Autoimmunity to deltaNp63alpha in chronic ulcerative stomatitis.

Chronic ulcerative stomatitis (CUS) is a recently described mucocutaneous condition in which patients experience chronic, painful, ulcerative lesions of the oral mucosa. CUS is diagnosed by immunofluorescence studies that demonstrate antinuclear antibodies. These autoantibodies are specific for a protein, deltaNp63alpha, which is normally expressed in basal cell nuclei of stratified squamous epithelia. The purpose of this study was to characterize the autoimmune response in CUS. Protein antigens were produced by in vitro transcription/translation of polymerase chain-reaction (PCR)-amplified cDNAs. We used immunoblotting and immunoprecipitation experiments with serum from CUS patients to examine the (1) antibody isotype, (2) immunogenic functional domains of the deltaNp63alpha antigen, and (3) cross-reactivity with homologous p53, p73, and p63 proteins. Results demonstrate CUS patient antibodies to deltaNp63alpha, and 52% of cases have circulating IgA isotype antibodies. The N-terminal and DNA-binding domains are the immunodominant regions, and antibody cross-reactivity with p53, p63, and p73 isoforms is limited.

Using immunofluorescence in the diagnosis of chronic ulcerative lesions of the oral mucosa.

Chronic ulcerative diseases of the oral mucosa are commonly seen in clinical practice. On clinical and histological appearance, the lesions may be hard to differentiate from each other. The establishment of definite diagnosis is essential because the patient may require different management and have widely varying prognosis. Immunofluorescence studies aid greatly in the process of determining the diagnosis of a number of chronic ulcerative diseases. This article reviews these chronic ulcerative diseases, describing their clinical, microscopic, and immunofluorescence characteristics. Methods of diagnosis and management of the diseases also are discussed.

Risks of occupational exposure to latex gloves.

The extraordinary increase in latex glove use in dentistry within the past decade has created a potential occupational hazard in the form of adverse reactions to components found in these gloves. Reactions may range from dry, itchy skin to a life-threatening, anaphylactic response. Management of these conditions may be as simple as switching glove brands; but in the most severe cases, it may entail the need to create a latex-free environment for the safety of the affected health care worker. This article reviews the pathophysiology, epidemiology, diagnosis and management of these conditions and provides references for more in-depth reading on the subject.

Local inflammatory effects of composite resins.

Composite resins have been widely used as an anterior restorative material and often have been used to restore posterior teeth. However, composites occasionally can become embedded in oral soft tissues during finishing or shaping procedures, which can lead to persistent chronic inflammation. Limited evaluation in animal model systems has shown that this entrapment in soft tissues can sometimes lead to local inflammation in adjacent soft tissues. Consequently, finishing and polishing procedures should be performed, where practical.

Phenotypic characterization of mononuclear cells from gingiva associated with periodontitis and peri-implantitis.

The purpose of this study was to compare the phenotypic distribution of resident gingival mononuclear inflammatory cells from tissues associated with peri-implantitis and periodontitis. Inflamed gingiva was obtained from six patients during surgical removal of failed dental implants. Similarly, inflamed gingiva around teeth was obtained from eight patients with moderate to advanced periodontitis. Monoclonal antibodies were used to identify membrane antigens from CD4+ T-lymphocytes, CD4+/CD8(+)-activated T-lymphocytes, tissue macrophages, CD20+ B-lymphocytes, and MHC class II (Ia) antigens. Gingival inflammation associated with both dental implants and natural teeth was characterized by substantial numbers of CD4+ T-lymphocytes, resident macrophages, and B-lymphocytes. In addition, there was an abundance of HLA class II-positive mononuclear cells throughout most specimens. These results suggest that the gingival mononuclear inflammatory response in peri-implantitis and periodontitis is similar and support the hypothesis that similar inflammatory mechanisms are associated with both conditions.

The effect of methotrexate on the expression of a cysteine protease inhibitor (type 2 cystatin) in rat sebaceous glands.

