Arndt Guentsch

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria

INTRODUCTION During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. METHODS GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and alpha-defensins (HNP1-3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. RESULTS Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1-3 in samples from patients, compared to controls. The amount of HNP1-3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. CONCLUSION The lack of a correlation between LL-37, HNP1-3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.

Marginal and internal fit of pressed lithium disilicate partial crowns in vitro: A three-dimensional analysis of accuracy and reproducibility

OBJECTIVES The objective of this in vitro study was to visualize and to quantify the marginal and internal fit of heat-pressed ceramic restorations by a novel three-dimensional procedure. Accuracy and reproducibility of the employed measuring method were determined. METHODS An acrylic model of a lower left first molar was prepared to receive a partial crown and duplicated by single step dual viscosity impressions. Corresponding working casts were formed from Type IV die stone and indirect restorations were fabricated from heat-pressable lithium disilicate ceramics (IPS e.max Press, Ivoclar Vivadent AG, Schaan, Liechtenstein). The acrylic tooth model and the ceramic partial crowns were digitized by a structure light scanner with a measurement-uncertainty of 4 μm and subjected to computer-aided quality inspection. Visual discrepancies in marginal and internal fit were displayed with colors. For quantitative analysis, mean quadratic deviations (RMS) were computed and analyzed by Students t-test (n=5, α=0.05). RESULTS Mean RMS-values for accuracy (reproducibility) ranged from 34 (14) μm for internal areas to 78 (23) μm for marginal surfaces. Differences in accuracy (p=0.003) and reproducibility (p<0.001) were statistically significant. In general, areas with sharp internal line angles such as occlusal ridges and the preparation finish line exhibited oversized dimensions, whereas areas with rounded and soft internal line angles were undersized. SIGNIFICANCE The viability of a computer-aided and three-dimensional approach for assessing marginal and internal fit of indirect restorations was demonstrated. Thereby, the obtained results track complex form changes as they occur during laboratory processing.

Comparison of Gingival Crevicular Fluid Sampling Methods in Patients With Severe Chronic Periodontitis

BACKGROUND The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified. METHODS GCF was sampled from four sites per patient (one sample per quadrant using two samples per method) in 36 patients with chronic periodontitis. One week later, the procedure was repeated with alternative methods. Variables determined were loads of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and P. gingivalis, levels of interleukin-6 and -8, activity of neutrophil elastase, and level of Rgps. RESULTS The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from paper strips and paper points. Rgps were only detectable in high quantities by washing the periodontal pocket. The level of Rgps correlated with the load of P. gingivalis. CONCLUSIONS The use of paper strips was suitable for the simultaneous determination of microbial and immunologic parameters. Obtaining GCF by washing can be useful for special purposes. The gingipain concentration in periodontal pockets was directly determined to be ≤1.5 μM. This value indicated that most of the substrates of these proteases by in vitro assays identified until now can be easily degraded in P. gingivalis-infected sites.

Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans

BACKGROUND AND OBJECTIVE This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. MATERIAL AND METHODS Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patients own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol-dependent and isoluminol-dependent chemiluminescence. RESULTS Polymorphonuclear neutrophils from patients with chronic (62.16 +/- 19.39%) and aggressive (43.26 +/- 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 +/- 19.87%) at the 30-min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. CONCLUSION High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.

Impact of digital impression techniques on the adaption of ceramic partial crowns in vitro.

OBJECTIVES To investigate the effects, digital impression procedures can have on the three-dimensional fit of ceramic partial crowns in vitro. METHODS An acrylic model of a mandibular first molar was prepared to receive a partial coverage all-ceramic crown (mesio-occlusal-distal inlay preparation with reduction of all cusps and rounded shoulder finish line of buccal wall). Digital impressions were taken using iTero (ITE), cara TRIOS (TRI), CEREC AC with Bluecam (CBC), and Lava COS (COS) systems, before restorations were designed and machined from lithium disilicate blanks. Both the preparation and the restorations were digitised using an optical reference-scanner. Data were entered into quality inspection software, which superimposed the records (best-fit-algorithm), calculated fit-discrepancies for every pixel, and colour-coded the results to aid visualisation. Furthermore, mean quadratic deviations (RMS) were computed and analysed statistically with a one-way ANOVA. Scheffés procedure was applied for multiple comparisons (n=5, α=0.05). RESULTS Mean marginal (internal) discrepancies were: ITE 90 (92) μm, TRI 128 (106) μm, CBC 146 (84) μm, and COS 109 (93) μm. Differences among impression systems were statistically significant at p<0.001 (p=0.039). Qualitatively, partial crowns were undersized especially around cusp tips or the occluso-approximal isthmus. By contrast, potential high-spots could be detected along the preparation finishline and at central occlusal boxes. CONCLUSIONS Marginal and internal fit of milled lithium disilicate partial crowns depended on the employed digital impression technique. CLINICAL SIGNIFICANCE The investigated digital impression procedures demonstrated significant fit discrepancies. However, all fabricated restorations showed acceptable marginal and internal gap sizes, when considering clinically relevant thresholds reported in the literature.

Differential impairment of vascularization and angiogenesis in bisphosphonate-associated osteonecrosis of the jaw-related mucoperiosteal tissue

OBJECTIVES Impaired vascularization in the etiopathology of aminobisphosphonate-associated osteonecrosis of the jaw (BONJ) is assumed, but evidence is lacking. This immunohistochemical study differentiated vascularization and angiogenesis in BONJ-adjacent mucoperiosteal tissue. STUDY DESIGN Twenty BONJ (after zoledronate treatment) and 20 control mucoperiosteal tissue samples were processed with an autostaining-based alkaline phosphatase-antialkaline phosphatase staining kit. Vascularization was assessed by CD31 staining and angiogenesis-related neovessels by CD105 staining. The ratio of stained capillary area to total area of visible field was assessed. Statistics included Bonferroni adjustment. RESULTS CD31-stained microvessels were detected in each section and CD105-stained neovessels in each control. BONJ-adjacent mucoperiosteal tissue showed significantly fewer CD105-positive vessels in capillary areas (P < .05) than control samples. CD31-stained capillary area was not significantly reduced in mucoperiosteal BONJ-samples. CONCLUSIONS Angiogenesis is impaired in BONJ-related mucoperiosteal tissue, but vascularization remains unaffected. Vessel remodeling and neovessel formation is delayed in BONJ, resulting in impaired tissue regeneration of bisphosphonate-exposed oral mucosa.

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG1 was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG3 was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC‐ and isothermal titration calorimetry‐based assays followed by Hill plots, revealed non‐Michaelis‐Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG1 cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin‐dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.—Vincents, B., Guentsch, A., Kostolowska, D., von Pawel‐Rammingen, U., Eick, S., Potempa, J., Abrahamson, M. Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis. FASEB J. 25, 3741–3750 (2011). www.fasebj.org

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis

BackgroundLoss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor.AimWe hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood.MethodsTo confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients.ResultsAlthough neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37.ConclusionsCollectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease.