Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine proteases purified from Porphyromonas gingivalis.

INTRODUCTION: Periodontitis is a common infectious disease to which Porphyromonas gingivalis has been closely linked, in which the attachment tissues of the teeth and their alveolar bone housing are destroyed. We conducted a study to determine if immunization using a purified antigen could alter the onset and progression of the disease. METHODS: Using the ligature-induced model of periodontitis in Macaca fascicularis, we immunized five animals with cysteine protease purified from P. gingivalis and used an additional five animals as controls. Alveolar bone loss was measured by digital subtraction radiography. RESULTS: Immunization induced high titers of specific immunoglobuin G serum antibodies that were opsonic. Total bacterial load, levels of P. gingivalis in subgingival plaque and levels of prostaglandin E(2) in gingival crevicular fluid were significantly reduced. Onset and progression of alveolar bone loss was inhibited by approximately 50%. No manifestations of toxicity were observed. CONCLUSIONS: Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.

Differential display analysis of Porphyromonas gingivalis gene activation response to heat and oxidative stress.

BACKGROUND/AIMS: The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress. METHODS: We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction. RESULTS: We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5 degrees C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities. CONCLUSION: These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

Educating the next generation of dentists.

A new vision for dental education is being shaped by (1) future dentists' need to keep pace with rapid developments in medicine and biomedical science, and changing health-care delivery mechanisms that will transform dentistry; (2) dissatisfaction with unintended, undesirable consequences of traditional approaches to dental education that do not integrate biomedical/clinical, behavioral, ethical, and population perspectives in clinical practice, and fail to cultivate the skills and values which support lifelong learning; (3) new developments in cognitive science demonstrating learning environments' need to be more student-centered, to emphasize active learning strategies, and teach students to use the scientific method to resolve clinical problems. These changes will require efforts in faculty development, and have implications for the administration and function of dental schools.

Alternative medicine. Achieving balance between herbal remedies and medical therapy.

The case patient was taking multiple herbal preparations as well as the prescription hypnotic zolpidem. The combination was probably increasing the patient's confusion, agitation, and aggression. The treatment team reached a compromise with the daughter after providing her with education and support. They continued the wheat germ oil and a multivitamin supplement, which appeared safe, even if of limited value. The patient continued taking valproate, 125 mg bid, which reduced her physical aggression and improved resistance to care. All other herbal remedies and zolpidem were discontinued. Balancing traditional therapies with requests for herbal remedies is a common challenge for physicians. The most successful intervention occurs when doctors familiarize themselves with herbal preparations and educate patients and families about the treatments.

Suicide in late life. Identifying and managing at-risk older patients.

The patient in this case displayed multiple risk factors for suicide, including depression, loneliness following recent widowhood, use of alcohol, and distribution of possessions to other people. In retrospect, it was found that he was stockpiling old medications and refused to comply with his plan of care. The patient's outcome was tragic despite numerous attempts to provide a support system to meet his needs. Although he appeared to improve with therapy, the patient may have had a suicidal agenda that he managed to hide from everyone, including his family and multiple healthcare professionals. The patient's daughter was offered bereavement counseling services, which she appreciated.

Tutor and student perceptions of the tutor's role in problem-based learning.

The role of the tutor in problem-based learning (PBL) differs dramatically from the traditional teaching role with which dental students and faculty are familiar. In this investigation, the dimensionality and complexity of the tutor role were assessed by first- and second-year dental students and by the tutors themselves using a twenty-four-item Likert-type questionnaire that served as one component of a comprehensive web-based assessment tool, the PBL-Evaluator. Evaluations were completed after each PBL case over a period of one and a half years by one class of students and for half a year by a second class. Exploratory principal components analyses of the responses to the questionnaire revealed a more complex factor structure for tutors than for students. While a five-factor solution was required for the tutors, a simpler two-factor solution sufficed for both groups of first-year students, and a three-factor solution was necessary for the second-year students. Although tutors displayed a more nuanced perspective on their performance than did students, items related to tutor modeling of professional conduct within tutorials consistently emerged as important for both.

Is it Alzheimer's? Neuropsychological testing helps to clarify diagnostic puzzle.

