RESUMEN
BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) type 3 is an autosomal recessive disorder arising from mutations in the ATP-binding cassette subfamily B member 4 (ABCB4) gene. This gene encodes multidrug resistance protein-3 (MDR3) that acts as a hepatocanalicular floppase that transports phosphatidylcholine from the inner to the outer canalicular membrane. In the absence of phosphatidylcholine, the detergent activity of bile salts is amplified and this leads to cholangiopathy, bile duct loss and biliary cirrhosis. Patients usually present in infancy or childhood and often progress to end-stage liver disease before adulthood. CASE PRESENTATION: We report a 32-year-old female who required cadaveric liver transplantation at the age of 17 for cryptogenic cirrhosis. When the patient developed chronic ductopenia in the allograft 15 years later, we hypothesized that the patient's original disease was due to a deficiency of a biliary transport protein and the ductopenia could be explained by an autoimmune response to neoantigen that was not previously encountered by the immune system. We therefore performed genetic analyses and immunohistochemistry of the native liver, which led to a diagnosis of PFIC3. However, there was no evidence of humoral immune response to the MDR3 and therefore, we assumed that the ductopenia observed in the allograft was likely due to chronic rejection rather than autoimmune disease in the allograft. CONCLUSIONS: Teenage patients referred for liver transplantation with cryptogenic liver disease should undergo work up for PFIC3. An accurate diagnosis of PFIC 3 is key for optimal management, therapeutic intervention, and avoidance of complications before the onset of end-stage liver disease.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Colestasis Intrahepática/inmunología , Fibrosis/inmunología , Trasplante de Hígado , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Adulto , Transporte Biológico , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Colestasis Intrahepática/cirugía , Femenino , Fibrosis/genética , Fibrosis/patología , Fibrosis/cirugía , Expresión Génica , Genes Recesivos , Heterocigoto , Humanos , Hígado/inmunología , Hígado/patología , Hígado/cirugía , Fosfatidilcolinas/metabolismo , Factores de TiempoRESUMEN
Multidrug resistance is one of the leading causes of chemotherapy failure in cancer patients. Early detection and capture of drug-resistant tumor cells can facilitate the monitoring of the therapy process and improve the prognosis of patients. In this study, novel P-glycoprotein (P-gp) antibody modified porous hydrogel particles are proposed for drug-resistant tumor cells capture. The hydrogel particles employ a highly biocompatible hydrogel, methacrylate gelatin (GelMA), as the carrier and replicate from the silica colloidal crystal beads. By the modification of P-gp antibody probes on their surfaces, the hydrogel particles are endowed with the ability to capture drug-resistant tumor cells, which overexpress specific components of P-gp on their membranes. Additionally, the acquired ordered porous nanostructure of the particles can provide not only more surface area for antibody immobilization but also a nanopatterned platform for highly efficient target cell capture. The above advantages make the porous hydrogel particles ideal for efficient capture and detection of the drug-resistant tumor cells, which can be expected to facilitate the point-of-care pharmacotherapy and promisingly improve the patient outcomes.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Anticuerpos Antineoplásicos/química , Resistencia a Antineoplásicos , Hidrogeles/química , Neoplasias/patología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/metabolismo , Anticuerpos Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Gelatina/química , Humanos , Células K562 , Metacrilatos/química , Nanoestructuras/química , Neoplasias/metabolismo , Porosidad , Dióxido de Silicio/químicaRESUMEN
Typhoid fever is a multisystemic illness caused by Salmonella enterica serovars Typhi and is resistant to most antibiotics and drugs. The resistance is conferred through multidrug resistance (MDR) proteins, which efflux most antibiotics and other drugs. We predicted potential candidate B-cell and T-cell epitopes using bio- and immune-informatics tools in the 11 MDR proteins - EmrA, EmrB, EmrD, MdtA, MdtB, MdtC, MdtG, MdtH, MdtK, MdtL and TolC. The antigenic potential of the MDR proteins was calculated using VaxiJen server. The B-cell and T-cell epitopes of the MDR proteins were predicted using BCPred and ProPredI and ProPred respectively. The binding affinities of the predicted T-cell epitopes were estimated using T-epitope designer and MHCPred tools. 