Elevated salivary and synovial fluid beta2-microglobulin in Sjogren's syndrome and rheumatoid arthritis.

Beta2-Microglobulin is normally present in low concentrations in serum and other bodily fluids. By use of a radioimmunoassay, elevated concentrations of beta2--microglobulin were found in saliva and synovial fluid from patients with Sjogren's syndrome and rheumatoid arthritis, autoimmune inflammatory diseases that attack and destroy the salivary glands and articular tissues, respectively. Elevated beta2-microglobulin concentrations decreased in the saliva of two patients who simultaneously showed a clinical response to systemic treatment. Measurement of beta2-microglobulin in inflammatory fluids may offer a simple method of quantifying local activity in autoimmune states.

Decreased autologous mixed lymphocyte reaction in Sjögren's syndrome.

The autologous mixed lymphocyte reaction (AMLR) measures the response of peripheral blood T cells to antigens present on the surface of non-T cells. The AMLR was studied in 25 patients with Sjögren's syndrome (SS). The AMLR was decreased in 15 of 25 (60%) of patients with SS (5,272 +/- 6,738 cpm vs. 14,396 +/- 10,092 cpm for the normal controls, P < 0.001). The AMLR was decreased in 8 of 15 patients with only glandular disease who were not on any systemic medications. Patients with SS and associated disease had lower responses than patients with SS alone. Two patients with pseudolymphoma had absent response. The decreased AMLR correlated with a decreased response to concanavalin A, suggesting a possible abnormality of a T cell subpopulation. There was no correlation between the decreased AMLR and age, focus score, serum immunoglobulin concentration, the titer of antilymphocyte antibody, or phytohemagglutinin response. In allogeneic MLR, SS non-T cells and macrophages stimulated normal allogeneic T cells less well than normal non-T cells and macrophages, suggesting a possible abnormality in the cells that stimulate in the cells that stimulate in the allogeneic MLR.

T and B lymphocytes in peripheral blood and tissue lesions in Sjögren's syndrome.

Lymphocyte heterogeneity was studied in peripheral blood and salivary gland lesions in 24 patients with Sjögren's syndrome. Peripheral blood B cells, measured by immunofluorescence with specific antiserum to immunoglobulins or by rosette assay with complementcoated erythrocytes, were increased in most patients. Peripheral blood T cells, measured by immunofluorescence with rabbit antiserum to human thymocytes or by rosette assay with sheep erythrocytes, were reduced in eight patients. Three had associated rheumatoid arthritis, two had a generalized lymphoproliferative disorder, and one each had scleroderma, systemic lupus erythematosus, and neuropathy. The salivary gland lymphocytic infiltrates present in labial biopsy specimens were compared in 10 patients using an indirect immunofluorescent method with anti-human T cell serum and a quantitative focus-scoring method. In general, there was a correlation between the number of T cells and the extent of the infiltrate. Striking accumulations of T cells were present in some patients, but clusters of presumed B cells were also seen. These results indicate an increase in peripheral blood B cells in most patients, a decrease in T cells in some, and a mixed T and B cell infiltrate in the salivary gland lesions.

Human mucosal Langerhans cells: postmortem identification of regional variations in oral mucosa.

Modified ATPase histochemistry was used to identify and count Langerhans cells (LC) in autopsy tissue from 8 oral mucosal sites, 8-20 h postmortem. The specificity of the ATPase method was confirmed on serial sections with indirect immunofluorescence using monoclonal antibodies OKT6 and antihuman Ia. Average LC counts on ATPase-stained epithelial sheets from each of the 8 sites ranged from 160-550 LC/mm2. Nonkeratinized mucosae of the soft palate, ventral tongue, lip, and floor of the mouth had the highest counts (mean +/- SD, 508 +/- 110 LC/mm2, n = 24), and keratinized mucosae of the hard palate and gingiva had the lowest counts (201 +/- 97 LC/mm2; n = 8). LC frequency was variable in 2 sites: In the dorsal tongue, LC occurred on only one side of filiform papillae and were absent from regularly recurring areas of interpapillary epithelium. In the cheek mucosa, LC clustered around connective tissue papillae and their numbers showed marked individual variation (130-650 LC/mm2). The number of LC in nonkeratinized oral mucosa is approximately the same as in skin, but keratinized oral mucosa has fewer LC. The frequency of oral mucosal LC varies inversely with the degree of keratinization. There are regions of the oral mucosa that have no LC.

