Gail C. Brammar

Kgp and RgpB, but Not RgpA, Are Important for Porphyromonas gingivalis Virulence in the Murine Periodontitis Model

ABSTRACT The contributions of three proteinase genes (rgpA, rgpB, and kgp) to the virulence of Porphyromonas gingivalis W50 were investigated in the murine periodontitis model. Mice were orally inoculated with eight doses (1 × 1010 cells per dose) of rgpA, rgpB, kgp, rgpA rgpB, or rgpA rgpB kgp isogenic mutants, and the level of alveolar bone loss, immune response induced, and number of bacterial cells per half maxilla were compared with those of animals inoculated with wild-type P. gingivalis. The kgp, rgpB, rgpA rgpB, and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) less bone loss than the rgpA isogenic mutant and the wild type did, and the virulence of the rgpA isogenic mutant and the wild type were not significantly different. Mice inoculated with the wild type or the rgpA isogenic mutant exhibited significantly (P < 0.01) more P. gingivalis cells per half maxilla than mice inoculated with rgpB, kgp, rgpA rgpB, and rgpA rgpB kgp isogenic mutants or nonchallenged mice did, as determined using real-time PCR. A significant positive correlation was found between the number of P. gingivalis cells detected per half maxilla and the amount of alveolar bone loss induced. Enzyme-linked immunosorbent assay results showed that each isogenic mutant and the wild type induced a predominant P. gingivalis antigen-specific immunoglobulin G3 (IgG3) response. Furthermore, the kgp and rgpA rgpB kgp isogenic mutants induced significantly (P < 0.05) lower IgG3 antibody responses than the responses induced by the wild type or the rgpA, rgpB, and rgpA rgpB isogenic mutants. The results suggest that the order in which the proteinases contribute to the virulence of P. gingivalis in the murine periodontitis model is Kgp ≥ RgpB ≫ RgpA.

Macrophage Depletion Abates Porphyromonas gingivalis–Induced Alveolar Bone Resorption in Mice

The role of the macrophage in the immunopathology of periodontitis has not been well defined. In this study, we show that intraoral inoculation of mice with Porphyromonas gingivalis resulted in infection, alveolar bone resorption, and a significant increase in F4/80+ macrophages in gingival and submandibular lymph node tissues. Macrophage depletion using clodronate-liposomes resulted in a significant reduction in F4/80+ macrophage infiltration of gingival and submandibular lymph node tissues and significantly (p < 0.01) less P. gingivalis–induced bone resorption compared with controls in BALB/c and C57BL/6 mice. In both mouse strains, the P. gingivalis–specific IgG Ab subclass and serum cytokine [IL-4, IL-10, IFN-γ, and IL-12 (p70)] responses were significantly (p < 0.01) lower in the macrophage-depleted groups. Macrophage depletion resulted in a significant reduction in the level of P. gingivalis infection, and the level of P. gingivalis infection was significantly correlated with the level of alveolar bone resorption. M1 macrophages (CD86+), rather than M2 macrophages (CD206+), were the dominant macrophage phenotype of the gingival infiltrate in response to P. gingivalis infection. P. gingivalis induced a significant (p < 0.01) increase in NO production and a small increase in urea concentration, as well as a significant increase in the secretion of IL-1β, IL-6, IL-10, IL-12 (p70), eotaxin, G-CSF, GM-CSF, macrophage chemoattractant protein-1, macrophage inflammatory protein-α and -β, and TNF-α in isolated murine macrophages. In conclusion, P. gingivalis infection induced infiltration of functional/inflammatory M1 macrophages into gingival tissue and alveolar bone resorption. Macrophage depletion reduced P. gingivalis infection and alveolar bone resorption by modulating the host immune response.

Renal Processing of Albumin in Diabetes and Hypertension in Rats

Background/Aims: Recent studies show that albuminuria may be the result of changes in post-glomerular cellular uptake and processing of albumin. This study aims to determine whether this processing is disrupted in diabetes and/or hypertension. Methods: Diabetes (d) was induced using streptozotocin in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) and studied after 8, 16 and 24 weeks of disease. Intact albumin excretion was determined by radioimmunoassay. Total albumin was determined by [14C]albumin. Lysosomal activity was determined by dextran sulfate desulfation. Renal TGF-β1 and transforming growth factor-β1 inducible gene-h3 mRNA (βig-h3) expression was determined by real time RT-PCR. Results: SHR-c rats exhibited an increase in intact albuminuria without significant change in total albumin excretion (intact plus albumin-derived peptides). For WKY-d rats, intact albuminuria developed initially, followed by an increase in total albumin excretion primarily in the form of albumin peptides (peptiduria). SHR-d rats exhibited both increases in peptiduria and intact albuminuria. There was no increase in glomerular permeability at 24 weeks for polydisperse [3H]Ficoll in all groups. Increased renal TGF-β1 and βig-h3 expression was correlated with a decrease in dextran sulfate desulfation and increased intact albuminuria independent of peptiduria. Conclusion: Increased albumin excretion in hypertension and/or diabetes is manifested in different forms independent of glomerular permeability.