The purpose of this study was to assess whether rat cystatin S, a cysteine proteinase inhibitor, is present in rat sebaceous glands, and to measure the effects of methotrexate on the expression of cystatin in these glands. With methotrexate treatment, the number of skin sebaceous cells expressing cystatin increased from 13.9% to 34.3% (P < .05). A smaller increase (from 15.3% to 23.9%; P = .1) was observed in Zymbal sebaceous glands. Type 2 cystatin could not be detected in the major salivary glands, nor in trachea, lung, stomach, small intestine, large intestine, spleen, liver, kidney, or pancreas, in any of the rats given either saline or methotrexate. Our results suggest that type 2 cystatin is a constituent of normal sebaceous glands, and that the amount of cystatin present in these glands increases with methotrexate administration. We speculate that, in addition to the protective functions ascribed to sebaceous lipids, sebum may augment the physical barrier of skin through secretion of cysteine proteinases that may be pharmacologically modulated.

Localized cellular inflammatory responses to subcutaneously implanted dental mercury.

Previous reports have demonstrated mercury accumulation and toxicity in oral tissues following exposure to mercury vapor from dental amalgam restorations. In the present study, inflammatory responses to subcutaneously administered mercury were assessed histopathologically and immunocytochemically in a rat model system. A panel of six well-characterized monoclonal antibodies specific for monocytes, macrophage subsets, T and B lymphocytes, and major histocompatibility complex (MHC) class II (la) determinants was used to quantitate alterations in mononuclear cell subsets in situ at time intervals from 2 d to 8 wk. The results revealed acute inflammatory cell infiltration at 2 and 3 d, followed by chronic inflammation that persisted after 8 wk. The numbers of monocytes, resident macrophage subsets, and mononuclear cells expressing la antigen were significantly different from control tissues at 1-2 wk. The numbers of resident macrophages remained significantly higher even after 8 wk. These data showed that in situ mercury accumulation can lead to altered expression of MHC class II determinants with persistent chronic inflammation and shifts in mononuclear cell subpopulations.

Effect of protoporphyrin IX limitation on porphyromonas gingivalis.

Porphyromonas gingivalis has been shown to require hemin or hemoglobin for in vitro growth. We have previously shown that protoporphyrin IX and inorganic iron can replace the hemin requirement, suggesting that the hemin requirement of this microorganism is actually a porphyrin requirement. We examined the effect of protoporphyrin IX limitation to P. gingivalis strain A7A1-28 in the presence of sufficient iron on growth characteristics, proteolytic enzyme production, virulence in a mouse abscess model, and expression of membrane proteins. Bacterial cells were grown in medium varying between 0 to 5 microM reduced growth by at least 50%. Protoporphyrin IX availability did not affect proteolytic enzyme production or virulence in a mouse abscess model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane preparations demonstrated that protoporphyrin IX limitation induced the expression of new proteins at 42, 34, 30, 29, and 18 kDa and suppressed the production of proteins at 47, 27, 17, and 15 kDa. These studies suggest that in vivo protoporphyrin availability may modulate membrane protein expression and in turn affect host immune responses against P. gingivalis.

Cellular inflammatory responses to implanted dental materials.

Cellular inflammatory responses to subcutaneous implantation of amalgam and composite resins were assessed in rats by use of histologic and immunocytochemical methods 2 days to 8 weeks after implantation. Frozen and paraffin sections were obtained from subcutaneous tissues associated with amalgam and composite resin suspensions. The amalgam induced mild inflammation with proliferation of few resident macrophages, but implantation of composite resins was associated with an influx of monocytes, increased numbers of resident connective tissue macrophages, and abnormal major histocompatibility antigen class II (Ia antigen) expression. The data suggest that composite resins may produce a a more pronounced inflammatory response than dental amalgams do when incorporated in soft tissues.

Mononuclear cells in salivary glands of normal and isoproterenol-treated rats.