Localization of basement membrane components in mucous membrane pemphigoid.

We examined the distribution of laminin, type IV collagen, and fibronectin in subepithelial vesicles of oral mucous membrane pemphigoid (MMP). Indirect immunofluorescence staining of these macromolecules was performed on 10 frozen biopsy specimens of oral MMP. We found type IV collagen in the connective tissue floor and laminin in the epithelial roof of these lesions. Our results suggest that the inflammatory injury in oral MMP may disrupt the interaction of laminin with type IV collagen in the basement membrane zone.

Absence of Langerhans cells in oral hairy leukoplakia, an AIDS-associated lesion.

Oral hairy leukoplakia (HL) is a recently described manifestation of human immunodeficiency virus (HIV) infection in which Epstein-Barr virus (EBV) has been shown to replicate. To seek evidence for a local defect in mucosal immunity, we assessed the presence of epithelial Langerhans cells (LC) in these lesions and in autologous nonlesional mucosa. We used monoclonal antibodies against HLA-DR, HLA-DQ, and T6 antigens to identify LC in biopsy specimens of HL from 23 homosexual men. In all lesion specimens, LC either were not detected or were present only in greatly reduced numbers with at least 1 of the antibodies. In nonlesional oral mucosa from the same patients, LC were detected with all 3 antibodies in 11/12 specimens (92%) and were found in approximately normal numbers with at least 1 antibody. There was close correlation between the absence of LC and positive staining for EBV, human papillomavirus antigens, and candidal hyphae in the epithelium. We conclude that LC are absent or greatly reduced in the lesions of HL. Absence of normal LC function may be important in the pathogenesis of HL and may reflect an event in the pathogenesis of other features of the acquired immune deficiency syndrome.

Cytokine expression in labial salivary glands from patients with primary Sjögren's syndrome.

The presence and distribution of 7 cytokines was examined immunohistologically in labial salivary gland (LSG) specimens from patients with primary Sjögren's syndrome (SS) and control subjects. The cytokines interleukin (IL)-1 beta IL-6, tumor necrosis factor (TNF) alpha and interferon (IFN) gamma were identified in defined parts of LSG from patients but not in the corresponding parts of control glands: (a) LSG acinar epithelium expressed IL-1 beta, (b) blood vessels located in both normal LSG stroma and within lymphocytic infiltrates expressed IL-1 beta, IL-6 and IFN gamma, and (c) lymphocytic infiltrates expressed IL-1 beta, IL-6 and TNF alpha. All four cytokines were expressed by salivary ducts within both patient and control specimens, but with generally greater intensity in patients. IL-1 alpha, IL-4 and TNF beta (lymphotoxin) could not be detected in any of the specimens from patients or controls. The locations of cytokines in LSG suggests possible mechanisms of immunologically mediated parenchymal damage in primary SS.

Salivary and oral components of Sjögren's syndrome.

The intraoral symptoms and signs of SS are not specific to SS, being shared with other conditions in which salivary function is diminished. The decrease in saliva causes chronic oral discomfort and functional problems and predisposes patients to dental caries and oral candidiasis. Many methods have been used to assess SGs in SS objectively, but at present a labial salivary gland biopsy specimen showing focal lymphocytic sialadenitis provides the best diagnostic criterion for the salivary component of SS, in terms of its disease specificity, convenience, availability, and low risk. The treatment of xerostomia in patients with SS consists of (1) preventing new and recurrent dental caries by frequent and regular application of topical fluoride, careful dental supervision, and avoidance of sucrose and other metabolizable carbohydrates between meals; (2) reducing oral symptoms by diagnosing and treating oral candidiasis (repeatedly if necessary); and (3) attempting to replace lost saliva by stimulating salivary secretion with physiologic sialogogues or pilocarpine, or if adequate amounts of saliva cannot be stimulated, using some form of saliva substitute, especially for patients wearing complete dentures.

