RESUMEN
BACKGROUND: In situ vaccination-induced local inflammatory response resulted in the establishment of a pool of tissue-resident memory T (TRM) cells and new vessels after the resolution of inflammation. TRM cells have received increasing attention; however, the role of new vessels in protective response is still unknown. MATERIALS AND METHODS: We performed the laparotomy to access the stomach and injected alum-based vaccine into the gastric subserous layer (GSL). At 28 days post vaccination, a parabiosis mouse model along with depletion of anti-CD90.2 antibody was employed to explore the function of perivascular lymphocyte clusters in recall responses. The composition of the gastric lymphocyte clusters was analyzed by immunofluorescence staining. Antibody responses were detected using ELISA. Gastric lymphocytes were analyzed using flow cytometry. RESULTS: GSL vaccination induced the formation of new vessels in the inflamed region. These new vessels were different from native vessels in that they were generally accompanied by perivascular lymphocyte clusters that mainly consisted of CD90-expressing cells. Additionally, histological analysis revealed the presence of CD4+ and CD8+ T cells in the perivascular lymphocyte clusters. Administration of a dose of an anti-CD90.2 antibody to GSL-vaccinated mice resolved these clusters. The efficacy of protection was compared in the parabiosis mice. Upon challenge, the presence of perivascular lymphocyte clusters was responsible for the fast recall response, as depletion of these clusters by CD90.2 antibody administration resulted in decreased expressions of VCAM-1, Madcam-1, and TNF-α, as well as lower recruitment of proinflammatory immune cells, decreased antibody levels, and poor protection. CONCLUSIONS: Our research demonstrates that in situ vaccination-induced regional inflammatory response contributes to optimal recall response not only by establishing a CD4+ TRM pool but also by creating an "expressway," i.e., perivascular lymphocyte cluster.
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Vacunas Bacterianas/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Vacunación/métodos , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Vacunas Bacterianas/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Mucosa Gástrica/citología , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Humanos , Memoria Inmunológica , Inyecciones Intralesiones , Ratones , Antígenos Thy-1/antagonistas & inhibidores , Antígenos Thy-1/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Tissue-resident memory T cells accelerate the clearance of pathogens during recall response. However, whether CD4+ TRM cells themselves can provide gastric immunity is unclear. MATERIALS AND METHODS: We established a parabiosis model between the enhanced green fluorescent protein and wild-type mice that the circulation system was shared, and the wild-type partner was vaccinated with H pylori vaccine composed of CCF and silk fibroin in gastric subserous layer to induce gastric EGFP+ CD4+ TRM cells. Antigen-specific EGFP+ CD4+ T cells and proliferous TRM cells were analyzed by flow cytometry. The colonization of H pylori was detected by quantitative real-time PCR. EGFP+ CD4+ TRM cells and the inflammation of the stomach were observed by histology. RESULTS: A parabiosis animal model was employed to identify the cells that introduced by vaccination in GSL. Antigen-specific EGFP+ CD4+ T cells could be detected at day 7 post-vaccination. Thirty days later, EGFP+ CD4+ TRM cells were established with a phenotype of CD69+ CD103- . Of note, we found that when circulating lymphocytes were depleted by FTY720 administration, these TRM cells could proliferate in situ and differentiate into effector Th1 cells after H pylori challenge. A decrease in H pylori colonization was observed in the vaccinated mice but not unvaccinated mice. Further, we found that although FTY720 was administrated, mounted pro-inflammatory myeloid cells still emerged in the stomach of the vaccinated mice, which might contribute to the reduction of H pylori colonization. CONCLUSIONS: Our study reveals that H pylori vaccine-induced CD4+ TRM cells can proliferate and differentiate in situ to enhance gastric local immunity during recall response.