Ramipril prevents microtubular changes in proximal tubules from streptozotocin diabetic rats

SUMMARY:  This study has investigated the microtubular cytoskeleton in rat glomerular and proximal tubule cells in experimental diabetes. The effect of treatment with ramipril on the relationship between microtubule organization and albuminuria in diabetes has also been examined. Diabetes was induced in male Sprague–Dawley rats by administration of streptozotocin (50 mg/kg, i.v.). Rats were treated with or without ramipril in their drinking water for 12 weeks. Diabetes was characterized by an increase in blood glucose level, glomerular filtration rate, and albumin excretion rate. Treatment of diabetic rats with ramipril did not affect glycaemic control, but reduced systolic blood pressure and prevented the rise in albuminuria and glomerular filtration rate. Immunohistochemistry was performed by using the ARK Peroxidase method with α‐tubulin antibody. The regular, grainy staining pattern of the microtubules present in the renal proximal tubules from control kidneys was altered in diabetic animals, and appeared fragmented and striated. This was prevented by treatment with ramipril. Quantitative morphometric analysis revealed an increase in the percent proportional staining for α‐tubulin in the proximal tubules of untreated diabetic rats (33.3 ± 3.3%, n = 8, P < 0.05 vs control) compared with control rats (11.7 ± 1.7%, n = 6), which was reduced by ramipril treatment (26.7 ± 2.1%, n = 6, P < 0.05 vs untreated diabetic). Staining for α‐tubulin in glomerular cells was unchanged in all groups. There was no significant difference in renal α‐tubulin expression among all groups, as determined by real‐time reverse transcription–polymerase chain reaction. These results raise the possibility that diabetes‐induced changes in microtubules in the renal proximal tubules may contribute, in part, to the increase in albuminuria observed in diabetes.

Additive effects of hypertension and diabetes on renal cortical expression of PKC-α and -ε and α-tubulin but not PKC-β1 and -β2

Objective This study examined the separate and combined effects of hypertension and diabetes on renal cortical expression of protein kinase C (PKC) isoforms -β1, -β2, -α and -&epsis;, to determine whether albuminuria is the result of an increase in the expression of one or a combination of PKC isoforms. Corresponding changes in renal microtubules were also assessed. Methods Diabetes (D) was induced in Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR) by streptozotocin. After 24 weeks, PKC expression was determined by Western blot and microtubules were assessed by immunohistochemistry for α-tubulin protein. Results Diabetes was characterized by significant increases in glycated haemoglobin (HbA1c) as compared to controls (C). There was a significant increase of three- to four-fold in PKC protein content for all four isoforms in renal cortex from SHR-C and WKY-D, and similar and significant levels of albuminuria (∼10 mg/24 h) observed in these groups in comparison to WKY-C (∼1 mg/24 h). Interestingly, PKC-α and -&epsis; but not PKC-β1 and -β2 protein content was doubled in SHR-D, and albuminuria increased tenfold (∼100 mg/24 h) in comparison to SHR-C and WKY-D. These changes were paralleled by a significant decrease in α-tubulin protein content of ∼50% in SHR-C and ∼33% in WKY-D compared to WKY-C, with a further decrease of ∼67% in SHR-D compared to WKY-C. Conclusion These findings indicate that PKC expression can be increased by either diabetes or hypertension, and that there are further specific increases in the expression of PKC isoforms -α and -&epsis; in the model of combined diabetes and hypertension. In addition, the degree of disruption in microtubular cytoskeleton appears to be correlated with PKC activation and levels of albuminuria.

Rapid Chair-Side Test for Detection of Porphyromonas gingivalis

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.

GM-CSF and uPA are required for Porphyromonas gingivalis-induced alveolar bone loss in a mouse periodontitis model.