Electrical stimulation of salivary flow in patients with Sjögren's syndrome.

Patients with the salivary component of Sjögren's syndrome (SS) develop chronic xerostomia, which causes oral symptoms and functional impairment in approximate proportion to its severity. The purpose of this double-blind study was to determine whether an electrical stimulus applied to the tongue and hard palate by a battery-operated device (SAL II, Biosonics, Inc.) could stimulate salivary flow in subjects with generally severe SS. Twenty-nine patients with the salivary component of SS (diagnosed as the presence of focal chronic sialadenitis in a labial salivary gland biopsy specimen with a focus score of greater than 1 focus/4 mm2) were randomly assigned active or placebo devices, which they used for three minutes, three times a day for four weeks. Whole saliva flow rates were measured at weeks 0, 2, and 4 by collection of whole saliva both before and after stimulation with the device. Twenty-four subjects completed the study. The change in mean post-stimulation flow rate from week 0 to week 4 was greater for the 13 subjects using an active device (0.08 +/- S.D. 0.08 g/2 min, to 0.24 +/- 0.33 g/2 min) than for the 11 subjects using a placebo device (0.11 +/- 0.15 g/2 min, to 0.08 +/- 0.18 g/2 min) (p = 0.04). However, the magnitude of the mean difference was small, because three subjects using active devices responded and others did not. Only five subjects, all using active devices, reported a subjective increase in the amount of their saliva. The results of this study indicate that some SS patients with residual salivary flow show a significant response to electrical stimulation, but others with low or absent whole saliva flow rates do not respond.

LETS protein in normal and pathological human oral epithelium.

The distribution of LETS protein in human oral mucosa was studied by indirect immunofluorescence. Normal epithelium showed surface staining. Intracellular staining occurred in epithelial cell cytoplasm in lichen planus and pemphigoid. Surface staining was absent in discoid lupus erythematosus. In pemphigus, intercellular staining was seen near areas of acantholysis.

Tear lysozyme activity in frozen Schirmer strips and salivary gland biopsy as parameters of lacrimal gland function.

PURPOSE: The lysozyme concentration in human tears is an important parameter for tear gland function. The decline of lysozyme in tears reflects lacrimal gland destruction. In Sjögren's patients, lacrimal gland destruction parallels labial salivary gland destruction. The objective of this study was to determine whether human tear lysozyme that was frozen on Schirmer strips at -20 degrees C for several years maintained activity and whether there was a linear relation with inflammatory changes in labial salivary glands. METHODS: A total of 200 frozen Schirmer strips were processed. They were collected from 20 randomly selected patients each year of five consecutive years, all attending the UCSF Sjögren's Clinic. The tear lysozyme in the Schirmer strips was measured by a colorimetric assay. The average lysozyme concentration each year was calculated and compared. One third of the patients underwent labial salivary gland biopsy. The correlation was calculated between the tear lysozyme concentration and the lymphocytic focus scores in biopsy specimens. RESULTS: No significant difference of average lysozyme concentration in the Schirmer strips was found when the five different years of collection were compared. The linear relation between the tear lysozyme concentrations and the focus score in labial salivary gland biopsies showed a coefficient of r = -0.41. The linear relation between other diagnostic measurements, like Schirmer test, tear breakup time, or rose bengal staining pattern, and the focus score was lower. CONCLUSIONS: Human tear lysozyme in Schirmer strips can be stored at -20 degrees C for at least five years. There is little difference in lysozyme activity of frozen compared to unfrozen specimens. The lysozyme concentration in tears correlates better with the lymphocytic focus score in labial salivary gland biopsy than does clinical assessment and is therefore a parameter for the actual degree of tear gland destruction.

Advanced diagnostic methods in oral and maxillofacial pathology. Part I: molecular methods.

