RESUMEN
Background. The Centers for Disease Control and Prevention (CDC) recommends testing and linkage to care for persons most likely infected with hepatitis C virus (HCV), including persons with human immunodeficiency virus. We explored facilitators and barriers to integrating HCV point-of-care (POC) testing into standard operations at an urban STD clinic. Methods. The OraQuick HCV rapid antibody test was integrated at the Denver Metro Health Clinic (DMHC). All clients with at least one risk factor were offered the POC test. Research staff conducted interviews with clients (three HCV positive and nine HCV negative). Focus groups were conducted with triage staff, providers, and linkage-to-care counselors. Results. Clients were pleased with the ease of use and rapid return of results from the HCV POC test. Integrating the test into this setting required more time but was not overly burdensome. While counseling messages were clear to staff, clients retained little knowledge of hepatitis C infection or factors related to risk. Barriers to integrating the HCV POC test into clinic operations were loss to follow-up and access to care. Conclusion. DMHC successfully integrated HCV POC testing and piloted a HCV linkage-to-care program. Providing testing opportunities at STD clinics could increase identification of persons with HCV infection.
RESUMEN
BACKGROUND: Approximately 4.1 million Americans are estimated to have been infected with hepatitis C virus (HCV), 45-85% of whom are unaware of their infection. Persons who inject drugs (PWID) account for 55.8% of all persons with HCV antibody (anti-HCV) in the U.S. PWID have limited access to healthcare and are infrequently tested for anti-HCV using conventional laboratory assays. OBJECTIVE: To evaluate performance characteristics (sensitivity and specificity) of three, pre-market rapid point-of-care tests (one oral fluid and two finger-stick assays) from two manufacturers (Chembio and MedMira) in settings providing services to young adult PWID in San Diego, CA. STUDY DESIGN: Behavioral risk assessment surveys and testing for HCV were conducted among persons who reported injection drug use (IDU) within the past 6 months as part of the Study to Assess Hepatitis C Risk (STAHR) among PWID aged 18-40 years in 2009-2010. Sensitivity and specificity of the rapid anti-HCV assays were evaluated among STAHR participants, using two commonly used testing algorithms. RESULTS: Variability in sensitivity (76.6-97.1%) and specificity (99.0-100.0%) was found across assays. The highest sensitivity achieved for the Chembio finger-stick blood, Chembio oral fluid and MedMira finger-stick blood tests was 97.1%, 85.4% and 80.0% respectively; the highest specificity was 99.0%, 100.0% and 100.0%, respectively. In multivariate analysis false negative anti-HCV results were associated with female sex for the MedMira blood assay. CONCLUSIONS: Sensitive anti-HCV rapid assays are appropriate and feasible for high-prevalence, high-risk populations such as young PWID.
Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/diagnóstico , Virología/métodos , Adolescente , Adulto , Femenino , Humanos , Inmunoensayo/métodos , Masculino , Sistemas de Atención de Punto , Sensibilidad y Especificidad , Abuso de Sustancias por Vía Intravenosa/complicaciones , Factores de Tiempo , Estados Unidos , Adulto JovenRESUMEN
This study examines cytotoxicity of poly-methylmethacrylate (PMMA)-based dental temporary filling resin to dental pulp cells, and the potential amelioration of the toxicity with an anti-oxidant amino-acid, N-acetyl cysteine (NAC). Dental pulp cells extracted from rat maxillary incisors were cultured on the resin material with or without NAC incorporation, or on the polystyrene. The cultures were supplied with osteoblastic media, containing dexamethasone. Forty five percent of cells on the PMMA dental resin were necrotic at 24h after seeding. However, this percentage was reduced to 27% by incorporating NAC in the resin, which was the level equivalent to that in the culture on polystyrene. The culture on the untreated resin was found to be negative for alkaline phosphate (ALP) activity at days 5 and 10 or von Kossa mineralized nodule formation at day 20. In contrast, some areas of the cultures on NAC-incorporated resin substrates were ALP and von Kossa positive. Collagen I and dentin sialoprotein genes were barely expressed in day 7 culture on the untreated resin. However, those genes were expressed in the culture on the resin with NAC. These results suggest that the decreased cell viability and the nearly completely suppressed odontoblast-like cell phenotype of dental pulp cells cultured on PMMA dental resin can be salvaged to a biologically significant degree by the incorporation of NAC in the resin.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Resinas Compuestas/toxicidad , Pulpa Dental/efectos de los fármacos , Polimetil Metacrilato/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Resinas Compuestas/química , Citoprotección , Pulpa Dental/citología , Pulpa Dental/metabolismo , Restauración Dental Provisional , Proteínas de la Matriz Extracelular , Expresión Génica , Masculino , Odontoblastos/metabolismo , Fosfoproteínas , Precursores de Proteínas/biosíntesis , Ratas , Ratas Sprague-Dawley , SialoglicoproteínasRESUMEN
Potential risks of the use of resin-based restorative materials include direct damage to the pulp cells and the induction of hypersensitivity reactions in patients. In this study, we tested the hypothesis that N-acetyl cysteine (NAC) inhibits resin toxicity and restores the function of pulp cells. Analysis of our data demonstrates toxicity of composite resins on pulp cells in both an in vivo rat and an ex vivo human model system. Moreover, cells that survive after the placement of composites are weaker, and they are induced to undergo cell death when exposed to 2-hydroxyethyl methacrylate (HEMA). The toxic effect of composites on pulp cells is neutralized by NAC. Therefore, NAC protects the cells from damage induced by clinically relevant levels of restorative materials, in both rat and human model systems. The addition of N-acetyl cysteine prior to or concomitant with the application of restorative materials may be beneficial for the health and safety of dental patients.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Muerte Celular/efectos de los fármacos , Resinas Compuestas/toxicidad , Pulpa Dental/efectos de los fármacos , Animales , Pulpa Dental/citología , Restauración Dental Permanente/efectos adversos , Humanos , Masculino , Metacrilatos/toxicidad , Tercer Molar , Ratas , Ratas Sprague-Dawley , Cementos de Resina/toxicidad , Células del Estroma/efectos de los fármacosRESUMEN
Despite its proven cytotoxicity, poly-methyl methacrylate (PMMA) resin is one of the most frequently and extensively used materials in dental practice. This study hypothesized that an anti-oxidant amino acid, N-acetyl cysteine (NAC), has the potential to detoxify this material. Ten percent of the rat dental pulp cells were viable when cultured on the PMMA resin for 24 hours, while over 70% of the cells were viable on the NAC-added resin. Nearly all suppressed alkaline phosphatase activity, matrix mineralizing capability, and odontoblastic gene expression, such as dentin sialoprotein, on the untreated control resin was recovered by NAC in a concentration-dependent manner. A Ca/P ratio of 1.65 was found in the extracellular matrix of cultures on NAC-added resin, while that in the untreated resin culture was 0.70. The addition of NAC to PMMA resin significantly ameliorated its cytotoxicity to the dental pulp cells and restored their odontoblast-like cell phenotype to a biologically significant degree.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Pulpa Dental/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Polimetil Metacrilato/toxicidad , Acetilcisteína/administración & dosificación , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/efectos de los fármacos , Animales , Antioxidantes/administración & dosificación , Apoptosis/efectos de los fármacos , Biotransformación , Calcificación Fisiológica/efectos de los fármacos , Calcio/análisis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/antagonistas & inhibidores , Colágeno Tipo I/efectos de los fármacos , Pulpa Dental/citología , Relación Dosis-Respuesta a Droga , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular , Depuradores de Radicales Libres/administración & dosificación , Masculino , Odontoblastos/efectos de los fármacos , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/efectos de los fármacos , Fósforo/análisis , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , SialoglicoproteínasRESUMEN
The GroEL chaperonin has the ability to behave as an unfoldase, repeatedly denaturing proteins upon binding, which in turn can free them from kinetic traps and increase their folding rates. The complex formed by GroEL+GroES+ATP can also act as an infinite dilution cage, enclosing proteins within a protective container where they can fold without danger of aggregation. Controversy remains over which of these two properties is more critical to the GroEL/ES chaperonin's function. We probe the importance of the unfoldase nature of GroEL under conditions where aggregation is the predominant protein degradation pathway. We consider the effect of a hypothetical mutation to GroEL which increases the cycle frequency of GroEL/ES by increasing the rate of hydrolysis of GroEL-bound ATP. Using a simple kinetic model, we show that this modified chaperonin would be self-defeating: any potential reduction in folding time would be negated by an increase in time spent in the bulk, causing an increase in aggregation and a net decrease in protein folding yields.
