Generic tools for conditionally altering protein abundance and phenotypes on demand.

Conditional gene expression and modulating protein stability under physiological conditions are important tools in biomedical research. They led to a thorough understanding of the roles of many proteins in living organisms. Current protocols allow for manipulating levels of DNA, mRNA, and of functional proteins. Modulating concentrations of proteins of interest, their post-translational processing, and their targeted depletion or accumulation are based on a variety of underlying molecular modes of action. Several available tools allow a direct as well as rapid and reversible variation right on the spot, i.e., on the level of the active form of a gene product. The methods and protocols discussed here include inducible and tissue-specific promoter systems as well as portable degrons derived from instable donor sequences. These are either constitutively active or dormant so that they can be triggered by exogenous or developmental cues. Many of the described techniques here directly influencing the protein stability are established in yeast, cell culture and in vitro systems only, whereas the indirectly working promoter-based tools are also commonly used in higher eukaryotes. Our major goal is to link current concepts of conditionally modulating a protein of interest's activity and/or abundance and approaches for generating cell and tissue types on demand in living, multicellular organisms with special emphasis on plants.

Prognostic model for tooth survival in patients treated for periodontitis.

BACKGROUND: In patients with periodontitis, a quantitative prognostic assessment is needed in order to make evidence-based decisions about retaining teeth or extracting and replacing them with a dental prosthesis. METHODS: One hundred and ninety eight patients receiving active periodontal treatment in 1989 or 1990 and complying with supportive periodontal therapy (SPT) over an average of 11.8+/-2.3 years were included in the study. A generalized linear model was established and fitted via generalized estimating equations to identify predictors for tooth loss during SPT. RESULTS: Of the 4559 teeth present at baseline, 166 (3.6%) were extracted during active treatment and 249 (5.5%) during SPT. Baseline findings of diabetes mellitus (OR=4.17), reduced alveolar bone levels (OR=1.04 for each 1% increment), increased tooth mobility (III versus 0: OR=5.52), multiple roots (OR=1.82), and non-vital pulp (OR=2.24) were significant (p<0.05) predictors for tooth loss during SPT. Based on these parameters, a prognostic model was constructed that provides estimates of tooth survival probability when periodontal therapy is rendered. CONCLUSION: Using a multivariate approach, a prognostic model was developed that may be of value for clinical decision making.

Change of antibiotic susceptibility following periodontal therapy. A pilot study in aggressive periodontal disease.

BACKGROUND: The hypothesis was tested that bacterial susceptibilities in aggressive periodontitis change upon administration of systemic antibiotics as adjuncts to periodontal therapy. METHODS: In 23 subjects (average age 38.9+/-6.7 years) with aggressive periodontitis, microbial parameters were assessed prior to and 1 year after completion of comprehensive mechanical/surgical and systemic antimicrobial therapy. Following identification of five selected pathogens with the Rapid ID 32 A system, their susceptibilities towards amoxicillin/clavulanate potassium, metronidazole, and tetracycline were examined with the E-test. Antibiotics were administered according to the test results, and the minimal inhibitory concentrations (MIC90) were reevaluated after 1 year. Statistical analysis was performed on a patient basis, with the site data used for evaluation of the MIC levels. RESULTS: Bacterial MIC levels remained constant among the three antibiotic treatment groups compared with baseline. Mean MIC90 values ranged from <0.02 to 0.11 microg/ml (amoxicillin/clavulanate potassium), <0.02 to 0.27 microg/ml (metronidazole), and <0.02 to 0.11 microg/ml (tetracycline). Observed changes in susceptibility were attributed to the elimination of single bacterial taxa in the subgingival environment after antibiotic therapy. There were no statistically significant differences in clinical parameters among the treatment groups. Single tetracycline MICs were 1.5- to 6-fold enhanced compared to amoxicillin/clavulanate potassium and metronidazole. CONCLUSION: The periodontal pathogens investigated prior to and 1 year after periodontal therapy are tested sensitive to the antimicrobial agents. In aggressive periodontitis, changes in bacterial susceptibility upon the administration of systemic antibiotics are associated with the limited number of isolates tested following therapy.

The microflora recovered from the outer-surfaces of the Frialit-2 implanto-prosthetic connector.

The aim of the present investigation was to examine the outer-surface microbiota of the prosthetic connector of Frialit-2 implants, and to compare the microbial findings with the peri-implant parameters 2 years after functional loading. In 16 implant-treated patients (55.8 +/- 9.5 years) the outer-surface micro-organisms of the prosthetic connectors were determined in 32 Frialit-2 implants. The functional loading time of the prosthetic suprastructures was 24.1 +/- 13.8 months on average. After removal of the implant-supported restoration, microbial samples were obtained from the outer-surfaces of the Frialit-2 prosthetic connector. The microbial plaque samples were specified on CDC-blood agar as percentages of the total cultivable flora. Actinobacillus actinomycetemcomitans was semiquantitively determined on TSBV-agar in CFU/ml. The microbial plaque samples were dominated by Actinomyces israelii (68.8%), Eubacterium lentum (56.3%) and Veillonella parvula (43.8%) with proportions ranking between 3.9% (V. parvula) and 11.1% (A. israelii). The most frequently detected gram-negative microorganisms were Fusobacterium nucleatum (87.5%), Porphyromonas gingivalis (81.3%), and Peptostreptococcus micros (68.8%) with enhanced proportions for P. gingivalis (11.4%) and P. micros (11.4%). No statistical significant correlation could be established between the microbiota present on the outer-surfaces of the F2-connector and the peri-implant parameters examined. The outer-surface microflora recovered from the implanto-prosthetic-connector of Frialit-2 implants reveals a colonization with gram-positive bacteria and potentially harmful gram-negative micro-organisms that were frequently detected, but present at low levels. After 2 years of restorative loading, the outer-surface microbial colonization is compatible with peri-implant soft tissue health.