The purpose of this study was to analyse the phenotypical distribution of resident cells of the mononuclear phagocyte system in rat salivary glands, and to determine whether isoproterenol induces alterations in macrophage and lymphocyte surface-marker expression. Frozen sections of gland tissues were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigens (OX6), CD5-positive T lymphocytes (OX19), and rat B lymphocytes (OX33). Double-labelling techniques were used to detect the coexpression of ED1/ED2 and OX6/ED2 mononuclear cell markers in the major salivary glands. ED2-positive macrophages were predominant in all three major glands, ranging from 96 cells/0.87 mm2 field in the parotid gland to 165 cells/0.87 mm2 in the submandibular. OX19-positive T lymphocytes were rarely observed in submandibular and parotid glands but represented a distinguishing feature of the sublingual. Moderate numbers of ED3-positive macrophages also were detected in sublingual tissues. In the submandibular and parotid glands, isoproterenol resulted in a decrease in ED2-positive cells, but ED2-positive macrophages increased in sublingual glands with isoproterenol. Isoproterenol resulted in a decrease in MHC class II antigen expression on submandibular and sublingual mononuclear cells but an induction of Ia antigen in the parotid gland. Double labelling revealed that isoproterenol induced coexpression of ED1/ED2 markers on mononuclear cells in the submandibular glands, but ED1/ED2-positive cells were absent from other glands. However, coexpression of MHC class II markers on ED2-positive cells in the sublingual and parotid glands of normal rats was frequently observed, with isoproterenol decreasing coexpression in the sublingual gland and increasing it in the parotid. B lymphocytes were not detected in any of the glands examined. These findings indicate that important differences exist in normal resident mononuclear cell subsets among the major salivary glands of the rat. The differential effects of isoproterenol on inflammatory cells may reflect important differences in local salivary gland immunoregulation. Although salivary gland inflammation induced by isoproterenol does not appear to result from immune mechanisms, the rich population of T lymphocytes and ED3-positive macrophages, and presence of MHC class II antigens, suggest that the sublingual gland may function as an immune organ and have a role in mucosal immunity.

Phenotypic characterization of mononuclear cells following anorganic bovine bone implantation in rats.

The purpose of this study was to measure inflammatory changes associated with implantation of anorganic bovine bone and bovine bone/collagen composite grafts, and to compare the response to that obtained following grafting with hydroxyapatite. Anorganic bovine bone, either with or without bovine collagen, as well as granular and block forms of synthetic hydroxyapatite, were implanted subcutaneously in Wistar rats. Saline and turpentine oil were used as controls. Biopsies were obtained after 3 days and at 1, 2, 4, 6, and 8 weeks. A panel of 6 monoclonal antibodies was used to detect monocytes, several distinct macrophage subsets, Ia-antigen expression, and T- and B-lymphocytes. Cells identified by each antibody were counted after immunocytochemical staining, and sera obtained 6 weeks after grafting were used in immunoblotting assays to detect antibodies to bovine serum proteins and collagen. Anorganic bovine bone, bovine bone/collagen, and hydroxyapatite all produced a transient macrophage infiltrate that was maximum 3 days after implantation, but resolved to normal levels within 6 to 8 weeks. Lymphocyte infiltration was not elicited by any bovine graft material, and antibodies to bovine serum proteins or type I collagen were not detected in any of the animals examined. These data indicate that a systemic or local immune response does not develop following implantation with anorganic bovine bone or with anorganic bovine bone/collagen materials. It appears appropriate to explore further the merits of these materials for periodontal regenerative procedures.

Membrane protein expression by Actinobacillus actinomycetemcomitans in response to iron availability.

Iron-limited growth conditions under anaerobiosis were established for Actinobacillus actinomycetemcomitans strains Y4, JP2, and 75 by use of the ferrous ion chelator 2,2'-dipyridyl. Growth inhibition was reversible with both ferrous and ferric iron sources. Sarcosyl-insoluble membrane fractions of iron-stressed anaerobic A. actinomycetemcomitans cultures revealed a similar iron-repressible protein of approximately 70 kDa in A. actinomycetemcomitans strains Y4, JP2, and 75. This 70-kDa protein was recognized by serum from localized juvenile periodontitis patients and a periodontally healthy subject. This suggested that the 70-kDa iron-repressible protein may be expressed in vivo. When A. actinomycetemcomitans was grown under aerobic conditions, the ferric iron chelator, ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) was utilized for growth limitation. EDDA inhibition was reversible in strain Y4 with ferrous and ferric iron sources. An iron-repressible protein of approximately 70 kDa was also noted in iron-stressed aerobic cultures. The 70-kDa protein may be involved in iron transport by A. actinomycetemcomitans. Preliminary experiments were performed to examine potential iron transport systems for A. actinomycetemcomitans. Production of the two most common chemical types of siderophore was not detected in A. actinomycetemcomitans culture supernatants. Iron-starved A. actinomycetemcomitans cells did not bind transferrin or lactoferrin in a dot blot assay.