Asunto(s)
Chaperonina 10/química , Chaperonina 10/ultraestructura , Chaperonina 60/química , Chaperonina 60/ultraestructura , Modelos Químicos , Modelos Moleculares , Sitios de Unión , Chaperoninas/química , Chaperoninas/ultraestructura , Dimerización , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión Proteica , Conformación Proteica , Pliegue de ProteínaRESUMEN
Mounting effective anti-tumor immune responses against tumors by both the innate and adaptive immune effectors is important for the clearance of tumors. However, accumulated evidence indicates that immune responses that should otherwise suppress or eliminate transformed cells are themselves suppressed by the function of tumor cells in a variety of cancer patients, including those with oral cancers. Signaling abnormalities, spontaneous apoptosis, and reduced proliferation and function of circulating natural killer cells (NK), T-cells, dendritic cells (DC), and tumor-infiltrating lymphocytes (TILs) have been documented previously in oral cancer patients. Several mechanisms have been proposed for the functional deficiencies of tumor-associated immune cells in oral cancer patients. Both soluble factors and contact-mediated immunosuppression by the tumor cells have been implicated in the inhibition of immune cell function and the progression of tumors. More recently, elevated levels and function of key transcription factors in tumor cells, particularly NFkappaB and STAT3, have been shown to mediate immune suppression in the tumor microenvironment. This review will focus on these emerging mechanisms of immunosuppression in oral cancers.
Asunto(s)
Tolerancia Inmunológica/inmunología , Neoplasias de la Boca/inmunología , Apoptosis/inmunología , Células Dendríticas/inmunología , Humanos , Inmunidad Celular/inmunología , Células Asesinas Naturales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , FN-kappa B/inmunología , Factor de Transcripción STAT3/inmunología , Transducción de Señal/inmunologíaRESUMEN
A variety of small cageless chaperones have been discovered that can assist protein folding without the consumption of ATP. These include mini-chaperones (catalytically active fragments of larger chaperones), as well as small proteins such as alpha-casein and detergents acting as "artificial chaperones." These chaperones all possess exposed hydrophobic patches on their surface that act as recognition sites for misfolded proteins. They lack the complexity of chaperonins (that encapsulate proteins in their inner rings) and their study can offer insight into the minimal requirements for chaperone function. We use molecular dynamics simulations to investigate how a cageless chaperone, modeled as a sphere of tunable hydrophobicity, can assist folding of a substrate protein. We find that under steady-state (non-stress) conditions, cageless chaperones that bind to a single substrate protein increase folding yields by reducing the time the substrate spends in an aggregation-prone state in a dual manner: (a) by competing for aggregation-prone hydrophobic sites on the surface of a protein, hence reducing the time the protein spends unprotected in the bulk and (b) by accelerating folding rates of the protein. In both cases, the chaperone must bind to and hold the protein loosely enough to allow the protein to change its conformation and fold while bound. Loose binding may enable small cageless chaperones to help proteins fold and avoid aggregation under steady-state conditions, even at low concentrations, without the consumption of ATP.