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Vacunas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Memoria Inmunológica , Animales , Vacunas Bacterianas/administración & dosificación , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Tissue-resident memory T (Trm) cells are enriched at the sites of previous infection and required for enhanced protective immunity. However, the emergence of Trm cells and their roles in providing protection are unclear in the field of Helicobacter pylori (H. pylori) vaccinology. Here, our results suggest that conventional vaccine strategies are unable to establish a measurable antigen (Ag)-specific memory cell pool in stomach; in comparison, gastric subserous injection of mice with micro-dose of Alum-based H. pylori vaccine can induce a pool of local CD4+ Trm cells. Regional recruitment of Ag-specific CD4+ T cells depends on the engagement of Ag and adjuvant-induced inflammation. Prior subcutaneous vaccination enhanced this recruitment. A stable pool of Ag-specific CD4+ T cells can be detected for 240 days. Two weeks of FTY720 administration in immune mice suggests that these cells do not experience the recirculation. Immunohistochemistry results show that close to the vaccination site, abundant CD4+T cells locate on epithelial niches, independent of lymphocyte cluster. Paradigmatically, Ag-specific CD4+ T cells with a phenotype of CD69+CD103- are preferential on lymphocytes isolated from epithelium. Upon Helicobacter infection, CD4+ Trm cells orchestrate a swift recall response with the recruitment of circulating antigen-specific Th1/Th17 cells to trigger a tissue-wide pathogen clearance. This study investigates the vaccine-induced gastric CD4+ Trm cells in a mice model, and highlights the need for designing a vaccine strategy against H. pylori by establishing the protective CD4+ Trm cells.
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Vacunas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Linfocitos T Reguladores/fisiología , Compuestos de Alumbre , Animales , Resistencia a la Enfermedad , Femenino , Humanos , Memoria Inmunológica , Activación de Linfocitos , Ratones , Membrana Serosa/metabolismo , VacunaciónRESUMEN
Heat-killed probiotics or microbial autologous components show multiple activities on modulating host immune responses towards tolerance or vice versus aggressiveness. Gram-positive enhancer matrix particles (GEMs), the non-genetically modified particles which composed of the cell wall derived from Lactococcus lactis (L. lactis), were used as a typical microbial molecule to investigate the mechanism of opposite immune responses generated in disparate scenarios. The results of stool 16S rRNA Illumina sequencing suggested that the overwhelming number of mice pre-administered with GEMs showed the expansion of Bacteroidetes but contraction of Verrucomicrobia. Co-administration GEMs and antibiotics could preserve the microbial diversity, even though the abundance of gut microbes was largely depleted by antibiotics. Additionally, dendritic cells (DCs) from mice receiving GEMs rather than DCs that in vitro treated with GEMs induced the expansion of regulatory T cells (Tregs), witnessing the critical role of gut flora alteration. Importantly, this alteration provided protection to alleviate dextran sulfate sodium (DSS)-induced intestinal inflammation. On the other hand, in the context of Helicobacter felis (H. felis) infection, the mice pre-administrated with GEMs exhibited a comparably potent gastric immunity with the elevated expression of IFN-γ, IL-17, and multiple anti-microbial factors, leading to the reduced burden of H. felis. However, tolerance for both DSS-induced intestinal inflammation and immunity against H. felis was depleted in a mice model lacking of transforming growth factor-ß1 (TGF-ß1) in myeloid cells. These findings suggest that GEMs can modulate host immune responses bidirectionally according to context, and may serve as a supplement for antibiotic treatment.
