Proliferative verrucous leukoplakia of the gingiva.

OBJECTIVE: The purpose of this study was to describe the clinical-pathologic features of what appears to be a gingival form of proliferative verrucous leukoplakia. STUDY DESIGN: Ten adult patients with recurrent and histologically progressive gingival leukoplakias who were diagnosed and treated at the University of California, San Francisco between 1994 and 1999, comprised the subject group for this investigation. Clinical and microscopic features were reviewed. Proliferation indices and p53 expression were evaluated immunohistochemically, and the presence of human papillomavirus (HPV) DNA was determined by using polymerase chain reaction (PCR) amplification. RESULTS: Lesions presented as solitary or regional flat/papillary/verrucal leukoplakias of the free and attached gingiva (tooth-bearing areas only). With time, flat lesions developed a papillary or verruciform profile. Although lesions were recurrent, they were confined to the gingiva, and multiple lesions did not develop. Half the patients used tobacco, and HPV could not be detected by using PCR. Microscopically, 6 cases began as hyperkeratotic lesions, and 4 initially exhibited a psoriasiform pattern with a marked inflammatory component. With recurrences, the lesions became progressively atypical histologically. The proliferation indices for these lesions showed modest increases over normal epithelium, and positive p53 staining was evident in 4 of 10 cases, indicating a disruption of the keratinocyte cell cycle in these lesions. The mechanism associated with the positive p53 staining (protein binding to wild type p53 versus mutation of the p53 gene) was not determined. Lesions recurred after conservative scalpel or laser excision, and many developed into verrucous or squamous cell carcinoma. CONCLUSIONS: Proliferative verrucous leukoplakia of the gingiva (PVLG) appears to be a subset of oral proliferative verrucous leukoplakia. It can be characterized as a solitary, recurring, progressive white patch that develops a verruciform architecture and may not be associated with HPV. PVLG has an unpredictable course and is at risk for development into verrucous or squamous cell carcinoma. Currently, there is no way to determine or predict which gingival white lesions will follow the clinical course described for this group of patients with PVLG.

Heat shock (stress) proteins and gamma delta T lymphocytes in oral lichen planus.

OBJECTIVE: Heat shock proteins (Hsps), a highly conserved class of protective cellular proteins that are produced under various conditions of environmental challenge, have been implicated as the antigenic stimulus in autoimmune diseases. Because lichen planus (LP) appears to be an autoimmune or hyperimmune condition (mediated by T cells), Hsps may have a role in the pathogenesis of this disease. We believe that if keratinocyte Hsps are antigenic targets of a cellular immune response, upregulation of these proteins should be demonstrable in tissue sections. STUDY DESIGN: Immunohistochemistry was used to evaluate expression of several families of Hsps in oral lichen planus tissues. The number and distribution of gamma delta T cells, a subset of T lymphocytes with an immune surveillance function that may contribute to autoimmunity, were also evaluated. Monoclonal antibodies to Hsps 27, 60, 70, 90, gamma delta receptor, and CD3 (pan-T lymphocyte marker) were incubated with frozen sections of LP (n = 22) and normal oral mucosa (n = 17) followed by an avidin-biotin-peroxidase labeling method. Antibodies to bacterial Hsps (GroEL and DnaK) were used as negative controls, and antibody to constitutive eukaryotic Hsp (Hsc70) was used as a positive control. RESULTS: In six cases of LP, basal keratinocytes stained intensely for Hsp27, whereas controls showed only slight staining. Otherwise LP and normal tissues showed comparable positive staining of upper level keratinocytes with anti-Hsp27. Subjective increases in antibody staining were noted for Hsp60 in LP, which was due in part to staining of infiltrating lymphocytes and in part to keratinocyte expression. Normal tissues showed weak basal cell antibody staining for Hsp60. Hsp70 staining was observed at a less intense level in LP than in controls. Except for more intense basement membrane staining with anti-Hsp90 antibody in gingiva and palate, no differences in the occurrence of this protein were found. Absolute numbers of gamma delta T cells were increased in LP when compared with those in control specimens (n = 10 vs n = 1, respectively, per high-power field). However, gamma delta T cells represented less than 1% of the CD3+ lymphocytes. CONCLUSIONS: It was concluded that normal oral mucosa expresses Hsps 27, 60, 70, and 90 and contains few gamma delta T cells. Although the expression of Hsps was altered in LP, the differences demonstrated were slight and were therefore inconclusive. The Hsps expressed in LP could have contributed to the persistence or chronicity of the disease, or they could have simply reflected cellular injury.

Hemin uptake in Porphyromonas gingivalis: Omp26 is a hemin-binding surface protein.