Asunto(s)
Chaperonas Moleculares/química , Pliegue de Proteína , Simulación por Computador , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Unión Proteica , TermodinámicaRESUMEN
The periodontal pathogen Fusobacterium nucleatum induces apoptosis in lymphocytes. We previously identified the autotransporter protein Fap2 in F. nucleatum strain PK1594 that induced apoptosis in lymphocytes when expressed in Escherichia coli. In this study, we identified protein homologs of Fap2 in the transformable F. nucleatum strain ATCC 23726, to determine their role in the induction of apoptosis in lymphocytes. We used a new gene-inactivation vector conferring thiamphenicol resistance (pHS31) to construct a mutant deficient in one of the homologs, aim1. Transcriptional analyses demonstrated disruption of aim1 expression, and phenotypic analyses revealed a 41% decrease in the ability of the mutant to induce apoptosis in Jurkat cells, as compared with the parental strain. These studies demonstrate, in the native host cell background, the contribution of aim1 to F. nucleatum induction of apoptosis and, to the best of our knowledge, represent the first report of a genetically defined and phenotypically characterized mutation in F. nucleatum.
Asunto(s)
Apoptosis/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , ADN Bacteriano , Fusobacterium nucleatum/química , Marcación de Gen , Genes Bacterianos , Humanos , Células Jurkat , Proteínas de Transporte de Membrana/fisiología , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Plásmidos , Linfocitos T/microbiología , Transcripción GenéticaRESUMEN
Mechanisms by which the resin monomer 2-hydroxyethyl methacrylate (HEMA) induces hypersensitivity reactions in humans are not well-established, nor have the direct effects of HEMA on cell death been fully characterized. The objective of this study was to establish whether HEMA is capable of inducing apoptotic cell death, and whether differences exist in the levels of apoptotic death induced by HEMA in cells obtained from healthy individuals and from patients with established HEMA hypersensitivity. HEMA induced apoptotic death in Peripheral Blood Mononuclear Cells (PBMCs) obtained from both healthy and HEMA-sensitized patients and in the murine RAW cells in a dose-dependent manner. However, induction of cell death by HEMA was lower in PBMCs obtained from patients in comparison with healthy individuals. Studies reported in this paper demonstrate that HEMA induces apoptotic death, and that decreased susceptibility of lymphocytes to HEMA-mediated death might be an important mechanism for the generation and persistence of hypersensitivity reactions in patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Resinas Compuestas/efectos adversos , Hipersensibilidad/inmunología , Metacrilatos/efectos adversos , Monocitos/efectos de los fármacos , Animales , Apoptosis/inmunología , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Análisis por Apareamiento , Ratones , Polietilenglicoles/efectos adversos , Ácidos Polimetacrílicos/efectos adversosRESUMEN
Recent experiments suggest that the folding of certain proteins can take place entirely within a chaperonin-like cavity. These substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. Rather, they undergo only a single binding event, followed by sequestration into the chaperonin cage. The present work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within this chaperonin cavity. The chaperonin interior is modeled by a sphere with a lining of tunable degree of hydrophobicity. We demonstrate that a moderately hydrophobic environment, similar to the interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations support a mechanism by which the moderately hydrophobic chaperonin environment provides an alternate pathway to the native state through a transiently bound intermediate state.