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Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/microbiología , Infecciones por Helicobacter/inmunología , Lactococcus lactis/fisiología , Células Mieloides/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Células Dendríticas/inmunología , Femenino , Microbioma Gastrointestinal , Helicobacter/fisiología , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Humanos , Lactococcus lactis/genética , Ratones Endogámicos C57BL , Probióticos/administración & dosificación , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
BACKGROUND: Astragalus membranaceus is a traditional Chinese medicine that has a long history of medical applications. It is of interest to investigate the functional components of A. membranaceus waste with regard to its development and utilization and increasing resource utilization. RESULTS: The protein AMWP was isolated from the A. membranaceus waste. This protein was further purified by DEAE-cellulose-52 chromatography and Sephadex G-200 size-exclusion chromatography to obtain three fractions, named AMWPDG2, AMWPDG4 and AMWPDG6. Then, their immunomodulatory activities were evaluated by using cell model experiments. The results indicated that the protein fractions could significantly increase the proliferation of splenic lymphocytes, peritoneal macrophages and bone-marrow-derived cells (BMDCs). AMWPDG2 showed the highest immunocompetence. AMWPDG2, AMWPDG4 and AMWPDG6 not only significantly improved the phagocytosis and immunomodulatory factors (interleukin (IL)-6, tumor necrosis factor-α, nitric oxide, hydrogen peroxide) secretion of peritoneal macrophages, but also promoted the expression of inflammatory cytokines (IL-6, IL-12 p40, IL-1ß, IL-1α) and chemokines (CXCL1, CCL3) in BMDCs. CONCLUSION: Taken together, these results indicated that three protein fractions from the A. membranaceus waste might be a potential natural immunomodulator. Moreover, it also provided the theoretical basis for further researching the mechanism of AMWPDG2, AMWPDG4 and AMWPDG6 on improving the immune response. © 2019 Society of Chemical Industry.
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Astragalus propinquus/química , Medicamentos Herbarios Chinos/farmacología , Factores Inmunológicos/farmacología , Residuos/análisis , Animales , Células Cultivadas , Quimiocinas/genética , Quimiocinas/inmunología , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/aislamiento & purificación , Factores Inmunológicos/química , Factores Inmunológicos/aislamiento & purificación , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos ICR , Fagocitosis/efectos de los fármacosRESUMEN
BACKGROUND: Toxic adjuvant is considered as an indispensable constituent for oral Helicobacter pylori (H. pylori) vaccines. However, the elaborate role of toxic adjuvant in the initiation of adaptive immune response is largely undescribed. MATERIALS AND METHODS: We employed an acid-resistant HP55/PLGA nanoparticles (NPs) delivery system encapsulating three antigens (Hsp, Nap, and Lpp20) from H. pylori and accompanied with three adjuvants (LPS, CpG, and chimeric flagellum (CF)) to explore the underlying mechanism of the adjuvant constituent. H. pylori-specific antibody responses were detected by ELISA. Gastric inflammatory and Th1/Th17 responses were analyzed by flow cytometry. Expressions of inflammatory cytokines were measured by quantitative real-time PCR. RESULTS: In bone marrow-derived dendritic cells' (BMDCs) model, the addition of toxic adjuvants is responsible for the proinflammatory function, but not the mature phenotype of BMDCs. In vivo, intestinal loop injection with NPs + LPS, rather than NPs alone, altered the dendritic cell (DC) phenotypes in mesenteric lymph nodes and drove a local proinflammatory microenvironment. In a prophylactic vaccination model, mice immunized with NPs + adjuvants significantly reduced the gastric colonization of H. pylori, induced antigen-specific antibody responses and Th1/Th17 cell responses. After H. pylori challenge, these mice showed potent recall responses involving both neutrophil and inflammatory monocyte infiltration. Additionally, TLR4 knockout mice were immunized with NPs + LPS and NPs + CF, respectively; only the recipients of NPs + CF orchestrated a protective response to control bacterial infection. CONCLUSIONS: Our study indicated that toxic adjuvants within oral H.pylori vaccines altered the function and phenotype of dendritic cells and facilitated the establishment of proinflammatory microenvironment to initiate adaptive immune responses.