The practice of pathology is currently undergoing significant change, in large part due to advances in the analysis of DNA, RNA, and proteins in tissues. These advances have permitted improved biologic insights into many developmental, inflammatory, metabolic, infectious, and neoplastic diseases. Moreover, molecular analysis has also led to improvements in accuracy of disease diagnosis and classification. It is likely that, in the future, these methods will increasingly enter into the day-to-day diagnosis and management of patients. The pathologist will continue to play a fundamental role in diagnosis and will likely be in a pivotal position to guide the implementation and interpretation of these tests as they move from the research laboratory into diagnostic pathology. The purpose of this 2-part series is to provide an overview of the principles and applications of current molecular biologic and immunologic tests. Part I will discuss the biologic fundamentals of DNA, RNA, and proteins and the methods that are currently available or likely to become available to the pathologist in the next several years for their isolation and analysis in tissue biopsies.

Apoptosis-associated proteins in oral lymphomas from HIV-positive patients.

Extranodal oral lymphomas, seen with increasing frequency in HIV infection, may have dysfunctional apoptotic mechanisms that favor tumor progression. The purpose of this study was to evaluate extranodal lymphomas from HIV-positive patients for expression of apoptosis-associated proteins. Correlations were made with 10 histologically comparable extranodal lymphomas from HIV-negative patients and 6 hyperplastic lymph nodes from otherwise healthy young adults. Formalin-fixed tissue sections were immunohistochemically stained for apoptosis-associated proteins (Bcl-2, Bcl-x, Bax, Bak, p53, MDM2, BHRF). In situ hybridization was also done on deparaffinized sections for Epstein-Barr virus EBER mRNA. Eighteen consecutive oral lymphomas were studied in HIV/AIDS-positive patients. Four of 5 intermediate-grade lymphomas expressed Bcl-2 to a greater degree than did high-grade lymphomas (4 of 13). Most lymphomas were positive for Bcl-x and Bax, and few expressed Bak. The staining patterns for these proteins were similar to those seen in HIV-negative patients. Staining patterns were relatively consistent in the hyperplastic lymph nodes, whereas such patterns were irregular in lymphomas. Positive p53 staining was seen in 11 of 18 HIV-positive cases; 9 of these were also MDM2-positive. Double stains suggested that both p53 and MDM2 proteins were expressed in the same cells in these nine cases. Epstein-Barr virus-EBER mRNA was detected in 14 of 18 cases and in 3 of 10 cases from HIV-negative patients. BHRF staining was evident in only a few cells of three HIV-positive lymphomas. The irregular expression of Bcl-2, Bcl-x, Bax, and Bak in oral lymphomas indicates dysfunctional apoptotic mechanisms in these tumors. Bcl-2 staining differs with tumor grade. Positive staining for p53 and MDM2 proteins is a notable feature of lymphomas in HIV-positive patients and may relate to binding of MDM2 to wild-type p53. Epstein-Barr virus is more commonly associated with oral lymphomas in HIV-positive patients, although the Epstein-Barr virus-produced protein BHRF, which has Bcl-2-like activity, is minimally expressed.

Oral mucosal melanomas: the WESTOP Banff workshop proceedings. Western Society of Teachers of Oral Pathology.

A workshop to discuss primary oral melanomas was convened at the annual Western Society of Teachers of Oral Pathology meeting in Bannf, Alberta, Canada. Fifty oral melanomas, identified from the files of the participants, were reviewed in order to better understand the clinical features, histologic spectrum, and natural history of these perplexing lesions. Results confirmed that oral melanomas occur in adults almost three times more frequently in men than women and have a decided predilection for the palate and gingiva. Some lesions exhibit a clinically detectable and prolonged in situ growth phase, whereas others seem to lack this property and exhibit only or predominantly invasive characteristics. Recurrences, metastases, and death from tumor were characteristic of the follow-up of a limited number of patients. Until definitive prospective data are collected that elucidate natural history, oral mucosal melanomas should be tracked separately from cutaneous lesions. All oral pigmented lesions that are not clinically diagnostic should be biopsied. Lesions with equivocal histopathologic features might be referred to as "atypical melanocytic proliferation" and should be excised. Recognition of lesions in an early in situ phase and aggressive treatment should have a favorable effect on prognosis. To enhance future or prospective study of these rare neoplasms, guidelines for reporting oral melanomas are suggested.

Oral mucosal lesions found in smokeless tobacco users.