Asunto(s)
Chaperoninas/química , Pliegue de Proteína , Interacciones Hidrofóbicas e Hidrofílicas , Procesos Estocásticos , TermodinámicaRESUMEN
Oral epithelium may play a regulatory role in local immune responses when interacting with bacteria. The present study was undertaken to investigate the effects of selected bacterial pathogens found in periodontal and endodontic infections on oral epithelial cells. Expression of cell surface molecules (major histocompatibility complex (MHC) Class II, CD54, CD70, CD80 and CD86) and secretion of inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha) in response to selected bacterial challenge were examined on an immortalized oral epithelial cell line, HOK-18A and a skin epithelial cell line, HaCaT. Actinomyces viscosus, Actinomyces israelii, Fusobacterium nucleatum lipopolysaccharide (LPS) or primary human periradicular exudate from a granuloma were co-cultured with epithelial cells for 4 or 24 h. Subsequently, cell surface expression of MHC Class II, CD54, CD70, CD80 and CD86, along with pro-inflammatory cytokine levels were determined using flow cytometry, ELISA and RT-PCR. Results indicated that the selected oral bacteria have greater effects on oral versus skin epithelial cells. F. nucleatum increased MHC Class II and CD54 (ICAM-1) cell surface expression on HOK-18A and HaCaT cells. A. israelii also had enhancing effects on the expression of CD54 and MHC Class II. A. israelii and LPS induced a 2.8-fold (P < 0.001) and 4.4-fold (P < 0.005) TNF-alpha secretion, respectively, while F. nucleatum and LPS induced a 10-fold (P < 0.0004) and 6-fold (P < 0.01) IL-1beta secretion, respectively by HOK-18A. Interestingly, CD70, CD80, and CD86 were generally decreased upon bacteria and LPS challenge on HOK-18A. The effects of increased MHC Class II and decreased CD70 were also evident with challenge of human periradicular exudate on HOK-18A. The implications of the study are unique in that oral epithelial cells may play both activating and inhibitory roles in the host immune response towards infection by oral bacteria. We introduce a concept of 'dormancy' where the differential expression of key cell surface antigens on oral epithelial cells may keep the recruited immune effector cells in a state of unresponsiveness, thus contributing to the long term quiescent period observed in many periodontal and endodontic lesions.
Asunto(s)
Actinomyces/inmunología , Antígenos CD/análisis , Citocinas/análisis , Fusobacterium nucleatum/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Mucosa Bucal/inmunología , Adulto , Antígeno B7-1/análisis , Antígeno B7-2 , Ligando CD27 , Línea Celular , Células Epiteliales/inmunología , Exudados y Transudados , Femenino , Humanos , Mediadores de Inflamación/análisis , Molécula 1 de Adhesión Intercelular/análisis , Interleucina-1/análisis , Interleucina-6/análisis , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/análisis , Proteínas de la Membrana/análisis , Granuloma Periapical/inmunología , Piel/inmunología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (TAsunto(s)
Chaperonina 60/química
, Chaperonina 60/metabolismo
, Pliegue de Proteína
, Proteínas/química
, Termodinámica
, Simulación por Computador
, Interacciones Hidrofóbicas e Hidrofílicas
, Cinética
, Modelos Moleculares
, Proteínas/metabolismo
, Temperatura
RESUMEN
The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.
Asunto(s)
Resinas Compuestas/metabolismo , Resinas Compuestas/toxicidad , Polietilenglicoles/metabolismo , Polietilenglicoles/toxicidad , Ácidos Polimetacrílicos/metabolismo , Ácidos Polimetacrílicos/toxicidad , Animales , Resinas Compuestas/administración & dosificación , Femenino , Cobayas , Inyecciones Intravenosas , Inyecciones Subcutáneas , Intubación Gastrointestinal , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/administración & dosificación , Ácidos Polimetacrílicos/administración & dosificación , Distribución TisularRESUMEN
Addition of antibodies to major histocompatibility complex class I (MHC class I) and F(c) gamma RIII (CD16) antigens resulted in the synergistic augmentation of natural killer (NK) cell death, and the loss of NK cell cytotoxic function. The binding of anti-CD16 and anti-class I antibodies to the same population of NK cells is required for the synergistic augmentation of NK cell death. Moreover, the addition of antibodies to leukocyte function antigen-1 (LFA-1), which significantly inhibited the phorbol 12-myristate 13-acetate (PMA) and ionomycin mediated NK cell death, had no effect on NK cell death mediated by anti-CD16 and anti-class I antibodies. The increase in NK cell death was associated with an increase in tumor necrosis factor-alpha (TNF-alpha) secretion, and concomitant inhibition of nuclear factor kappa B (NFkappaB) activation and the induction of c-jun N-terminal kinase (JNK) activity in NK cells treated with the combination of anti-class I and anti-CD16 antibodies. Furthermore, the inhibition of NFkappaB activation in anti-CD16 and anti-class I antibody treated NK cells was paralleled with a significant increase in inhibitor of kappa B (IkappaB) protein expression. Overexpression of IkappaB super-repressor in YT, a NK cell line, caused significant up-regulation of TNF-alpha, PMA and ionomycin and Fusobacterium nucleatum mediated NK cell death. Overall, our studies suggest an important regulatory role for NFkappaB and JNK activities in MHC class I mediated NK cell death.