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Vacunas Bacterianas/inmunología , Células Dendríticas/metabolismo , Helicobacter pylori/inmunología , Adyuvantes Inmunológicos , Animales , Células de la Médula Ósea/citología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Células TH1/metabolismo , Células Th17/metabolismoRESUMEN
The immunogenicity of oral subunit vaccines is poor partly as a result of the harsh milieu of the gastrointestinal (GI) tract. For some pathogens that restrictedly inhabit the GI tract, a vaccine that works in situ may provide more potent protection than vaccines that operate parenterally. Yet, no appropriate delivery system is available for oral subunit vaccines. In this study, we designed HP55/poly( n-butylcyanoacrylate) (PBCA) nanoparticles (NPs) to carry Helicobacter pylori ( H. pylori) subunit vaccine CCF for oral administration in a prophylactic mice model. These NPs, which are synthesized using an interfacial polymerization method, protected the CCF antigen not only from the acidic pH in simulated gastric fluid (SGF, pH 1.2) but also from the proteolysis in simulated intestinal fluid (SIF, pH 7.4). Oral vaccination of mice with HP55/PBCA-CCF NPs promoted the production of serum antigen-specific antibodies, mucosal secretory IgA, and proinflammatory cytokines. Moreover, a Th1/Th17 response and augmented lymphocytes were found in the gastric tissue of HP55/PBCA-CCF NP-immunized mice, which might eventually limit H. pylori colonization. Collectively, these results indicate that HP55/PBCA NPs are promising carriers against the severe situation of the GI tract and thereby may be further utilized for other orally administrated vaccines or drugs.
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Vacunas Bacterianas/inmunología , Portadores de Fármacos/química , Infecciones por Helicobacter/terapia , Inmunogenicidad Vacunal , Administración Oral , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/metabolismo , Cianoacrilatos/química , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Masculino , Metilcelulosa/análogos & derivados , Metilcelulosa/química , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Proteolisis , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/metabolismoRESUMEN
BACKGROUND: Gram-positive enhancer matrix particles (GEM) produced by Lactococcus lactis can enhance vaccine-induced immune response. However, the mechanism under which this adjuvant mounts the efficacy of orally administered vaccines remains unexplored. MATERIALS AND METHODS: We used a prophylactic mice model to investigate the mechanism of GEM-adjuvanted vaccination. Helicobacter pylori urease-specific antibody response was monitored and detected in murine serum by ELISA. Urease-specific splenic cytokine profile was examined. Gastric inflammatory responses were measured on day 43 or 71 by quantitative real-time PCR, flow cytometry and histology. RESULTS: We found that GEM enhanced the efficiency of oral H. pylori vaccine by promoting innate immunity. The vaccine CUE-GEM composed of GEM particles and recombinant antigen CTB-UE provided protection of immunized mice against H. pylori insult. The protective response was associated with induction of postimmunization gastritis and local Th1/Th17 cell-medicated immune response. We showed that innate inflammatory responses including neutrophil chemokines CXCL1-2, neutrophils, and antimicrobial proteins S100A8 and MUC1 were significantly elevated. Within all infected mice, S100A8 and MUC1 levels were negatively correlated with H. pylori burden. Strikingly, mice receiving GEM also show reduction of colonization, possibly through natural host response pathways to recruit CD4+ T cells and promote S100A8 expression. CONCLUSIONS: These findings suggest that GEM-based vaccine may impact Th1/Th17 immunity to orchestrate innate immune response against H. pylori infection.
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Adyuvantes Inmunológicos/administración & dosificación , Vacunas Bacterianas/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Inmunidad Innata , Lactococcus lactis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/aislamiento & purificación , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Mucosa Gástrica/patología , Infecciones por Helicobacter/patología , Leucocitos Mononucleares/inmunología , Masculino , Ratones Endogámicos BALB C , Ureasa/inmunologíaRESUMEN
The present project was designed to optimize the microemulsion (ME) formulation of oil in water (O/W) for dexamethasone acetate (DA), and examine its impact on DA percutaneous permeation. The saturated solubility of DA in different oils, surfactants and co-surfactants was tested. The ratio of surfactant to co-surfactant was selected by constructing pseudo three phase diagrams to investigate the maximal microemulsion area. In vitro permeation studies of DA from microemulsion and suspension were performed to optimize the formulation further. Differential scanning calorimetry(DSC) and attenuated total reflection flourier transformed infrared spectroscopy (ATR-FTIR) were performed to investigate the mechanism of microemulsion action on skin. The optimized formulation was composed of oleic acid/Labrasol/propylene glycol/water with 8/45/15/32 (w/w), and the DA loading was 0.75% (w/w). The permeation enhancement of microemusion was 6.00-fold as that of suspension, and the DA from microemulsion retained in the skin was 4.79-fold as that of suspension. DSC and ATR-FTIR results suggested that microemulsion could affect the intercellular lipid lamellae and keratin of the stratum corneum. The barrier function of stratum corneum was disordered by the microemulsion so that the dermal drug delivery was enhanced. Therefore, the optimized microemulsion enhanced DA percutaneous permeation significantly through the interaction of microemulsion with skin, microemulsion is a promising approach for DA percutaneous delivery.
