Evidence-based clinical practice guideline for the evaluation of potentially malignant disorders in the oral cavity: a report of the American Dental Association

BACKGROUND: An expert panel convened by the American Dental Association (ADA) Council on Scientific Affairs and the Center for Evidence-Based Dentistry conducted a systematic review and formulated clinical recommendations to inform primary care clinicians about the potential use of adjuncts as triage tools for the evaluation of lesions, including potentially malignant disorders (PMDs), in the oral cavity. TYPES OF STUDIES REVIEWED: This is an update of the ADA's 2010 recommendations on the early diagnosis of PMDs and oral squamous cell carcinoma. The authors conducted a systematic search of the literature in MEDLINE and Embase via Ovid and the Cochrane Central Register of Controlled Trials to identify randomized controlled trials and diagnostic test accuracy studies. The authors used the Grading of Recommendations Assessment, Development and Evaluation approach to assess the certainty in the evidence and to move from the evidence to the decisions. RESULTS: The panel formulated 1 good practice statement and 6 clinical recommendations that concluded that no available adjuncts demonstrated sufficient diagnostic test accuracy to support their routine use as triage tools during the evaluation of lesions in the oral cavity. For patients seeking care for suspicious lesions, immediate performance of a biopsy or referral to a specialist remains the single most important recommendation for clinical practice. In exceptional cases, when patients decline a biopsy or live in rural areas with limited access to care, the panel suggested that cytologic testing may be used to initiate the diagnostic process until a biopsy can be performed (conditional recommendation, low-quality evidence). CONCLUSIONS AND PRACTICAL IMPLICATIONS: The authors urge clinicians to remain alert and take diligent action when they identify a PMD. The authors emphasize the need for counseling because patients may delay diagnosis because of anxiety and denial.(AU)

Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis.

Adult periodontitis is a chronic inflammatory disease that affects over 49 million people in the USA alone. Porphyromonas (formerly Bacteroides) gingivalis, a Gram-negative anaerobe, has a diverse repertoire of virulence factors that may be involved in the induction or progression of periodontitis.

Changes in the localization of catalase during differentiation of neutrophilic granulocytes.

The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Rapid fluorometric quantification of monocyte attachment in tissue culture wells.

A simple fluorometric assay that permits rapid quantification of attachment of monocytes or macrophages in tissue culture wells is described. Using 4,6-diamidino-2-phenylindole (DAPI) as a specific fluorochrome marker for DNA, we observed a dose-dependent increase with strong linear correlation in fluorescent emission over a broad range of DNA concentrations. Measurements of the DNA content of the human monocytic cell line THP-1 demonstrated a linear correlation between fluorescence intensity and cell number from 5 x 10(4) to 1 x 10(6) cells, with an estimated average DNA content of 7.5 pg DNA per cell. While untreated THP-1 cells were not detectably adherent, PMA induction for 24 h results in 57-76% adherence to plastic surface. This method was found to be useful for measuring the number of peripheral blood monocytes separated from lymphocytes by attachment. 16 subjects were sampled and the standard deviation of each individual did not exceed 10%. The number of attached cells was between 10-16% of the total mononuclear cells. Fluorescence measurement of DNA with DAPI permits rapid and accurate determination of cell numbers and appears useful in the quantification of adherent populations such as myelocytic cells and cell lines.

Functional differences in human neutrophils isolated pre- and post-prandially.

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid-rich meal. There was up to 2-fold greater superoxide generation in response to agonists in leukocytes obtained post-prandially; the maximum increase was observed about 4 h after eating and followed the peak (2-4 h) in serum triglycerides. Neutrophils isolated post-prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

Antibody-dependent alternate pathway of complement activation in opsonophagocytosis of Porphyromonas gingivalis.

It has been suggested that the ability of Porphyromonas gingivalis to proteolyse complement, as well as its production of a capsule, contributes to resistance to phagocytosis by polymorphonuclear leukocytes. In this report, the opsonic role of serum complement and its activation pathways were investigated, using individual sera heat treated or depleted of factors B, C2, and C1q and the divalent cations Mg2+ and Ca2+. A fluorochrome microassay was used to quantitate phagocytosis of P. gingivalis A7436 by human polymorphonuclear leukocytes. Heat treatment of rabbit antiserum to P. gingivalis (RaPg) (56 degrees C, 30 min) resulted in a reduction in phagocytosis from 100% to 55% +/- 5%, while heat treatment of chronic adult periodontal disease serum abrogated phagocytosis. The heat-labile activity of RaPg was fully restored with MgEGTA-chelated rabbit serum but not EDTA- or EGTA-chelated rabbit serum. The addition of serum depleted of factor B but not C2 or C1q restored most of the heat-labile activity; however, the factor B-depleted serum was suspect, due to low-level opsonization of zymosan (inhibitable by EDTA but not MgEGTA). Adding C1q at 80 micrograms/ml to serum depleted of C1q restored much but not all of the activity lost through heat treatment or through depletion of C1q. A large part of opsonic activity with C2- and C1q-depleted sera was enhanced by the addition of 4 x 10(-3) M Mg2+. The data indicate that although opsonophagocytosis of P. gingivalis A7436 is dependent on the classical complement pathway, a significant contribution is made by an antibody-dependent alternate pathway.

Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone.

Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen.

Superior leukocyte separation with a discontinuous one-step Ficoll-Hypaque gradient for the isolation of human neutrophils.

A discontinuous gradient system composed of two commercially available Ficoll-Hypaque mixtures (Mono-Poly resolving medium (MPRM) and Histopaque 1.077) is described for the purification of mononuclear and polymorphonuclear leukocytes from human blood. Like the original one-step Hypaque-Ficoll procedure (Ferrante and Thong, 1978), a single centrifugation at 500 X g for 30 min resulted in the formation of two distinct leukocyte fractions. In contrast to MPRM alone, the discontinuous system (MPRM-HP) was capable of resolving leukocyte fractions from blood volumes as small as 1 ml with excellent purity and yield. MPRM-HP was also compatible with a wider range of anticoagulants and permitted fractionation of specimens resistant to MPRM.