10, 7, 5, 12, 14, 21, 26, 3, 3 and 3 B-cell epitopes were identified in EmrA, EmrB, EmrD, TolC, MdtA, MdtB, MdtC, MdtG, MdtH and MdtL respectively. We predicted 9 T-cell epitopes - YVSRRAVQP (EmrA), FGVANAISI (EmrB), MVNSQVKQA and YQGGMVNSQ (TolC), WDRTNSHKL (MdtA), FLRNIPTAI (MdtB), YVEQLGVTG (MdtG), VKWMYAIEA (MdtH) and LAHTNTVTL (MdtL) capable of eliciting both humoral and adaptive immune responses. These T-cell epitopes specifically bind to HLA alleles - DRB1*0101 and DRB1*0401. This is the first report of epitope prediction in the MDR proteins of S. Typhi. Taken together, these results indicate the MDR proteins - EmrA, MdtA and TolC are the most suitable vaccine candidates for S. Typhi. The findings of our study on the MDR proteins prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of typhoid fever.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/análisis , Epítopos/química , Salmonella typhi/química , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Antibacterianos/química , Antibacterianos/farmacología , Epítopos/efectos de los fármacos , Epítopos/inmunología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Salmonella typhi/efectos de los fármacos , Salmonella typhi/inmunologíaRESUMEN
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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Colitis/inmunología , Enfermedad de Crohn/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Receptores de Hidrocarburo de Aril/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Animales , Antiinflamatorios/inmunología , Antiinflamatorios/farmacología , Apirasa/genética , Apirasa/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Bilirrubina/inmunología , Bilirrubina/farmacología , Hipoxia de la Célula , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/genética , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Sulfato de Dextran/administración & dosificación , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Membrana Mucosa/inmunología , Membrana Mucosa/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Cultivo Primario de Células , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Receptores de Hidrocarburo de Aril/genética , Ritonavir/farmacología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Bullous pemphigoid (BP) constitutes the most prevalent disease in the group of bullous dermatoses with the autoimmune background. Some authors suggest that certain cytokines (IL-2, IFN-γ) may be transported by P-glycoprotein (P-gp), the product of the ABCB1 gene. ABCB1 polymorphism might affect not only the effectiveness of treatment with drugs that are P-gp substrates but also contribute to the development of diseases, including BP. In the present work, we resolved to conduct a haplotype analysis of ABCB1 in patients with BP and to answer the question of whether any of the haplotypes are able to affect the incidence of this entity. The study involved 71 patients with BP and 100 healthy volunteers. Determination of polymorphisms 1236C > T and 3435C > T in ABCB1 was carried out with the PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method. The 2677G > T/A ABCB1 polymorphism was analyzed with the allele-specific PCR method. It was observed that the 1236T-2677G-3435T haplotype occurred with a statistically significantly lower frequency in patients with BP than in controls (1.4 vs. 10.0%). Carriers of this haplotype were also shown to have had a low relative risk for BP (OR = 0.13, p = 0.003). Haplotype analysis of ABCB1 conducted in patients with BP demonstrated that the 1236T-2677G-3435T haplotype may protect against development of this entity.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Predisposición Genética a la Enfermedad , Penfigoide Ampolloso/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Penfigoide Ampolloso/epidemiología , Penfigoide Ampolloso/inmunología , Polimorfismo de Longitud del Fragmento de Restricción , Adulto JovenRESUMEN
BACKGROUND: The ultimate emergence of multidrug resistance remains a severe limitation of chemotherapy treatment for patients with cancer. The best-characterized cause of drug resistance involves the overexpression of P-glycoprotein (Pgp), which decreases the intracellular accumulation of chemotherapeutic agents in drug-resistant cancer cells. Thus, Pgp has become an attractive potential target for treating chemotherapy-resistant cancer, but the outcomes of using chemotherapy in combination with Pgp inhibitors in clinical trials to date have been disappointing. OBJECTIVE: We herein examine the relationship between Pgp and drug resistance and update the strategies for overcoming drug resistance by targeting Pgp, with a special focus on the recent progress in the area of preventing the development of drug resistance by targeting Pgp both in vitro and in vivo. Given the essential roles of drug-resistant cancer models in these investigations, commonly used approaches for establishing drug-resistant models in the laboratory are also addressed. CONCLUSION: Considering the roles of Pgp in normal physiological conditions and its appreciated roles in detoxification, the currently available Pgp inhibitors undoubtedly cannot be used to reverse drug resistance in the clinic. Although agents that target Pgp to prevent and/or reverse drug resistance are not beneficial at the doses used in the laboratory when administered to patients with cancer who are enrolled in clinical trials, compounds targeting Pgp are widely acknowledged to be promising for circumventing drug resistance.