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Dexametasona/análogos & derivados , Sistemas de Liberación de Medicamentos , Emulsiones/química , Absorción Cutánea , Administración Cutánea , Animales , Dexametasona/farmacología , Composición de Medicamentos , Glicéridos , Aceites , Ácido Oléico , Permeabilidad , Propilenglicol , Piel , Solubilidad , Tensoactivos , AguaRESUMEN
Approaches to efficiently and accurately define the pharmacokinetics (PK) of large sets of small molecules in rodents have been previously described. Likewise, a variety of methods for determining brain tissue distribution (BTD) have been reported for use in the discovery of therapeutics targeting the central nervous system (CNS). Herein we describe a novel cassette approach to efficiently obtain concurrent PK and BTD data from a dose of up to five compounds in one rat over 24 h. In conjunction with fraction unbound (fu) data obtained in plasma and brain homogenate, this approach serves as an efficient means to determine compound unbound brain:unbound plasma partition coefficients (K p,uu), thereby providing insight to compounds bearing poor permeability and/or active transporter activity impacting their permeation of the blood-brain barrier (BBB). This integrated approach was utilized in a lead optimization effort towards the discovery of CNS-penetrant allosteric modulators of a seven-transmembrane (7TM) receptor target. Rat PK and brain distribution was rapidly obtained for 70 compounds and correlated to data obtained from in vitro assessments. Two compounds that were evaluated in cassette and discrete studies, displayed agreement in PK (compound 1: cassette CLp = 1.6 mL min(-1) kg(-1), discrete CLp = 1.6 mL min(-1) kg(-1); compound 2: cassette CLp = 11 mL min(-1) kg(-1), discrete CLp = 8.1 mL min(-1) kg(-1)) and BTD (compound 1: cassette K p = 0.11, discrete K p = 0.09; compound 2: cassette K p < 0.05, discrete K p = 0.04). The resulting data were used to guide medicinal chemistry efforts and to enable the progression of optimized compounds to in vivo pharmacodynamic assessments.
RESUMEN
In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [(3)H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 µM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [(14)C]AA diminished by about 50-70% relative to control in the presence of 200 µM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors.
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Ácido Ascórbico/análogos & derivados , Inhibidores de la Proteasa del VIH/síntesis química , Terapia Molecular Dirigida/métodos , Profármacos/metabolismo , Saquinavir/análogos & derivados , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Ácido Ascórbico/análisis , Ácido Ascórbico/síntesis química , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Transporte Biológico , Células CACO-2 , Línea Celular , Citocromo P-450 CYP3A , Composición de Medicamentos , Estabilidad de Medicamentos , Epitelio/metabolismo , Inhibidores de la Proteasa del VIH/análisis , Inhibidores de la Proteasa del VIH/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Microsomas Hepáticos/metabolismo , Permeabilidad , Profármacos/síntesis química , Ratas , Saquinavir/análisis , Saquinavir/síntesis química , Saquinavir/química , Saquinavir/metabolismo , Sodio/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Solubilidad , Vitaminas/metabolismoRESUMEN
HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Aminoácidos Neutros/metabolismo , Transporte Biológico/efectos de los fármacos , HEPES/farmacología , Adenosina Trifosfato/metabolismo , Animales , Células CACO-2 , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ciclosporina/metabolismo , Perros , Ácido Glutámico/metabolismo , Humanos , Lopinavir , Fenilalanina/metabolismo , Pirimidinonas/metabolismo , Ritonavir/metabolismo , Verapamilo/farmacologíaRESUMEN
The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [(14)C]ascorbic acid ([(14)C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di-substituted benzenes. Transepithelial transport of [(14)C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase-mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a K(m) of 83.2muM and V(max) of 94.2pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy-dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca(2+)/CaM mediated pathways. [(14)C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group, i.e., halogen- and nitro-substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A-B) as well as basal to apical side (B-A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile-substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs.
