Pneumocystis activates human alveolar macrophage NF-kappaB signaling through mannose receptors

Alveolar macrophages (AM) represent important effector cells in the innate immune response to the AIDS-related pathogen Pneumocystis, but the early AM host defense signaling events are poorly defined. Using AM from healthy individuals, we showed in the present study that Pneumocystis organisms stimulate AM NF-kappaB p50 and p65 nuclear translocation in a time-dependent and multiplicity-of-infection-dependent manner as determined by electrophoretic mobility shift assay and immunofluorescence microscopy and that NF-kappaB nuclear translocation is associated with I-kappaB phosphorylation. Importantly, competitive inhibition of mannose receptor and targeted short interfering RNA-mediated gene suppression of mannose receptor mRNA and protein is associated with complete elimination of NF-kappaB nuclear translocation in response to Pneumocystis. Furthermore, human immunodeficiency virus (HIV) infection of AM (as a model human disease state of reduced AM mannose receptor expression and function) inhibits Pneumocystis-mediated NF-kappaB nuclear translocation and is associated with reduced I-kappaB phosphorylation and reduced interleukin-8 (IL-8) release. In contrast, NF-kappaB nuclear translocation and IL-8 release in response to lipopolysaccharide are intact in AM from both healthy and HIV-infected individuals, indicating that the observed impairment is not a global disturbance of the NF-kappaB pathway. Thus, in addition to phagocytic and endocytic effector functions, the present study identifies mannose receptors as pattern recognition receptors capable of NF-kappaB activation in response to infectious non-self challenge. AM mannose receptor-mediated NF-kappaB activation may represent an important mechanism of the host cell response to Pneumocystis, and altered NF-kappaB activation in the context of HIV infection may impair a critical innate immune signaling response and may contribute to pathogenesis of opportunistic lung infections.     

The future is here: Integrating genetics into the pediatric pulmonary clinic

Recognition of underlying genetic etiologies of disease is increasing at an exponential rate, likely due to greater access to and lower cost of genetic testing. Monogenic causes of disease, or conditions resulting from a mutation or mutations in a single gene, are now well recognized in every subspecialty, including pediatric pulmonary medicine; thus, it is important to consider genetic conditions when evaluating children with respiratory disease. In the pediatric pulmonary clinic, genetic testing should be considered when multiple family members present with similar or related clinical features and when individuals have unusual clinical presentations, such as early‐onset disease or complex, syndromic features. This review provides a practical guide for genetic diagnosis in the pediatric pulmonary setting, including a review of genetic concepts, considerations for test selection and results in interpretation, as well as an overview of genetic differential diagnoses for common pediatric pulmonary phenotypes. Genetic conditions that commonly present to the pediatric pulmonary clinic are reviewed in a companion article by Yonker et al.     

Changes in gingival crevicular fluid matrix metalloproteinase-8 levels during periodontal treatment and maintenance

OBJECTIVES: The aim of this study was to investigate the effect of scaling and root planing (SRP) and the maintenance phase of treatment on the gingival crevicular fluid (GCF) matrix metalloproteinase-8 (MMP-8) levels. MATERIALS AND METHODS: Clinical measurements and GCF samples were taken from four sites in 20 adult periodontitis patients before and after SRP and during a 3-month maintenance phase of treatment. MMP-8 levels were measured from GCF samples by time-resolved immunofluorometric assay (IFMA) with monoclonal antibodies. RESULTS: SRP improved the clinical indices as would be predicted, 6.1 mm (SD = 1.4) at baseline compared with 4.3 mm (SD = 1.6) post-treatment (P < 0.001). Attachment level (AL) reduced but not significantly between these two visits 13.4 mm (SD = 2.4) compared with 12.8 mm (SD = 2.4) (P < 0.08) post therapy. GCF MMP-8 levels reduced after initial treatment from 33.8 micro g/30 s sample to 23.5 micro g/30 s, which just failed to reach statistical significance (P = 0.07). However, when MMP-8 levels were expressed as a concentration, the differences following initial therapy were significant (54.1 ng/ micro L at baseline compared with 34.2 ng/micro L post treatment; P < 0.005). The difference, however, between the baseline MMP-8 levels (33.8 ng/30 s) and the final visit (16 ng/30 s) following maintenance was markedly significant (P < 0.001) for both absolute amounts and on a concentration basis. CONCLUSION: In conclusion, clinical improvement following SRP was associated with significant reductions in MMP-8 levels. The GCF concentration of MMP-8 decreased after initial therapy but reduced even more dramatically (approximately 50%) following a 3-month period of maintenance (P < 0.001).     