A 26-kDa outer membrane protein (Omp26) has been proposed to play a role in hemin acquisition by Porphyromonas gingivalis (T. E. Bramanti and S. C. Holt, J. Bacteriol. 174:5827-5839, 1992). We studied [55Fe]hemin uptake in P. gingivalis grown under conditions of hemin starvation (Omp26 expressed on the outer membrane surface) and hemin excess (Omp26 not expressed on surface). [55Fe]hemin uptake occurred rapidly in hemin-starved cells which incorporated up to 70% of total [55Fe]hemin within 3 min. P. gingivalis grown under hemin-starved conditions or treated with the iron chelator 2,2'-bipyridyl to induce an iron stress took up six times more [55Fe]hemin than hemin-excess-grown cells. Polyclonal monospecific anti-Omp26 antibody added to hemin-starved cells inhibited [55Fe]hemin uptake by more than 50%, whereas preimmune serum had no effect. [55Fe]hemin uptake in hemin-starved P. gingivalis was inhibited (36 to 67%) in the presence of equimolar amounts of unlabeled hemin, protoporphyrin IX, zinz protoporphyrin, and Congo red dye but was not inhibited in the presence of non-hemin-containing iron sources. Heat shock treatment (45 degrees C) of hemin-excess-grown P. gingivalis (which cases translocation of Omp26 to the surface) increased [55Fe]hemin uptake by threefold after 3 min in comparison with cells grown at 37 degrees C. However, no [55Fe] hemin uptake beyond 3 min was observed in either hemin-excess-grown or hemin-starved cells exposed to heat shock. In experiments using heterobifunctional cross-linker analysis, hemin and selected porphyrins were cross-linked to Omp26 in hemin-starved P. gingivalis, but no cross-linking was seen with hemin-excess-grown cells. However, cross-linking of hemin to Omp26 was observed after heat shock treatment of hemin-excess-grown cells. Finally, anti-Omp26 antibody inhibited cross-linked of hemin to Omp26. These findings indicate that hemin binding and transport into P.gingivalis cell mediated by Omp26.

Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane.

We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.

Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis: identification of a novel 26 kDa hemin-repressible surface protein.

Porphyromonas gingivalis is capable of in vitro growth when iron sources are either complexed to hemin or host iron transport proteins, or exist in an inorganic form. This study examined the effect of these iron sources on outer membrane protein (OMP) expression in P. gingivalis W50. Hemin (iron) starved P. gingivalis was transferred into growth medium containing hemin, hemoglobin, hemin-saturated human serum albumin, hemin-free human serum albumin, transferrin, lactoferrin, or inorganic iron. Surface proteins were identified by 125I-labeling and resolved by SDS-PAGE and autoradiography. When grown under hemin starved conditions, P. gingivalis W50 and related strains expressed a major 26 kDa OMP, as revealed by 125I-autoradiography. Autoradiographic analysis demonstrated the absence of this 26 kDa OMP from the P. gingivalis surface in hemin-containing environments. Growth of P. gingivalis W50 in the presence of host iron transport proteins (hemin-free) or inorganic iron resulted in surface expression of a 26 kDa OMP. The presence of protoporphyrin IX or substitution of hemin-associated iron with zinc, resulted in continued surface expression of the 26 kDa OMP, indicating that repressibility of this OMP required the coordination of iron to the protoporphyrin IX molecule (i.e. hemin). A survey of 125I-labeled OMPs from several hemin starved P. gingivalis and related strains, demonstrated that a hemin-repressible 26 kDa OMP occurred only in P. gingivalis. We report here a newly described 26 kDa hemin-regulated surface protein occurring in several strains of P. gingivalis which is expressed on the cell surface in hemin starved conditions and is lost from the cell surface in response to an environment containing iron coordinated specifically to protoporphyrin IX (i.e. hemin).

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.

Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization.

Porphyromonas gingivalis W50, W83, A7A1-28, and ATCC 33277 were investigated for their abilities to lyse sheep, human, and rabbit erythrocytes. All of the P. gingivalis strains studied produced an active hemolytic activity during growth, with maximum activity occurring in late-exponential-early-stationary growth phase. The enzyme was cell bound and associated with the outer membrane. Fractionation of P. gingivalis W50 localized the putative hemolysin almost exclusively in the outer membrane fraction, with significant hemolytic activity concentrated in the outer membrane vesicles. Ca2+ and Mg2+ ions significantly increased the expression of hemolytic activity. Hemolytic activity was inhibited by proteinase K, trypsin, the proteinase inhibitors Na-P-tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenyl-hydrazone, and iodoacetate. KCN and sodium azide (NaN3) only partially inhibited P. gingivalis hemolytic activity, while antiserum to whole cells of each of the P. gingivalis strains had a significant inhibitory effect on hemolytic activity. The P. gingivalis W50 hemolysin was inhibited by cysteine, dithiothreitol, and glutathione at concentrations of at least 10 mM; at low concentrations (i.e., 2 mM), dithiothreitol did not completely inhibit hemolytic activity. Heating to temperatures above 55 degrees C resulted in an almost complete inhibition of hemolytic activity. The effect of heme limitation (i.e., iron) on hemolysin production indicated that either limitation or starvation for heme resulted in significantly increased hemolysin production compared with that of P. gingivalis grown in the presence of excess heme.