Asunto(s)
Apoptosis/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/citología , Proteínas Quinasas Activadas por Mitógenos/inmunología , FN-kappa B/inmunología , Receptores de IgG/inmunología , Transducción de Señal/inmunología , Anticuerpos Monoclonales/inmunología , Células Cultivadas , ADN/metabolismo , Activación Enzimática , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismoRESUMEN
Recently, we presented evidence for the role of MGN-3, an enzymatically modified arabinoxylan extracted from rice bran, in potent activation of human natural killer (NK) cell function in vivo and in vitro. In the current study, we examined the mechanism by which MGN-3 elevated NK cytotoxic activity. We did this by testing the action of MGN-3 on the levels of both tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) secretions and MGN-3 function on the expression of key cell surface receptors. Peripheral blood lymphocytes were treated with MGN-3 at concentrations of 0.1 mg/ml and 1 mg/ml, and supernatants were subjected to enzyme-linked immunosorbent assay. Results showed that MGN-3 is a potent TNF-alpha inducer. The effect was dose-dependent. MGN-3 concentration at 0.1 and 1 mg/ml increased TNF-alpha production by 22.8- and 47. 1-fold, respectively. MGN-3 also increased production of IFN-gamma but at lower levels as compared to TNF-alpha With respect to key cell surface receptors, MGN-3 increases the expression of CD69, an early activation antigen at 16 hours after treatment. Furthermore, the interleukin-2 receptor CD25 and the adhesion molecule ICAM-1 (CD54) were upregulated after treatment with MGN-3. Treating highly purified NK cells with MGN-3 also resulted in increased levels of TNF-alpha and IFN-gamma secretion in conjunction with augmentation of NK cell cytotoxic function. Furthermore, addition of MGN-3 to interleukin-2-activated NK cells resulted in a synergistic induction of TNF-alpha and IFN-gamma secretion. Overall, our data suggest that MGN-3, a novel biological response modifier, can be used as a safe alternative or as an adjuvant to the existing immunotherapeutic modalities.
Asunto(s)
Factores Inmunológicos/farmacología , Interferón gamma/biosíntesis , Células Asesinas Naturales/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Xilanos/farmacología , Adolescente , Adulto , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Células Cultivadas , Citotoxicidad Inmunológica , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-2/farmacología , Interleucina-2/fisiología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Probabilidad , Receptores de Interleucina-2/biosíntesis , Coloración y Etiquetado , Regulación hacia ArribaRESUMEN
We have developed new computational methods for displaying and analyzing members of protein superfamilies. These methods (MinRMS, AlignPlot and MSFviewer) integrate sequence and structural information and are implemented as separate but cooperating programs to our Chimera molecular modeling system. Integration of multiple sequence alignment information and three-dimensional structural representations enable researchers to generate hypotheses about the sequence-structure relationship. Structural superpositions can be generated and easily tuned to identify similarities around important characteristics such as active sites or ligand binding sites. Information related to the release of Chimera, MinRMS, AlignPlot and MSFviewer can be obtained at http:¿www.cgl.ucsf.edu/chimera.
