RESUMEN
Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis, and its intraoral levels have been shown to predict disease progression (activity). An accurate and sensitive chair-side (point of care) test to determine disease activity is critical for early intervention and clinical management of disease. This study aimed to develop a rapid, chair-side, saliva-based detection of P. gingivalis. Monoclonal antibodies (mAbs) to the A1-adhesin domain of the P. gingivalis RgpA-Kgp proteinase-adhesin complex were screened by enzyme-linked immunosorbent assay and microbial flow cytometry, with 2 mAbs shown to recognize all laboratory and clinical strains tested, without significantly cross-reacting with other oral bacteria tested. With these mAbs, an immunochromatographic device was produced and shown in preclinical studies to detect, in inoculated saliva, all P. gingivalis laboratory strains and clinical isolates tested. The device was able to detect ≥1 × 105 P. gingivalis cells/mL. In a patient age- and sex-matched control clinical cohort, P. gingivalis levels in saliva-as measured by real-time polymerase chain reaction-positively correlated with P. gingivalis levels in subgingival plaque ( r = 0.819, P < 0.01) and clinical parameters of disease ( r = 0.633, P < 0.01). A positive device result strongly correlated with P. gingivalis levels >1 × 105 cells/mL in saliva ( r = 0.778, P < 0.001) and subgingival plaque ( r = 0.715, P < 0.001) with sensitivity, specificity, positive/negative predictive values, and accuracy levels of 95.0%, 93.3%, 90.5%, 96.6%, and 94.0%, respectively. The device result also positively correlated ( r = 0.695, P < 0.01) with disease severity as measured by probing depth. Detection of P. gingivalis in saliva was found to be rapid, taking 3 min from sample collection.
Asunto(s)
Técnicas Bacteriológicas/instrumentación , Periodontitis Crónica/microbiología , Placa Dental/microbiología , Sistemas de Atención de Punto , Porphyromonas gingivalis/aislamiento & purificación , Saliva/microbiología , Adhesinas Bacterianas , Anticuerpos Monoclonales , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Valor Predictivo de las Pruebas , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Murine cytomegalovirus (MCMV) infection of BALB/c mice produces acute and chronic myocarditis similar to clinical disease in humans. In contrast, MCMV-infected C57BL/6 mice develop only mild acute myocarditis. We have investigated the effect of administration of the immunomodulator lipopolysaccharide (LPS) on the development of postviral myocarditis in mice. LPS exacerbated heart inflammation in both strains of MCMV-infected mice, with normally resistant C57BL/6 mice developing chronic myocarditis. Autoantibodies to cardiac myosin were enhanced with LPS treatment in both MCMV-infected mouse strains. LPS treatment also increased the production of TNF in the sera without affecting virus titers in the spleen, liver, or salivary glands, a target organ most affected during persistent virus infection. In LPS/MCMV-infected BALB/c mice, TNF, IL-6, and IL-10 levels were detected in cultures of heart infiltrating cells but not in splenocytes. Importantly, administration of the bioactive synthetic TNF peptide (amino acids 114-130) increased myocarditis in C57BL/6 mice, similar to that seen with LPS treatment. TNF peptide/MCMV-infected BALB/c and C57BL/6 mice showed distinct differences in the expression pattern of IFN-gamma, IL-10, and TNF. These data show that the disease may be partly regulated by TNF among other select cytokines and autoantibodies to cardiac myosin. The immunopathological nature of MCMV-induced myocarditis is thus highlighted.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Lipopolisacáridos/inmunología , Muromegalovirus/inmunología , Miocarditis/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Autoanticuerpos/sangre , Miosinas Cardíacas/inmunología , Células Cultivadas , Citocinas/biosíntesis , Infecciones por Herpesviridae/sangre , Infecciones por Herpesviridae/virología , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muromegalovirus/efectos de los fármacos , Muromegalovirus/fisiología , Miocarditis/sangre , Miocarditis/virología , Miocardio/citología , Péptidos/inmunología , Péptidos/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
The cardiovascular disease myocarditis is characterized by inflammation and necrosis of cardiac muscle. This disease has been associated with various viral etiologies, including cytomegalovirus (CMV). Murine CMV (MCMV) infection of adult BALB/c mice produces a disease with acute and chronic phases similar to that found in humans. In our murine model, we have investigated the therapeutic efficacy of antiviral drug administration on myocarditis. Two drugs commonly used for CMV treatment, ganciclovir and cidofovir, were subjected to trials, with both drugs showing potent antiviral activity against MCMV both in vitro and in vivo. The acute phase of myocarditis was significantly reduced when antiviral therapy commenced 24 h postinfection. Such treatment also reduced the severity of the chronic phase of myocarditis. In contrast, antiviral treatment commencing after the acute phase had no effect on chronic myocarditis. Reinfection of mice with MCMV caused exacerbation of myocardial inflammation. Such an increase in severity of myocarditis could be prevented with either ganciclovir or cidofovir treatment, but the preexisting inflammation and necrosis of the myocardium persisted. These data highlight possible therapeutic uses of antiviral drugs in viral myocarditis as well as further elucidating the pathogenic nature of the disease.