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Ácido Ascórbico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico Activo , Línea Celular , Permeabilidad de la Membrana Celular , Interpretación Estadística de Datos , Perros , Metabolismo Energético/fisiología , Humanos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Transducción de Señal/fisiología , Sodio/fisiología , Transportadores de Sodio Acoplados a la Vitamina C , Especificidad por SustratoRESUMEN
The primary objective of this study was to investigate the expression of a specialized carrier-mediated system for folic acid and to delineate its uptake mechanism and intracellular trafficking in a human derived retinoblastoma cell line (Y-79). Uptake of [3H]Folic acid was determined at various concentrations, pH, temperatures, in the absence of sodium and chloride ions and in the presence of structural analogs, methyltetrahydro folate (MTF) and methotrexate (MTX), vitamins, membrane transport and metabolic inhibitors to delineate the mechanism of uptake. Kinetics of uptake was studied in the presence of various intracellular regulatory pathways; protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK) and calcium-calmodulin modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of folate carrier systems. The uptake was found to be linear up to 30min. The rate of uptake followed saturation kinetics with apparent Km of 8.29+/-0.74nM, 17.03+/-1.98nM and 563.23+/-115.2nM and Vmax of 393.47+/-9.33, 757.58+/-26.21 and 653.17+/-31.7fmol/(minmg) protein for folic acid, MTF and MTX, respectively. The process was chloride, temperature and energy dependent but sodium and pH independent; inhibited by the structural analogs MTF and MTX but not by structurally unrelated vitamins. Membrane transport inhibitors did not affect the uptake of [3H]Folic acid, however endocytic inhibitor, colchicine, significantly inhibited the [3H]Folic acid uptake indicating the involvement of receptor mediated endocytosis process. PKC, PTK and Ca2+/calmodulin pathways appeared to play important roles in the regulation of folic acid uptake. Molecular evidence of the presence of folate receptor (FR) precursor was identified by RT-PCR analysis. This research work demonstrated, for the first time, the functional and molecular existence of a specialized high affinity carrier-mediated system for folic acid uptake, in human retinoblastoma cells.
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Ácido Fólico/administración & dosificación , Ácido Fólico/metabolismo , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Vitaminas/administración & dosificación , Vitaminas/metabolismo , Antimetabolitos/farmacología , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Interpretación Estadística de Datos , Portadores de Fármacos , Transferencia de Energía , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Proteínas de Transporte de Membrana/metabolismo , Proteína Quinasa C/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Especificidad por Sustrato , TemperaturaRESUMEN
The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing the MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [3H]biotin uptake and transport was delineated. Transepithelial permeability of the biotin-conjugated prodrug, i.e., biotin-saquinavir, was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the existence of SMVT in MDCK-MDR1 cells. Biotin uptake was Na+, pH, and temperature dependent, but energy independent. Uptake of biotin was found to be saturable with a Km of 13.0 microM, Vmax 21.5 of pmol min-1 (mg of protein)-1, and Kd of 0.12 microL min-1 (mg of protein)-1. Both apical and basal uptake and transepithelial transport of [3H]biotin showed that SMVT localized predominantly on the apical membrane of MDCK-MDR1 cells. [3H]Biotin uptake was inhibited by excess unlabeled biotin and its structural analogues, i.e., desthiolbiotin and valeric acid, and other vitamins such as lipoic acid and pantothenic acid, but not by acetic acid, benzoic acid, biotin methyl ester, and biocytin. Biotin-saquinavir caused lowering of [3H]biotin uptake, which indicates that it is recognized by SMVT. Apical to basal transport of [3H]biotin was also significantly inhibited in the presence of excess biotin or biotin-saquinavir. Transepithelial transport studies of biotin-saquinavir in MDCK-MDR1, wild type MDCK, and Caco-2 cells revealed that permeability of biotin-saquinavir was similar in all three cell lines. A band of SMVT mRNA at 862 bp was identified by RT-PCR. A sodium-dependent multivitamin transporter, SMVT, responsible for biotin uptake and transport, was identified and functionally characterized in MDCK-MDR1 cells. Therefore, the MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of biotin-conjugated prodrugs such as HIV protease inhibitors.