Asunto(s)
Proteínas/química , Alineación de Secuencia/métodos , Análisis de Secuencia/métodos , Programas Informáticos , Secuencia de Aminoácidos , Gráficos por Computador , Creatina Quinasa/química , Bases de Datos Factuales , Glutamato-Amoníaco Ligasa/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia/estadística & datos numéricos , Análisis de Secuencia/estadística & datos numéricosRESUMEN
The cytotoxic activity and survival of NK cells are negatively regulated in the presence of antibodies directed against the FCgammaRIII (CD16) surface receptor. The addition of MHC-class I monoclonal antibody (mAb) in combination with CD16 mAb resulted in a significant potentiation of the inhibition of NK cell cytotoxic function and both accelerated kinetics and synergistic induction of cell death in both resting and IL-2-treated human peripheral blood purified NK cells. The potentiating effect of class I MHC monoclonal antibody was specific as monoclonal antibodies directed against other cell surface antigens on NK (e.g., LFA-3, LFA-1, and CD56) had no effect. A correlation was observed between the levels of secreted TNF-alpha and the frequency of NK cell death and the addition of anti-TNF-alpha antibody inhibited NK cell death. The levels of death promoting gene products such as the Fas receptor and the Fas ligand were upregulated in NK cells in the presence of anti-class I and anti-CD16 antibodies. However, anti-Fas antibodies did not block cell death. These findings demonstrate that MHC-class I antigens regulate both TNF-alpha secretion and cell death of anti-CD16 mAb treated NK cells. These results also suggest that MHC-class I antigens regulate the function and survival of activated NK cells.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Superficie/biosíntesis , Apoptosis/inmunología , Biomarcadores , Supervivencia Celular/inmunología , Citotoxicidad Inmunológica , Sinergismo Farmacológico , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Células K562 , Lectinas Tipo C , Activación de Linfocitos , Receptores de IgG/inmunología , Receptores de Interleucina-2/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells. In this paper we present evidence for the immunosuppressive role of Fusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease. Our studies indicate that the immunosuppressive role of F. nucleatum is largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs). F. nucleatum treatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays. The ability of F. nucleatum to induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis. The data also indicated that F. nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-kappaB. Possible mechanisms of F. nucleatum's role in the pathogenesis of periodontal disease are discussed.
Asunto(s)
Apoptosis , Fusobacterium nucleatum/fisiología , Leucocitos Mononucleares/patología , Boca/microbiología , Neutrófilos/patología , Caspasa 1/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Hipersensibilidad/inmunología , Terapia de Inmunosupresión , Células Jurkat , FN-kappa B/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The interaction of natural killer (NK) cells with target cells, such as K562, results in NK functional inactivation and apoptosis. The role of NK-activating cytokines, IL-2, IL-12, and IL-15, in the regulation of NK inactivation and programmed cell death by target cells was examined. Purified natural killer cells were obtained from human peripheral blood and either co-incubated with K562 target cells and cytokines or the NK cells were pretreated with cytokines for 18 h prior to co-culture with K562 cells. Sorted NK cells were examined for cytotoxic activity and NK co-cultured with K562 were examined for cytokine secretion, phenotyping and DNA fragmentation. The cytotoxic activity was inhibited and was not alleviated by cytokine treatment. Whereas the cytokine treatment maintained NK cell viability for several days, NK cell viability was decreased significantly in the presence of K562 target cells. Downregulation of CD16 and upregulation of CD69 on NK cells were induced by K562 target cells and no modulation of these antigens was observed with cytokine treatment. A subpopulation of target-treated NK cells succumbed to cell death by apoptosis and cell death was not rescued by the activating cytokines. These findings demonstrate that target-induced functional inactivation and apoptosis of NK cells were not rescued by the activating cytokines IL-2, IL-12, and IL-15 regardless of whether the NK cells were pretreated with cytokines prior to exposure to K562 or the cytokines were added to the NK-K562 mixtures. These results also suggest that signals triggered by the target cells and resulting in NK cell anergy and apoptosis override cytokine-mediated signals for activation, cell proliferation, and survival.