PMN responses in chronic periodontal disease: evaluation by gingival crevicular fluid enzymes and elastase-alpha-1-proteinase inhibitor complex.

OBJECTIVES: In the present trial, the hypothesis was examined that the local PMN responses in untreated and treated chronic periodontitis can be differentiated by gingival crevicular fluid lysosomal enzyme activities and elastase-alpha-1-proteinase inhibitor complex. METHODS: In nine subjects (average age 49.2 +/- 7.1 years) with chronic periodontitis, clinical parameters and markers of the PMN-derived inflammatory tissue response in gingival crevicular fluid (GCF) were assessed before and 6 months after surgical periodontal therapy. Myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH) and cathepsin D (CD) were analyzed as indicators of the PMN-associated host tissue destruction, and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) as the major serum protein inactivating PMN elastase. The total activities of the lysosomal enzymes MPO and beta-NAH were evaluated spectrophotometrically, the CD levels by liquid scintillation counting with [14C] hemoglobin as substrate, and the total alpha-1-proteinase inhibitor complex using a sandwich-immunoassay. RESULTS: The clinical parameters revealed a statistical significant decrease at the 6-month reexamination. PD levels dropped from 5.40 to 2.88 mm (change 2.52 +/- 1.04 mm), the CAL scores from 6.67 to 4.43 mm (change 2.24 +/- 0.77 mm). The 30 s GCF volumes dropped from 129.8 to 68.6, displaying a change of 61.1 +/- 18.6, p

Resolution of crevicular fluid leukocyte activity in patients treated for aggressive periodontal disease.

BACKGROUND: Enhanced neutrophil responses play a critical role in the activation of the innate immune system and causation of aggressive periodontitis (AgP). The hypothesis that comprehensive periodontal treatment expedites resolution of amplified leukocyte activity and facilitates the reconstitution of periodontal health was tested. METHODS: Four different gingival crevicular fluid (GCF) markers from 14 patients were characterized prior to and at 3, 6, 12, 24, and 36 months after periodontal therapy. GCF myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), and beta-glucuronidase (beta-G) were determined spectrophotometrically, and cathepsin D (CD) by liquid scintillation counting using [14C] hemoglobin as substrate. The primary outcome was long-term stability of periodontal health. RESULTS: In untreated AgP, GCF markers were significantly amplified (MPO: 1.9-fold; beta-NAH: 1.3-fold; beta-G: 1.7-fold; CD: 4.7-fold). Following periodontal therapy, the leukocyte activity was significantly dampened (0.3- to 0.5-fold), and paralleled with a sustained improvement of periodontal health (P < 0.05). Thereafter and at 3 years, GCF leukocyte responses remained on a physiologic low level compatible to normal immune function. CONCLUSIONS: Comprehensive treatment of AP induces a downregulation of amplified crevicular neutrophil activity. The release of the innate immune system from exacerbating damage elicits a successful reconstitution of long-term periodontal health with no setbacks seen after 3 years.

Aggressive periodontitis: 5-year follow-up of treatment.

BACKGROUND: The hypothesis that in subjects with aggressive periodontitis, a long-term stability of periodontal health can be achieved following comprehensive mechanical/surgical and systemic antimicrobial therapy was tested in this prospective study. METHODS: Thirteen patients (36.9+/-7.4 years) with aggressive periodontitis were monitored before and up to 5 years following periodontal therapy. Clinical attachment levels (CAL) were assessed pretherapy, and at 3 months following completion of active periodontal therapy supplemented by amoxicillin plus metronidazole. All subjects were subsequently enrolled in a maintenance program and provided with supportive periodontal therapy with 3 to 4 appointments annually. Reexaminations were performed after 6 months and 1, 2, 3, 4, and 5 years. The data were analyzed using the method of generalized estimating equations (GEE) for CAL changes from baseline to the 3-month visit, and from completion of periodontal therapy to each annual visit up to the 5-year follow-up reappointment. RESULTS: During the 5-year study, all subjects strongly benefited from periodontal treatment. Between baseline and the 3-month reexamination, the CAL levels revealed a significant decrease of 2.23 mm (95% confidence interval [CI]: 1.77 to 2.69 mm; P < or =0.001). At the 5-year maintenance visit, the CAL changes ranged from -0.04 to +0.29 mm with no further statistically significant periodontal breakdown (P >0.05). Five years after surgery, 3.2% of the treated sites demonstrated a further CAL gain > or =3 mm. A stabilization (CAL -2 to +2 mm) occurred in 94.6% of the cases. The number of periodontal sites experiencing a breakdown varied from 5.3% at 6 months to 2.2% at 5 years. CONCLUSIONS: In aggressive periodontitis, comprehensive mechanical/surgical and antimicrobial therapy is an appropriate treatment regimen for long-term stabilization of periodontal health. In this study, periodontal disease progression was successfully arrested in 95% of the initially compromised lesions, while 2% to 5% experienced discrete or recurrent episodes of loss of periodontal support.