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and urokinase‐type plasminogen activator (uPA) can contribute to the progression of chronic inflammatory diseases with possible involvement of macrophages. In this study, we investigated the role of both GM‐CSF and uPA in Porphyromonas gingivalis‐induced experimental periodontitis using GM‐CSF−/− and uPA−/− mice. Intra‐oral inoculation of wild‐type (WT) C57BL/6 mice with P. gingivalis resulted in establishment of the pathogen in plaque and a significant increase in alveolar bone resorption. The infected mice also exhibited a CD11b+ CD86+ macrophage infiltrate into the gingival tissue, as well as P. gingivalis‐specific pro‐inflammatory cytokine and predominantly IgG2b antibody responses. In comparison, intra‐oral inoculation of P. gingivalis did not induce bone resorption and there was significantly less P. gingivalis recovered from plaque in GM‐CSF−/− and uPA−/− mice. Furthermore, P. gingivalis did not induce a macrophage gingival infiltrate or activate isolated peritoneal macrophages from the gene‐deficient mice. Pro‐inflammatory P. gingivalis‐specific T‐cell cytokine responses and serum interferon‐gamma (IFN‐γ) and IgG2b concentrations were significantly lower in GM‐CSF−/− mice. In uPA−/− mice, T‐cell responses were lower but serum IFN‐γ and IgG2b levels were comparable with WT mice levels. These results suggest that GM‐CSF and uPA are both involved in the progression of experimental periodontitis, possibly via a macrophage‐dependent mechanism(s).

A therapeutic Porphyromonas gingivalis gingipain vaccine induces neutralising IgG1 antibodies that protect against experimental periodontitis

Porphyromonas gingivalis infected mice with an established P. gingivalis-specific inflammatory immune response were protected from developing alveolar bone resorption by therapeutic vaccination with a chimera (KAS2-A1) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. Protection was characterised by an antigen-specific IgG1 isotype antibody and Th2 cell response. Adoptive transfer of KAS2-A1-specific IgG1 or IgG2 expressing B cells confirmed that IgG1-mediated protection. Furthermore, parenteral or intraoral administration of KAS2-A1-specific polyclonal antibodies protected against the development of P. gingivalis-induced bone resorption. The KAS2-A1-specific antibodies neutralised the gingipains by inhibiting: proteolytic activity, binding to host cells/proteins and co-aggregation with other periodontal bacteria. Combining key gingipain sequences into a chimera vaccine produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis.

Development and evaluation of a saliva-based chair-side diagnostic for the detection of Porphyromonas gingivalis.

Porphyromonas gingivalis is a key pathogen in the polymicrobial biofilm that is associated with the oral disease chronic periodontitis. A number of studies have shown that in humans the level of P. gingivalis in the polymicrobial biofilm is positively correlated with disease progression. The aim of this study was to develop a P. gingivalis diagnostic that has high specificity and sensitivity for P. gingivalis using a range of laboratory and clinical isolates and then compare the efficacy of the diagnostic with RTPCR using samples from chronic periodontitis patients and age- and sex-matched healthy controls. Key parameters for the kit were to use saliva as the biological fluid as this is a most convenient medium for chair-side sampling and to give a positive reading for the reported threshold for detection of 5×105 P. gingivalis cells/mL that indicates disease progression. We initially screened a range of monoclonal antibodies for recognition of the P. gingivalis conserved virulence factor RgpA-Kgp complex and identified two mAbs that could be used in a capture and detection ELISA system. These mAbs were used to formulate and manufacture the GC P. gingivalis saliva diagnostic kit used in the study. To validate the saliva kit, saliva (P. gingivalis free) was spiked with known concentrations of viable P. gingivalis whole cells of W50, 381, A7A1-28, and ATCC 33277; P. gingivalis clinical isolates; P. gingivalis vesicles; and the secreted form of the RgpA-Kgp complex. Laboratory findings indicated that the kit was able to detect all laboratory and clinical isolate strains of P. gingivalis at 5×104/mL to 5×105/mL. It was also able to detect the RgpA-Kgp complex and vesicles at 5×104 and 5×105 cell equivalent doses, respectively. Saliva and plaque were then collected from 50 subjects with moderate–severe chronic periodontitis and 50 age- and sex-matched subjects with healthy periodontium. Real-time PCR was utilised to analyse levels of P. gingivalis in both saliva and plaque. The saliva kit was found to give a positive result within 90 seconds. Using point bi-serial correlation analysis, a significant (p=0.04) correlation was found for detection of P. gingivalis using the saliva kit and P. gingivalis levels in saliva and plaque as determined by real-time PCR. A sensitivity of 92% and a specificity of 96% were found when compared to real-time PCR at a 105 P. gingivalis cell threshold. In conclusion, the P. gingivalis saliva kit was shown to be rapid and has a comparable detection capacity to real-time PCR. Thus, the P. gingivalis saliva diagnostic has the potential to be a simple and time-efficient chair-side diagnostic for the detection of P. gingivalis.