Asunto(s)
Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Silenciador del Gen , Humanos , Ratones , Terapia Molecular Dirigida , Nanopartículas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Overexpression of P-glycoprotein (Pgp) has been considered a primary cause for multidrug resistance in a variety of cancers for three decades. However, clinical translation of Pgp targeted therapeutics has been hindered by lack of patient preselection based on the Pgp presence in tumors. We aim to develop a molecularly targeted probe for imaging tumoral Pgp in vivo with positron emission tomography (PET) and fluorescence, and to provide a tool for preselecting the patients with tumoral Pgp expression. Thus, a Pgp monoclonal antibody 15D3 was chemically modified with IRDye800 (IR800) and DOTA chelator. The specificity of the antibody conjugates DOTA-Pab-IR800 was verified in Pgp-expressing 3T3-MDR1 and control 3T3 cells. After radiolabeling with 64Cu, the probe was applied in small animal PET imaging of Pgp in a mouse xenograft model of NCI/ADR-Res cells, which are chemoresistant through overexpression of Pgp. Quantification analysis of the PET images demonstrated that the tumor uptake of the radioactive probe was 9.9 ± 1.4, 12.1 ± 1.2, and 10.5 ± 1.0%ID/g at 4, 24, and 48 h post injection. The tumor-to-muscle ratio was 20.9 at 48 h post injection based on biodistribution studies. Fluorescence imaging was performed following PET experiments, and it demonstrated excellent tumor accumulation of this dual-modality probe in the NCI/ADR-Res tumors. Further, an image-guided surgery was successfully performed using the fluorescence modality of the probe, demonstrating potential utility of this probe in image-guided surgical removal of Pgp-positive drug resistant tumors in the patients. In conclusion, this study clearly demonstrated that the Pgp-targeted antibody probe, 64Cu-DOTA-Pab-IR800, could provide a promising diagnosis tool for detection of Pgp-expressing tumors in vivo.
Asunto(s)
Anticuerpos Monoclonales/química , Imagen Molecular/métodos , Sondas Moleculares/química , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Quelantes/química , Radioisótopos de Cobre/química , Resistencia a Antineoplásicos , Femenino , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Hibridomas , Indoles/química , Ratones , Ratones Desnudos , Sondas Moleculares/inmunología , Sondas Moleculares/farmacología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Neoplasias/patología , Imagen Óptica/métodos , Compuestos Organometálicos/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Linfocitos B/inmunología , Colitis Ulcerosa/inmunología , Neoplasias Colorrectales/inmunología , Regulación Neoplásica de la Expresión Génica , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Linfocitos B/patología , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/patología , Quimiotaxis , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Modelos Animales de Enfermedad , Epirregulina/genética , Epirregulina/inmunología , Genes de las Cadenas Ligeras de las Inmunoglobulinas/genética , Humanos , Interleucina-11/genética , Interleucina-11/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Masculino , Ratones , Ratones Noqueados , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/inmunología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/inmunologíaRESUMEN
The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Regulación Leucémica de la Expresión Génica , Memoria Inmunológica , Gripe Humana/prevención & control , Leucemia Mieloide Aguda/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Antibióticos Antineoplásicos/farmacología , Anticuerpos/farmacología , Transporte Biológico , Antígenos CD4/genética , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/inmunología , Daunorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Humanos , Inmunofenotipificación , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/inmunología , Gripe Humana/virología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/virología , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/crecimiento & desarrollo , Orthomyxoviridae/inmunología , Rodaminas/metabolismo , Rodaminas/farmacología , Transducción de Señal , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/patologíaRESUMEN
T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Depleción Linfocítica/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Metabolismo Energético , Enfermedad Injerto contra Huésped/inmunología , Humanos , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fármacos Fotosensibilizantes/aislamiento & purificación , Fármacos Fotosensibilizantes/farmacología , Trasplante HomólogoAsunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Policitemia Vera/genética , Mielofibrosis Primaria/genética , Pirazoles/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antineoplásicos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular/efectos de los fármacos , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Activación de Linfocitos/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/inmunología , Mutación , Proteínas de Neoplasias/inmunología , Nitrilos , Fitohemaglutininas/farmacología , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/inmunología , Policitemia Vera/patología , Cultivo Primario de Células , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/inmunología , Mielofibrosis Primaria/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Transducción de Señal , Resultado del TratamientoRESUMEN
The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-ß-cyclodextrin or other chemically modified ß-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Colesterol/metabolismo , Microdominios de Membrana/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Unión Competitiva , Epítopos , Microdominios de Membrana/efectos de los fármacos , Ratones , Células 3T3 NIH , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Transfección , beta-Ciclodextrinas/farmacologíaRESUMEN
Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.