Asunto(s)
Antivirales/uso terapéutico , Citosina/uso terapéutico , Ganciclovir/uso terapéutico , Infecciones por Herpesviridae/tratamiento farmacológico , Miocarditis/tratamiento farmacológico , Organofosfonatos , Compuestos Organofosforados/uso terapéutico , Animales , Cidofovir , Citosina/análogos & derivados , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Muromegalovirus/efectos de los fármacos , Miocarditis/virologíaRESUMEN
The beneficial effects of cyclo-oxygenase (COX) inhibitors in both colon cancer and adenomatous polyps suggest a role for the prostanoid pathway in epithelial malignancy. Although variable prostanoid synthesis in non-small cell lung cancer (NSCLC) has been demonstrated in freshly obtained tissue, COX messenger ribonucleic acid (mRNA) and protein localization in such tumours had not been investigated ex vivo. Thirty-four cases of primary NSCLC were examined for both constitutive (COX-1) and inducible COX (COX-2) by means of in situ hybridization and immunohistochemistry. COX-1 mRNA expression was absent or below the level of detection via in situ hybridization. COX-1 immunohistochemistry demonstrated uniform faint cytoplasmic staining in tumour cells and stromal inflammatory cells. Semiquantitative analysis of COX-2 expression in NSCLC demonstrated the highest levels of both mRNA and protein in adenocarcinoma cells (n=10, p<0.005 compared with large cell and squamous cell carcinoma), intermediate and variable expression in large cell carcinoma (n=11) and low or absent expression in squamous cell tumours (n=13). Levels of COX-2 expression in infiltrating inflammatory cells was the same in all tumour types. In conclusion, tumour cell cyclo-oxygenase-2 rather than cyclo-oxygenase-1 expression may account for the variable prostanoid production seen in non-small cell lung cancer, and primary lung adenocarcinoma expresses the highest levels of cyclooxygenase-2. Assessment of cyclo-oxygenase-2 expression ex vivo should be performed in studies examining the potential therapeutic effects of cyclo-oxygenase inhibitors in non-small cell lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Isoenzimas/genética , Neoplasias Pulmonares/enzimología , Prostaglandina-Endoperóxido Sintasas/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Pulmón/enzimología , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas de la Membrana , Pronóstico , ARN Mensajero/genéticaRESUMEN
Cyclo-oxygenase is the rate-limiting enzyme in the prostanoid pathway. Although expression of the inducible isoform of cyclo-oxygenase (COX-2) is associated with cytokine-mediated inflammation, recent evidence suggests a homeostatic role for epithelial COX-2 in the gastrointestinal tract. The aim of this study was to examine the expression and localization of COX-2 in human airway epithelium both in vivo and in vitro. Human airway specimens from patients undergoing lung resection surgery for primary lung tumours (n=10) or nasal mucosal resection for non-inflammatory nasal obstruction (n=5) were examined for COX-2 expression by in situ hybridization and immunohistochemistry. COX-2 expression was also studied in two human airway epithelial cell lines (BEAS-2B and A549) using reverse transcription polymerase chain reaction and Northern and Western blot analysis. COX-2 messenger ribonucleic acid (mRNA) and protein were localized to individual columnar epithelial cells and to airway resident inflammatory cells in 9/10 lower and 5/5 upper airway specimens. Expression of COX-2 did not correlate with evidence of airway inflammation. Focal expression of COX-2 mRNA and protein was observed in bronchus-associated lymphoid tissue. Both COX-2 mRNA and protein were detected in BEAS-2B and A549 cells cultured under standard conditions. In conclusion, expression of COX-2 in human airway epithelium occurs in the upper and lower airways, is widespread in airway epithelial and airway resident inflammatory cells in the absence of overt airway inflammation, and is detectable in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. These data suggest a potentially important homeostatic role for COX-2 in the regulation of human airway contractility, inflammation and immune responses.
Asunto(s)
Isoenzimas/metabolismo , Pulmón/enzimología , Mucosa Nasal/enzimología , Peroxidasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Adulto , Anciano , Northern Blotting , Western Blotting , Células Cultivadas , Ciclooxigenasa 2 , Células Epiteliales/enzimología , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Pulmón/citología , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Mucosa Nasal/citología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.
Asunto(s)
Glutamato-Cisteína Ligasa/metabolismo , Pulmón/enzimología , Recuento de Células , División Celular , Línea Celular , Medios de Cultivo , Células Epiteliales/citología , Células Epiteliales/enzimología , Glutatión/metabolismo , Humanos , Pulmón/citología , Factores de TiempoRESUMEN
The roles of the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-12, in murine cytomegalovirus (MCMV) disease were investigated in susceptible BALB/c and resistant C57BL/6 mice. MCMV infection induced IL-1 and TNF-alpha production by peritoneal cells from BALB/c mice, as demonstrated previously in C57BL/6 mice. Overt ill-health and viral replication in the spleens of BALB/c mice were increased by in vivo treatment with soluble TNF-alpha receptors to inhibit the activity of this cytokine, whilst antibodies to IL-12 had a similar but more restricted effect C57BL/6 mice were not affected by either treatment, suggesting TNF-alpha and IL-12 are not critical for natural killer cell-mediated restriction of viral replication in the spleen. Soluble TNF-alpha receptors and antibodies to IL-12 also enhanced MCMV titres and numbers of viral antigen-positive cells in the livers of BALB/c mice and TNF-alpha receptors have similar effects in C57BL/6 livers. In contrast, IL-1 receptors improved the health of MCMV-infected BALB/c mice and reduced viral replication and hepatitis at some time-points. Mechanisms which may underlie these changes are discussed.