Evaluation of the Humoral Immune Response to the Cytolethal Distending Toxin of \u3cem\u3eAggregatibacter Actinomycetemcomitans\u3c/em\u3e Y4 in Subjects With Localized Aggressive Periodontitis

Introduction:  Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls. Methods:  Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells. Results:  Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20–75%). Conclusions:  Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease

Differential Effect of the Cytolethal Distending Toxin of \u3cem\u3eActinobacillus actinomycetemcomitans\u3c/em\u3e on Co-Cultures of Human Oral Cells

The periodontal pathogen Actinobacillus actinomycetemcomitans expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G0/G1 or G2/M phase of the cell cycle. It was previously found that CDT failed to arrest the growth of human periodontal ligament fibroblasts (HPLFs) when grown in pure culture. In contrast, proliferation of an oral epithelial cell line was rapidly inhibited by the toxin. In this study, the feasibility of using mixed-cell cultures and cell-specific markers to evaluate the response of oral cells, when in heterogeneous populations, to CDT was established. Proliferation of epithelial cells was rapidly inhibited and the cells were selectively eliminated in co-culture with HPLFs or cementoblasts by 24–48 h post-intoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cell-specific immunolabelling with antibodies against simian virus 40 large T antigen and the Ab-1 surface antigen, respectively. These results demonstrated that the activities of potential virulence factors, such as CDT, from periodontal pathogens can be successfully examined in mixed-cell cultures. This approach is especially relevant to infectious diseases that affect tissues with a diverse cellular composition, such as the periodontium

Do Buffered Local Anesthetics Provide More Successful Anesthesia Over Non-Buffered Solutions in Patients Requiring Dental Therapy? – A Systematic Review & Meta-Analysis.

Background: The pH of commercially available local anesthetics (LAs) is purposefully low (pH 3–4). Decreasing the pH extends the shelf life of the solution and prevents its early oxidation. However, a low pH may produce a burning sensation on the injection site, a slower onset of anesthesia, and a decrease in its clinical efficacy. Buffering of local anesthetics (alkalinization) by adding sodium bicarbonate has been suggested to achieve better pain control, reduce the pain of injection and produce a faster onset of local anesthetics. The aim of this review is to utilize a systematic review to collate evidence on the use of buffering agents with local anesthetics and its effect on causing profound pulpal anesthesia in patients requiring dental therapy and its side effects. Methods: Electronic searches were conducted in MEDLINE, Scopus, Cochrane Library, and ClinicalTrials.gov, World Health Organization (WHO) International Trials Registry Platform, OpenGrey & Google Scholar beta. Hand searching of two books “Handbook of Local Anesthesia” & “Successful Local Anesthesia for Restorative Dentistry and Endodontics” was conducted. Also, the reference lists of all included and excluded studies were checked to identify any further trials. Weighted anesthesia success rates and 95% confidence intervals (CIs) were estimated and compared by using a random-effects model. Results: 14,011 studies were initially identified from the search; 5 double-blind, randomized clinical trials met the inclusion criteria. For combined studies, buffered local anesthetics were more likely than non-buffered solutions to achieve successful anesthesia (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.11–4.71; P = 0.0232; I2 = 66%). Conclusion: This systematic review of double-blind, randomized clinical trials comparing the use of buffered and non-buffered local anesthetics in patients requiring dental therapy provides level ‘A’ evidence that is based on the criteria given by the Strength of Recommendation Taxonomy (SORT). In conclusion, the present meta-analysis showed that in patients receiving dental therapy, buffered local anesthetics are more effective than non-buffered solutions when used for mandibular or maxillary anesthesia. Buffering local anesthetics has 2.29 times greater likelihood of achieving successful anesthesia

Characterization of Point Mutations in the \u3cem\u3ecdtA\u3c/em\u3e Gene of the Cytolethal Distending Toxin of \u3cem\u3eActinobacillus Actinomycetemcomitans\u3c/em\u3e

The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5′- and 3′-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function

Functional and Structural Characterization of Chimeras of a Bacterial Genotoxin and Human Type I DNAse

Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells

Neonatal Exposure to Thymotropic Gross Murine Leukemia Virus Induces Virus-Specific Immunologic Nonresponsiveness

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses

18F-FDG-PET/CT in Radiation Therapy-Induced Cerebellar Inflammation

ABSTRACT Background 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18FDG- PET/CT) is used in the clinical diagnosis and management of oncologic and inflammatory pathologies. It may also have utility in detecting tissue damage induced by radiotherapy (RT) used to treat various types of cancer. The aim of the present study was to use 18FDG-PET/CT to evaluate the effect of RT on the uptake of 18FDG by the cerebellum. Methods Thirty patients with head and neck cancer (HNC) were included in this retrospective study. The patients were treated with photon, proton, or combined photon/proton RT, in addition to chemotherapy. All patients received 18FDG- PET/CT imaging pre-treatment and 3 months post-treatment. The global mean standardized uptake value (SUVmean) of the cerebellum was determined for every patient by global assessment of 18FDG activity using OsiriX MD software. A two-tailed paired t-test was used to compare global SUVmean pre- and post-RT. Results The pre-treatment and post-treatment global SUVmean for the photon group were 5.26 and 5.51 (p: 0.42), respectively. As for the proton only group, the pre- and post-treatment global SUVmeans were 7.06 and 6.05, respectively. In the combined RT group, the pre- and post-treatment global SUVmeans were 6.14 and 6.19 respectively (p: 0.92). The differences between the pre- and post-treatment values failed to reach statistical significance for any of the treatment groups but it should be noted that there was a trend of increased 18FDG uptake in the cerebellum following photon therapy. This trend was not clear in the combined group. As for the proton group, p-value was not calculated as only two patients were included. Conclusion Although not statistically significant, the results showed an incremental increase in global SUVmean following treatment with photon RT likely reflecting the presence of mild radiation-induced inflammation in the cerebellum