Asunto(s)
Hepatocitos/enzimología , Hepatocitos/metabolismo , Espectrometría de Masas/métodos , Péptidos/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Afinidad de Anticuerpos , Hidrocarburo de Aril Hidroxilasas/inmunología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Atorvastatina/farmacocinética , Células Cultivadas , Cromatografía Liquida/métodos , Citocromo P-450 CYP3A/inmunología , Citocromo P-450 CYP3A/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Hepatocitos/citología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Péptidos/inmunología , Pravastatina/farmacocinética , Cultivo Primario de Células , Homología de Secuencia de AminoácidoRESUMEN
PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 µM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 µM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 µM verapamil and 50 µM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. ß-amyloid significantly reduced ABCB1 activity, and the ß-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.
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Receptor Toll-Like 4/inmunología , Malla Trabecular/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/agonistas , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Digoxina/farmacología , Fluoresceínas/metabolismo , Fluoresceínas/farmacología , Expresión Génica , Humanos , Ácido Hialurónico/antagonistas & inhibidores , Ácido Hialurónico/farmacología , Inmunidad Innata , Ácido Láctico/farmacología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Naloxona/farmacología , Células PC12 , Ratas , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Malla Trabecular/citología , Malla Trabecular/efectos de los fármacosRESUMEN
Intestinal mucositis represents the most common complication of intensive chemotherapy, which has a severe adverse impact on quality of life of cancer patients. However, the precise pathophysiology remains to be clarified, and there is so far no successful therapeutic intervention. In this study, we investigated the role of innate immunity through TLR signaling in modulating genotoxic chemotherapy-induced small intestinal injury in vitro and in vivo. Genetic deletion of TLR2, but not MD-2, in mice resulted in severe chemotherapy-induced intestinal mucositis in the proximal jejunum with villous atrophy, accumulation of damaged DNA, CD11b(+)-myeloid cell infiltration, and significant gene alterations in xenobiotic metabolism, including a decrease in ABCB1/multidrug resistance (MDR)1 p-glycoprotein (p-gp) expression. Functionally, stimulation of TLR2 induced synthesis and drug efflux activity of ABCB1/MDR1 p-gp in murine and human CD11b(+)-myeloid cells, thus inhibiting chemotherapy-mediated cytotoxicity. Conversely, TLR2 activation failed to protect small intestinal tissues genetically deficient in MDR1A against DNA-damaging drug-induced apoptosis. Gut microbiota depletion by antibiotics led to increased susceptibility to chemotherapy-induced mucosal injury in wild-type mice, which was suppressed by administration of a TLR2 ligand, preserving ABCB1/MDR1 p-gp expression. Findings were confirmed in a preclinical model of human chemotherapy-induced intestinal mucositis using duodenal biopsies by demonstrating that TLR2 activation limited the toxic-inflammatory reaction and maintained assembly of the drug transporter p-gp. In conclusion, this study identifies a novel molecular link between innate immunity and xenobiotic metabolism. TLR2 acts as a central regulator of xenobiotic defense via the multidrug transporter ABCB1/MDR1 p-gp. Targeting TLR2 may represent a novel therapeutic approach in chemotherapy-induced intestinal mucositis.