Humoral immune responses in periodontal disease may have mucosal and systemic immune features

The humoral immune response, especially IgG and IgA, is considered to be protective in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources. In order to understand better the local immunoglobulin production, we examined biopsy tissue from periodontitis lesions for the expression of IgM, IgG, IgA, IgE and in addition the IgG and IgA subclasses and J-chain by in situ hybridization. Tissues examined were superficial inflamed gingiva and the deeper granulation tissue from periodontal sites. These data confirm that IgM, and IgG and IgA subclass proteins and J-chain can be locally produced in the periodontitis tissues. IgG1 mRNA-expressing cells were predominant in the granulation tissues and in the gingiva, constituting approx. 65% of the total IgG-expressing plasma cells. There was a significantly increased proportion of IgA-expressing plasma cells in the gingiva compared with the granulation tissue (P < 0.01). Most of the IgA-expressing plasma cells were IgA1, but a greater proportion expressed IgA2 mRNA and J-chain mRNA in the gingival tissues (30.5% and 7.5%, respectively) than in the periodontal granulation tissues (19% and 0-4%, respectively). The J-chain or dimeric IgA2-expressing plasma cells were located adjacent to the epithelial cells, suggesting that this tissue demonstrates features consistent with a mucosal immune response. Furthermore, we were able to detect the secretory component in gingival and junctional epithelial cells, demonstrating that the periodontal epithelium shares features with mucosal epithelium. In contrast, deeper tissues had more plasma cells that expressed IgM, and less expressing IgA, a response which appears more akin to the systemic immune response. In conclusion, this study suggests that immune mechanisms involved in the pathogenesis of periodontitis may involve features of both the mucosal and systemic immune systems, dependent on tissue location.     

Failure to detect an association with IL1 genotypes in European Caucasians with generalised early onset periodontitis

OBJECTIVES: In order to elucidate the genetic background to EOP it is useful to investigate associations with genetic polymorphisms of immune response genes, whose products play a r le in the inflammatory process. The aim of this study was to examine IL1A and IL1B genetic polymorphisms in unrelated European white Caucasian patients with generalised early-onset periodontitis (GEOP). MATERIAL AND METHODS: Polymerase chain reaction (PCR) amplification of the ILIA (-889) gene and IL1B (+3953) gene (56 patients, 56 controls) was carried out and PCR products subjected to restriction fragment length polymorphism (RFLP) analysis. RESULTS: There were no significant differences between patients and controls for any of the genotype or allele frequencies investigated (p = 1.0). Smoking status was also included as a covariate but this did not alter the results. Furthermore, expression of the composite genotype described by Kornman and coworkers was also investigated in these subjects. No significant differences were found between patients and controls whether smoking was included as a covariate or not. CONCLUSION: The lack of any association between the IL1 polymorphisms and GEOP, in the population presented here, brings into doubt the usefulness of these candidate genes as markers of susceptibility to this form of periodontitis.     

Lactic acid is a potential virulence factor for group B Streptococcus

Group B Streptococcus (GBS) is a Gram-positive bacterium that causes sepsis and meningitis in neonates and infants. Although several GBS-associated virulence factors have been described, the mechanisms of GBS invasive disease are not well understood. To characterize additional virulence factors, a novel in vitro infection assay was developed using rat fetal lung explants. However, application of GBS to the system induced rapid lung tissue destruction associated with increased media acidity. Since lactic acid produced by other streptococci is an important virulence factor, we hypothesized that lactic acid contributed to the virulence of GBS. Spent growth media and neutralized-spent media were applied to explants and results indicated that neutralization of the media completely protected the tissue from degradation. These results were verified using multiple viability assays and with transformed cell lines. Furthermore, comparable spent media from Escherichia coli did not induce tissue cytotoxicity, suggesting that GBS produces organic acids in excess of other potential bacterial pathogens. Analysis of the spent media indicated that l-lactate levels reached ∼70 mM, indicating that lactic acid is a major constituent of the metabolic acid produced by GBS. Treatment of explants with lactic acid alone produced dose-dependent tissue degradation, indicating that lactic acid is independently sufficient to induce target-tissue cytotoxicity. Finally, both spent media and 23.6 mM lactic acid produced dramatic tissue autofluorescence; the basis for this is currently unknown. These studies demonstrate that GBS-produced lactic acid is a potential virulence factor and may contribute to GBS invasive disease.