The risk for oral mucosal lesions associated with use of smokeless tobacco among 1,109 professional baseball players during spring training in 1988 was investigated. Leukoplakia was very strongly associated with use of smokeless tobacco in this population of healthy young men. Of the 423 current smokeless tobacco users, 196 had leukoplakia compared to seven of the 493 nonusers (OR = 60.0, 95% CI = 40.5-88.8). The amount of smokeless tobacco used (in hours per day that smokeless tobacco was held in the mouth), recency of smokeless tobacco use (hours since last use), type (snuff versus chewing tobacco), and brand of snuff used were significantly associated with risk for leukoplakic lesions among smokeless tobacco users. Ninety-eight leukoplakic areas in 92 subjects were biopsied and examined microscopically. All lesions were benign, but one specimen had mild epithelial dysplasia. The long-term significance of leukoplakia in smokeless tobacco users and their relation to oral cancer is not clear.

Xerostomia--clinical evaluation and treatment in general practice.

Xerostomia is a common symptom with various causes that, if ignored, can lead to serious oral consequences. Clinical evaluation of patients complaining of dry mouth must include some additional history and specific examination of the salivary glands, oral mucosa, and teeth. Additional evaluation may include consultation with the patient's physician, request for microbial culture, or labial salivary gland biopsy. No one form of treatment for patients with chronic xerostomia is sufficient, but comprehensive treatment is effective in improving patient oral comfort and function and preventing unnecessary loss of teeth. This treatment must include ongoing dental caries prevention and treatment, salivary flow stimulation, recognition and treatment of oral candidiasis, selective use of saliva substitutes, and possible changes in the patients' prescription and nonprescription drug use.

Evaluation, differential diagnosis, and treatment of xerostomia.

The salivary component of Sjögren's syndrome (SS) is defined as xerostomia (dry mouth). However, xerostomia is a common symptom associated with quantitative and qualitative changes in saliva, which are generally referred to as salivary hypofunction. This can be caused by various systemic diseases (including SS), anticholinergic effects of many drugs, psychological conditions, and physiological changes. Chronic salivary hypofunction is clinically significant because it can cause oral dysfunction, dental destruction, and mucosal infection. Evaluating patients complaining of xerostomia requires particular attention to their current medications and physical examination of the major salivary glands, teeth, and oral mucosa. Based on that information and the differential diagnosis of salivary hypofunction, appropriate tests can then be selected to develop a final diagnosis. Effective treatment of patients with chronic salivary hypofunction requires a combination of: (1) ongoing dental decay prevention and treatment supervised by their dentist; (2) salivary flow stimulation; (3) recognition and treatment of chronic oral candidiasis; (4) selective use of saliva substitutes; and (5) prescription drug review.

American College of Rheumatology classification criteria for Sjögren's syndrome: a data-driven, expert consensus approach in the Sjögren's International Collaborative Clinical Alliance cohort.

OBJECTIVE: We propose new classification criteria for Sjögren's syndrome (SS), which are needed considering the emergence of biologic agents as potential treatments and their associated comorbidity. These criteria target individuals with signs/symptoms suggestive of SS. METHODS: Criteria are based on expert opinion elicited using the nominal group technique and analyses of data from the Sjögren's International Collaborative Clinical Alliance. Preliminary criteria validation included comparisons with classifications based on the American­European Consensus Group (AECG) criteria, a model-based "gold standard"obtained from latent class analysis (LCA) of data from a range of diagnostic tests, and a comparison with cases and controls collected from sources external to the population used for criteria development. RESULTS: Validation results indicate high levels of sensitivity and specificity for the criteria. Case definition requires at least 2 of the following 3: 1) positive serum anti-SSA and/or anti-SSB or (positive rheumatoid factor and antinuclear antibody titer >1:320), 2) ocular staining score >3, or 3) presence of focal lymphocytic sialadenitis with a focus score >1 focus/4 mm2 in labial salivary gland biopsy samples. Observed agreement with the AECG criteria is high when these are applied using all objective tests. However, AECG classification based on allowable substitutions of symptoms for objective tests results in poor agreement with the proposed and LCA-derived classifications. CONCLUSION: These classification criteria developed from registry data collected using standardized measures are based on objective tests. Validation indicates improved classification performance relative to existing alternatives, making them more suitable for application in situations where misclassification may present health risks.