This patient displayed a pattern of neuropsychological test results that indicated early Alzheimer's dementia. Using neuropsychological testing to make the diagnosis provided an answer for the patient, family, and psychiatrist, who all recognized a problem, and allowed for future planning. Recommendations for the patient included: psychoeducation and support for the patient and family assistance with planning for the future, including discussions of advance directives and financial management re-evaluation of the patient's ability to drive a motor vehicle a trial of a cholinesterase inhibitor such as donepezil or rivastigmine ongoing health maintenance, including periodic screening for depression and other psychiatric symptoms repeat neuropsychological assessment in 6 months to 1 year to assess rate of decline and confirm the diagnosis.

Serotonin syndrome. A common but often unrecognized psychiatric condition.

This case illustrates that compliance with the basic principles of geriatric pharmacology (start low, go slow, and avoid polypharmacy) might have prevented a clinical syndrome that could have caused serious complications. Fortunately, serotonin syndrome was recognized early, the offending agents were discontinued, and supportive treatment was provided. The patient was ultimately treated with mirtazapine, an antidepressant not associated with serotonergic effects.

Treatment of impulsivity and aggression in a patient with vascular dementia.

This case illustrates how treatment with divalproex sodium, an agent approved for use as an anticonvulsant and mood stabilizer, led to a reduction in overt aggression, diminished impulsivity, and improved functional status in a patient with vascular dementia. Improved receptivity to care and reduced hostility also led to an increased quality of life for the family caregivers. The daughter also benefited greatly from participation in a family support group.

St. John's wort and antidepressant drug interactions in the elderly.

There is increasing interest in and use of the herbal preparation St. John's wort. Hypericin, the major active ingredient, has many psychoactive properties. The agent is sold in the US as a nutritional supplement and is recommended for numerous conditions, including depression, anxiety, insomnia, and inflammation. We report a series of five cases of clinically diagnosed central serotonergic syndrome among elderly patients who combined prescription antidepressants with St. John's wort. Older adults are large consumers of both over-the-counter and prescription medications. They are particularly vulnerable to interactions between medications and products sold as nutritional or herbal supplements. St. John's wort requires further evaluation due to potential for drug interactions with central nervous system agents and for more definitive therapeutic indications.

Activities of the Porphyromonas gingivalis PrtP proteinase determined by construction of prtP-deficient mutants and expression of the gene in Bacteroides species.

PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-D-Tyr-L-Pro-L-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.

Immunization with Porphyromonas gingivalis cysteine protease: effects on experimental gingivitis and ligature-induced periodontitis in Macaca fascicularis.

Targeting bacterial virulence factors such as proteases for immunization may hold the key to limiting or preventing loss of attachment and alveolar bone in periodontal disease. This study examined the clinical, microbiological, and immununological responses following active immunization with a purified Porphyromonas gingivalis cysteine protease (porphypain-2) in the nonhuman primate (Nhp) Macaca fascicularis. One group of Nhp was immunized with porphypain-2 antigen while control Nhp received placebo injections. All Nhp were subjected to experimental gingivitis followed by ligature-induced periodontitis in a split-mouth design. An enzyme-linked immunosorbent assay demonstrated that immunization elicited a significantly elevated and specific IgG antibody response to both whole cell P. gingivalis (36-fold) and to porphypain-2 (194-fold). Checkerboard hybridization DNA analysis of subgingival plaque from ligated sextants demonstrated that 25% more Gram-negative anaerobic species became significantly elevated from baseline and at earlier timepoints in the control group than in the immununized group. Immunization with this protease did not suppress the emergence of P. gingivalis. Clinical indices showed few changes related to immunization. Alveolar bone density changes demonstrated a highly significant loss in ligated sextants compared to non-ligated sextants within the control group (P < 0.001), and a smaller but significant difference within the immunized group (P = 0.043). Comparison of ligated sextants only demonstrated more bone loss in the control group versus the immunized group (-13.07+/-9.51 versus -9.41+/-6.18; computer-assisted densitometric image analysis units +/- SD); the difference approached, but did not reach, significance. The results suggest that porphypain-2 may contribute to the pathogenic potential of the subgingival plaque microbiota in the Nhp model of ligature-induced periodontitis, and that active immunization with porphypain-2 appeared capable of altering this pathogenic response.