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Sistemas de Liberación de Medicamentos/estadística & datos numéricos , Simportadores/metabolismo , Animales , Biotina/farmacocinética , Células CACO-2/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Perros , Interacciones Farmacológicas , Estudios de Factibilidad , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Profármacos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saquinavir/farmacología , Sodio/farmacología , Especificidad por SustratoRESUMEN
The objective of this research was to investigate the presence of a specialized carrier-mediated system for biotin and delineate uptake mechanism and intracellular trafficking of biotin in the human derived retinoblastoma cell line (Y-79). Human derived retinoblastoma cell line, Y-79, was used for uptake studies. Uptake of [3H]Biotin was determined at various concentrations, pH, temperatures, in the absence of sodium and in the presence of other vitamins and metabolic inhibitors to delineate the mechanism of uptake. Uptake was determined in the presence of various intracellular regulatory pathways (protein kinase A & C, protein tyrosine kinase and calcium-calmodulin) modulators. Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the molecular identity of human sodium-dependent multivitamin transporter (hSMVT). Uptake of [3H]Biotin in Y-79 cells were found to be saturable at micromolar concentration range, with apparent Km of 8.53 microM and Vmax of 14.12 pmol/min/mg protein, but linear at nanomolar concentration range. Uptake was sodium, pH, temperature and energy-dependent, but chloride independent; inhibited by the structural analogue desthiobiotin, pantothenic acid and lipoic acid at milimolar concentrations and not at nanomolar concentrations. Uptake of [3H]Biotin was trans-stimulated by the intracellular biotin. Ca2+/calmodulin pathways appeared to play important roles in the regulation of riboflavin uptake in Y-79 cells via significant reduction in Vmax (66%) and Km (28%) of the uptake process. A human sodium-dependant multivitamin transporter, hSMVT, was identified by RT-PCR in Y-79. These studies demonstrated for the first time the existence of a human sodium dependant multivitamin transporter (hSMVT), a specialized carrier-mediated system for biotin uptake, in human derived retinoblastoma cells.
Asunto(s)
Biotina/metabolismo , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Simportadores/fisiología , Transporte Biológico , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TemperaturaRESUMEN
The purpose of this study was to synthesize a series of diester prodrugs of ganciclovir (GCV), for improving ocular and oral bioavailability and therapeutic activity. Solubility, logP, pH stability profile, in vitro antiviral activity, cytotoxicity, inhibition profile and ocular tissue hydrolysis of the GCV prodrugs were measured. Val-Val-GCV and Val-Gly-GCV diesters were found to exhibit greater aqueous stability compared to Val-GCV and Gly-Val-GCV while ocular tissue hydrolysis demonstrated Val-Gly-GCV and Gly-Val-GCV to be more stable. Val-Val-GCV and Val-GCV diesters were the most lipophilic compounds and were predicted to possess a partition coefficient 295- and 12-fold greater than that of GCV, respectively. All the prodrugs possess much higher aqueous solubility than the parent drug GCV. Ex vivo uptake in the rabbit eye indicates that the prodrugs have high uptake potential. The prodrugs showed no increase in cytotoxicity compared to GCV, instead there was a marked increase in their potency against human cytomegalovirus (HCMV) as well as HSV-1 and HSV-2. This should allow therapeutic response to be seen at a lower concentration that can be achieved more easily, than the drugs currently being used. In conclusion, the diester GCV prodrugs demonstrated excellent chemical stability, high aqueous solubility and markedly enhanced antiviral potency against the herpes viruses without any increase in cytotoxicity.