Cellular inflammatory responses to direct restorative composite resins.

As a result of shaping, finishing, or removal of dental composite resin restorations, resin particles may become trapped and embedded in oral tissues. However, tissue reactions induced by entrapped composite resin particles have not been thoroughly examined. To assess the soft tissue inflammatory response to composite restorative materials, three commercial dental composite resin suspensions were implanted subcutaneously in rats. Implantation resulted in a granulomatous inflammatory reaction that persisted 8 weeks after placement. The lesion was characterized by an influx of lymphocytes and the presence of fibroblasts and epithelioid cells. Composite resin particles have the potential to cause persistent inflammation if they are entrapped in oral tissues.

Induction of type 2 cystatin in rat submandibular glands by systemically administered agents.

An inducible type 2 cystatin has earlier been characterized in submandibular glands and kidneys of rats treated with isoproterenol, as well as in kidneys of rats with experimental renal disease. The purpose now was to determine whether giving agents that have systemic toxicity could also be associated with induction of cystatin in rat salivary glands. Female Wistar rats (200-250 g) were given isoproterenol, cyclocytidine, potassium dichromate or turpentine oil. After autopsy, the organs were sectioned, fixed in 10% formalin, and processed routinely. Paraffin sections were processed for both the peroxidase-antiperoxidase and the avidin-biotin-alkaline phosphatase immunocytochemical methods. The submandibular glands of rats given cyclocytidine had generalized, strong staining of acinar cells, as well as occasional weak staining within granular convoluted tubules. Animals given either potassium dichromate or turpentine oil exhibited moderate staining for cystatin in submandibular acini. Rats given isoproterenol as a positive control exhibited strong acinar staining throughout the submandibular gland, while the glands of untreated rats were unreactive. Inducible type 2 cystatin could not be detected in the parotid or sublingual glands, or in trachea, lung, stomach, small intestine, large intestine, spleen, liver and pancreas, after treatment with any of the systemic agents evaluated. The results indicate that elaboration of type 2 cystatin can be induced by a variety of systemically administered agents other than isoproterenol, and suggest that elaboration of type 2 cystatin may represent a more generalized response to tissue injury.

Induction of type 2 salivary cystatin in immunological and chemical kidney injury.

We have previously reported that isoproterenol induces type 2 salivary cystatin in both submandibular glands and kidney tubule cells of rats but not in any other organs examined. In the present study, we investigated whether this salivary protein is induced in other conditions that show kidney tubule injury. Immunocytochemistry, using a monospecific antiserum to this cystatin, revealed specific staining within the proximal tubule epithelium of the cortex as well as in the inner and outer stripe of the medulla of immunologically and chemically injured rats. Cystatin could not be detected in kidneys from healthy rats by means of immunocytochemistry. Weak staining was found in 3/3 kidneys of rats treated with turpentine and in 5/5 animals treated with potassium dichromate. In rats treated with puromycin, cystatin could not be demonstrated in 5/5 animals having proteinuria of less than 100 mg/24 h; however, moderate staining was observed in 4/5 puromycin-treated rats having proteinuria greater than 100 mg/24 h. In Heymann nephritis, cystatin was present in 7/31 kidneys with proteinuria lasting 6 to 15 weeks and in none (0/7) with proteinuria of shorter duration. Strong staining was also observed in 10/10 kidneys from rats with moderate-to-severe chronic serum sickness. This study shows that elaboration of type 2 cystatin in rats is not limited to salivary glands and, with our previous study, suggests that induction of this cysteine inhibitor may represent a local response to generalized tissue injury in both submandibular and renal tissues. These findings further demonstrate that induction of cystatin in salivary glands is not unique to these glands and suggest that induction of this cysteine proteinase inhibitor may represent a local response to tissue injury caused by diverse mechanisms.

