß), 12-fold (IL-6) and 41-fold (TNF-α) higher protein concentrations were observed in RAW 264.7 cells stimulated with BaeR, compared to control cells. Immunoblot analysis showed BaeR-induced phosphorylation of the MAPKs (ERK 1/2, JNK and p38 MAPK) in RAW 264.7 cells. Pharmacological inhibition of the three MAPKs using specific inhibitors resulted in significant reduction of BaeR-induced NO production and iNOS mRNA expression by inhibitors of JNK and p38 MAPK. Also, all inhibitors of the MAPKs significantly attenuated BaeR-induced IL-1ß, IL-6 and TNF-α at both transcript and protein levels with different degrees of inhibition. Taken together, our data suggest that BaeR is a putative inducer of inflammatory response and the MAPKs are involved in the process.
Assuntos
Interações Hospedeiro-Patógeno , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Salmonella paratyphi A/imunologia , Transdução de Sinais , Estresse Fisiológico , Transativadores/imunologia , Animais , Linhagem Celular , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Óxido Nítrico Sintase Tipo II/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6) structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9) mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations.
Assuntos
Antígenos , Atovaquona/administração & dosagem , Resistência a Múltiplos Medicamentos , Interações Hospedeiro-Parasita , Plasmodium falciparum , Variação Antigênica/efeitos dos fármacos , Variação Antigênica/genética , Antígenos/efeitos dos fármacos , Antígenos/genética , Citocromos b/genética , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Evolução Molecular , Genoma de Protozoário/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Malária Falciparum/genética , Malária Falciparum/imunologia , Mitose/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologiaRESUMO
PURPOSE: There is an urgent need to identify biomarkers for early diagnosis and prognosis of esophageal cancer. The present study was undertaken to test whether circulating autoantibodies to ATP-binding cassette C3 (ABCC3) transporter could serve as a biomarker for the malignant tumor. METHODS: An enzyme-linked immunosorbent assay approach was developed in-house to test circulating autoantibodies to ABCC3 in 114 patients with esophageal squamous cell carcinoma (ESCC) and 226 healthy subjects well matched in age and smoking history. RESULTS: Mann-Whitney U test showed that the IgA antibody levels were significantly higher in patients with ESCC than control subjects (Z = -4.226, P < 0.001) while the IgG antibody levels were not significantly different between the two groups (Z = -1.072, P = 0.284). The sensitivity against >95 % specificity was 13.2 % for the IgA assay with an inter-assay deviation of 13.0 and 7.9 % for the IgG assay with an inter-assay deviation of 9.4 %. CONCLUSIONS: This work suggests that circulating IgA autoantibody to ABCC3 may be a potential biomarker for ESCC, which could be used for early diagnosis and prognosis of the malignant tumor.