Factors in virulence expression and their role in periodontal disease pathogenesis.

The classic progression of the development of periodontitis with its associated formation of an inflammatory lesion is characterized by a highly reproducible microbiological progression of a Gram-positive microbiota to a highly pathogenic Gram-negative one. While this Gram-negative microbiota is estimated to consist of at least 300 different microbial species, it appears to consist of a very limited number of microbial species that are involved in the destruction of periodontal diseases. Among these "putative periodontopathic species" are members of the genera Porphyromonas, Bacteroides, Fusobacterium, Wolinella, Actinobacillus, Capnocytophaga, and Eikenella. While members of the genera Actinomyces and Streptococcus may not be directly involved in the microbial progression, these species do appear to be essential to the construction of the network of microbial species that comprise both the subgingival plaque matrix. The temporal fluctuation (emergence/disappearance) of members of this microbiota from the developing lesion appears to depend upon the physical interaction of the periodontal pocket inhabitants, as well as the utilization of the metabolic end-products of the respective species intimately involved in the disease progression. A concerted action of the end-products of prokaryotic metabolism and the destruction of host tissues through the action of a large number of excreted proteolytic enzymes from several of these periodontopathogens contribute directly to the periodontal disease process.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron-regulated outer membrane proteins in the periodontopathic bacterium, Bacteroides gingivalis.

Hemin has been implicated in the pathogenesis of the oral pathogen, Bacteroides gingivalis. In order to elucidate the role of hemin (iron) in the growth and expression of outer membrane proteins, B. gingivalis strain W50 was grown with and without hemin to induce iron-limitation. Cells grew slower under iron stress and growth was completely inhibited in the absence of added hemin. The outer membrane protein profiles of B. gingivalis grown under iron-replete and iron-restricted conditions were studied by extrinsic radiolabelling with [125I] and polyacrylamide gel-electrophoresis. The induction of 10 surface proteins, with apparent molecular weights of 26, 29, 50, 56, 58, 60, 62, 71, 77, and 80 Kd, was observed in B. gingivalis grown under iron-restricted conditions. These proteins were repressed under iron-replete conditions. We postulate the involvement of the iron-regulated proteins in hemin uptake and virulence in B. gingivalis.

Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277.

The chemistry and selected biological activity of lipopolysaccharide (LPS) from Bacteroides gingivalis strains W50, W83, and ATCC 33277 were compared, as well as the role of this molecule as a mediator of selected inflammatory responses. Chemically, the LPSs consisted of 47-58% Lipid A, 5-10% carbohydrate, 0.05% 3-deoxy 2-octulosonic acid, 0.3% heptose, 3.8-5.2% hexosamine, and 2% phosphate. Rhamnose represented the dominant sugar (26-36%), with lesser amounts of glucose (18-34%), galactose (18-25%), mannose (9-12%), glucosamine (7-11%), and galactosamine (2-5%). The major fatty acids were: 13-methyl-tetradecanoate (42-45%), 3-OH-heptadecanoate (21-23%), hexadecanoate (16-19%), and 12-methyl-tetradecanoate (6-8%). SDS-PAGE and sodium deoxycholate-PAGE revealed the LPS to be a smooth chemotype. Differences in migration patterns between the virulent and avirulent strain LPSs also occurred. C3H/HeN macrophages (Mø) exposed to 1 microgram/ml of LPS released 3.2-4.2 ng of prostaglandin E (PGE)/ml of supernatant, representing 236-278% of control. Interleukin-1 (IL-1) activity in C3H/HeN and C3H/HeJ Mø exposed to 50 micrograms of LPS/ml was 382-724% and 270-300% of control, respectively; similar Mø exposed to 10 micrograms of LPS/ml released 1.6-2.0 ng and 0.3-0.5 ng of tumor necrosis factor (TNF)/ml of supernatant, respectively. Maximum TNF release in C3H/HeN Mø occurred in response to 50 micrograms of LPS/ml, and was sustained for up to 96 hours. These results suggest that LPS from the B. gingivalis strains stimulate cytokine production from Mø which, in turn, may play a role in orchestrating the inflammatory response for the development of periodontal diseases.