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Antineoplásicos/efectos adversos , Mucositis/inmunología , Mucositis/microbiología , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Immunoblotting , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota , Mucositis/inducido químicamente , Células Mieloides/inmunología , Células Mieloides/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Toll-Like 2/inmunologíaRESUMEN
Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase ATP8B1 (familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter ABCB11 (bile salt export pump, BSEP), or ABCB4 (multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this "new" disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of "autoimmune BSEP disease."
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Transportadoras de Casetes de Unión a ATP/genética , Enfermedades Autoinmunes , Colestasis Intrahepática/genética , Colestasis Intrahepática/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Colestasis Intrahepática/diagnóstico , Colestasis Intrahepática/terapia , Progresión de la Enfermedad , Humanos , Trasplante de Hígado , Recurrencia , Índice de Severidad de la EnfermedadRESUMEN
ATP-binding cassette (ABC) transporters, including ABC-transporter B1 (ABCB1), extrude drugs, metabolites, and other compounds (such as mitotracker green (MTG)) from cells. Susceptibility of CD4(+) regulatory T (Treg) cells to the ABCB1-substrate cyclophosphamide (CPA) has been reported. Here, we characterized ABCB1 expression and function in human CD4(+) T-cell subsets. Naïve, central memory, and effector-memory CD4(+) T cells, but not Treg cells, effluxed MTG in an ABCB1-dependent manner. In line with this, ABCB1 mRNA and protein was expressed by nonregulatory CD4(+) T-cell subsets, but not Treg cells. In vitro, the ABCB1-substrate CPA was cytotoxic for Treg cells at a 100-fold lower dose than for nonregulatory counterparts, and, inversely, verapamil, an inhibitor of ABC transporters, increased CPA-toxicity in nonregulatory CD4(+) T cells but not Treg cells. Thus, Treg cells lack expression of ABCB1, rendering them selectively susceptible to CPA. Our findings provide mechanistic support for therapeutic strategies using CPA to boost anti-tumor immunity by selectively depleting Treg cells.
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Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Ciclofosfamida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Antineoplásicos Alquilantes/farmacocinética , Apoptosis/inmunología , Ciclofosfamida/farmacocinética , Citotoxinas/farmacocinética , Citotoxinas/farmacología , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Depleción Linfocítica/métodos , Masculino , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Linfocitos T Reguladores/citologíaRESUMEN
We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
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Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Anticuerpos Biespecíficos/biosíntesis , Complejo CD3/inmunología , Disulfuros/química , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Unión Competitiva , Línea Celular , Estabilidad de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , HumanosRESUMEN
To gain insight into the interaction of intracellular pathogens with host innate immune pathways, we performed an unbiased genetic screen of Listeria monocytogenes mutants that induced an enhanced or diminished host innate immune response. Here, we show that the major facilitator superfamily of bacterial multidrug resistance transporters (MDRs) controlled the magnitude of a host cytosolic surveillance pathway, leading to the production of several cytokines, including type I IFN. Mutations mapping to repressors of MDRs resulted in ectopic expression of their cognate transporters, leading to host responses that were increased up to 20-fold over wild-type bacteria, and a 20-fold decrease in bacterial growth in vivo. Mutation of one of the MDRs, MdrM, led to a 3-fold reduction in the IFN-beta response to L. monocytogenes infection, indicating a pivotal role for MdrM in activation of the host cytosolic surveillance system. Bacterial MDRs had previously been associated with resistance to antibiotics and other toxic compounds. This report links bacterial MDRs and host immunity. Understanding the mechanisms through which live pathogens activate innate immune signaling pathways should lead to the discovery of adjuvants, vaccines, and perhaps new classes of therapeutics. Indeed, we show that the mutants identified in this screen induced vastly altered type I IFN response in vivo as well.
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Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Proteínas Bacterianas/inmunología , Inmunidad Innata , Listeria monocytogenes/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Proteínas Bacterianas/genética , Citosol/inmunología , Genes Bacterianos , Genes MDR , Interferón beta/biosíntesis , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genéticaRESUMEN
AIM: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity. METHODS: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity. RESULTS: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively. CONCLUSION: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.