New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity.

AIMS: Expression of early (E) genes of human cytomegalovirus (HCMV) is stimulated cooperatively by the activities of host cell transcription factors and the viral immediate-early 2 (IE2) protein. Taking advantage of the IE2-dependent inducibility of E gene promoters, in this study, we generated cell-based assays in which the expression of the enhanced green fluorescence protein (EGFP) reporter gene was driven by the UL54 or UL112/113 E promoters. METHODS AND RESULTS: Cell clones derived from a stably transfected human cell line permissive to HCMV replication showed a specific and inducible dose- and time-dependent EGFP response to HCMV infection. The sensitivity of these indicator cells for detecting infectious particles of clinical isolates of HCMV was comparable to that of a conventional plaque assay. The HCMV-induced EGFP expression was completely prevented by treatment of indicator cells with fomivirsen, an antisense oligodeoxynucleotide designed to block IE2 expression, and this inhibitory activity was also observed when the IE2 protein alone was constitutively expressed in EGFP indicator cells. CONCLUSIONS: The EGFP-based cell assays have proved to be a rapid, sensitive, quantitative and specific system for detection of HCMV and selection of antivirals. SIGNIFICANCE AND IMPACT OF THE STUDY: These new cell-based assays can be exploited as functional assays to detect infectious HCMV particles, as well as to screen antiviral compounds that interfere with IE2 activity.

Debridement and local application of tetracycline-loaded fibres in the management of persistent periodontitis: results after 12 months.

BACKGROUNDS/AIMS: The aim of our study was to evaluate the clinical, radiological and microbiological response to the local delivery of tetracycline (TE) of sites with persistent periodontal lesions. MATERIALS AND METHODS: The study was conducted in a split-mouth design. Nineteen patients with at least four bilateral pockets 4-5 mm and bleeding on probing (BOP) were treated with scaling and root planing (SRP) plus TE fibres (test sites) or with SRP alone (control sites). Clinical and radiological measurements were taken at baseline, 6 months and 12 months post-operatively. Subgingival plaque samples were collected at baseline, at fibres removal, 6 and 12 months following treatment and analysed by polymerase chain reaction. RESULTS: Both treatments yielded a statistically significant (p<0.05) reduction of probing depth (2.05 and 1.21 mm), gain of clinical attachment level (1.71 and 0.53 mm) and reduction of BOP scores (23.68% and 57.89%) for TE and SRP groups, respectively, when comparing 12-month data with baseline. The differences between two groups were significant. The prevalence of Treponema denticola and Bacteroides forsythus decreased after therapy in both groups but only in the test sites Actinobacillus actinomycetemcomitans and Prevotella intermedia were not yield detected. The pathogens could be eliminated from five periodontal pockets by SRP alone, while 21 TE sites were not recolonized at 12 months. CONCLUSIONS: SRP plus TE fibres gave the greatest advantage in the treatment of periodontal persistent lesions at least 12 months following treatment.

Molecular approaches to the identification and treatment monitoring of periodontal pathogens.

Two different PCR-based molecular approaches, a commercial kit for detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola (Amplimedical "Paradonthosis") and a home-made multiplex PCR for A. actinomycetemcomitans, P. gingivalis and B. forsythus were compared for monitoring the efficacy of different dental treatments on localized persistent periodontal pockets. 44 sites were randomized in two treatment groups: mechanical treatment (22 control sites) and in conjunction with the application of tetracycline fibres (22 experimental sites). 40/44 sites were found positive with both tests for A. actinomycetemcomitans, P. gingivalis and B. forsythus pretheraphy. P. intermedia was detected alone in only three sites during the follow-up, while T. denticola. was always associated with the other pathogens. 20 sites were positive in conventional cultures for one to three of the pathogens. PCR-based approaches provided a sensitive and reliable method for identification and monitoring treatment of periodontal pathogens.

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation.

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.