Characterization of total membrane protein of Porphyromonas endodontalis.

Porphyromonas endodontalis strains ATCC 35406, HG 181, and HG 413 were cultured in an enriched broth medium, and growth curves were determined. Total membrane protein profiles of cells, harvested either from exponential phase or from stationary phase of growth, were studied by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The protein profiles indicated that the three strains shared three major proteins, designated A, B, and C, with molecular masses of 59, 43, and 41 kDa, respectively. In addition, the strains showed homology among the majority of the minor proteins. However, some minor proteins were unique for each strain. Two common minor proteins with molecular masses of 56 and 51 kDa were weakly expressed or absent in membrane preparations from cells of the early exponential phase, while they were present in membrane preparations from cells harvested in the late phase of growth. The major and minor membrane proteins may play a role in the interaction between P. endodontalis and the host. In addition, the membrane proteins may play an important role in the physiology of this endodontic pathogen.

Phenotypic characterization of resident macrophages in submandibular salivary glands of normal and isoproterenol-treated rats.

Macrophages exert a major effect in the stimulation of lymphocytes and the modulation of immunological responses. To determine the presence and phenotypic distribution of the resident cells of the mononuclear phagocyte system in submandibular glands, frozen sections were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with circulatory monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), Ia antigen (OX6), CD5-positive T lymphocytes (OX19) and rat B lymphocytes (OX33). Cells identified by each monoclonal antibody were quantified by averaging the number of positive cells in 10 consecutive random high-power fields. ED2 cells (165 cells/field) were predominant in normal rat submandibular gland, followed by lower numbers of OX6-positive cells (18 cells/field). Cells positive for the remaining markers were also present in smaller amounts. In submandibular glands, treatment of rats with isoproterenol resulted in an increase in ED1-positive cells (from 2 to 39 cells/field), but also in substantial decreases in the number of cells positive for the remaining cell markers. B cells were not detected in any of the submandibular glands examined. These data suggest that isoproterenol induces a mild inflammatory response within rat submandibular glands that is not observed in normal glands. This results in an increase in the relative number of infiltrating monocytes compared to the number of more mature tissue macrophages.

Human immunoglobulin G antibody response to iron-repressible and other membrane proteins of Porphyromonas (Bacteroides) gingivalis.

The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 microM alpha, alpha'-dypyridyl, a ferrous iron chelator. Iron-sufficient growth was achieved by growing bacteria in the same medium without alpha, alpha'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo.

Analysis of in vitro lymphoproliferative responses and antibody formation following subcutaneous injection of Actinobacillus actinomycetemcomitans and Wolinella recta in a murine model.

The potential of Wolinella recta and Actinobacillus actinomycetemcomitans to cause abscesses and induce an immune response was tested in BALB/c mice. Mice were injected subcutaneously with W. recta, A. actinomycetemcomitans or a mixture of these 2 microorganisms. Mice injected with A. actinomycetemcomitans alone, or with both organisms, demonstrated abscesses at the injection site 2 days later, from which pure cultures of A. actinomycetemcomitans were isolated. Mice injected with W. recta had small, flat abscesses at the injection site from which no bacteria could be cultured. W. recta was cultured from injection sites only when associated with A. actinomycetemcomitans. Mice developed positive serum IgG antibody responses to W. recta by 20 days post-injection but not to A. actinomycetemcomitans whether injected in pure culture or mixed infection. In vitro lymphoproliferative responses following injection of W. recta and/or A. actinomycetemcomitans resulted in increased lymphocyte reactivity in unstimulated cultures and decreased in vitro responses to phytohemagglutinin. In vitro lymphoproliferative responses to Escherichia coli LPS or Salmonella typhimurium LPS were depressed in mice injected with A. actinomycetemcomitans, but not in mice injected with W. recta.