Progress in understanding autoimmune exocrinopathy using the non-obese diabetic mouse: an update.

Sjögren's Syndrome (SS) is a chronic autoimmune disease characterized by histological and functional alterations of salivary and lacrimal glands that result in a severe dryness of the mouth and the eyes. The etiology of SS has remained undefined despite investigators' significant efforts to identify the mechanisms of initiation. Based on histopathology, several animal models are available--such as MRL/lpr, NZW/NZB, NFS/sld, graft vs. host, transgenic mouse expressing viral surface antigen, and the non-obese diabetic (NOD) mouse--for investigation of the etiology of SS. Biochemical and immunological similarities between human SS and autoimmune exocrinopathy (AEC) in the NOD mouse, including the loss of secretory function, establish the NOD mouse as an appropriate model to unravel the underlying pathophysiology of SS. Recently, several NOD congenic partner strains have been developed to investigate the roles of genetic intervals, cytokines, and autoantibodies in the disease pathogenesis. Studies on NOD-scid suggest that the pathogenesis of SS occurs in two phases: an asymptomatic phase, in which epithelial cells of exocrine tissues undergo dedifferentiation accompanied by elevated apoptosis; and a second phase in which autoaggression is mounted against target organ autoantigens, resulting in the activation of T- and B-cells, and the generation of autoantibodies. The presence of autoantibodies on the cell-surface signaling receptor, the muscarinic(3) receptor, in both SS patients and the NOD mice correlates with the hallmark clinical symptom of secretory dysfunction. Additionally, the NOD mouse model provides an important example of how both Th1 and Th2 cytokines, as well as non-immune genetic loci, are involved in the maintenance of and progression to the overt disease state. Ultimately, analysis of these data provides insight into potentially novel therapeutic interventions.

Abnormal organogenesis in salivary gland development may initiate adult onset of autoimmune exocrinopathy.

OBJECTIVES: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues. METHODS: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice. RESULTS: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein. DISCUSSION: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity.

Polarized expression of G protein-coupled receptors and an all-or-none discharge of Ca2+ pools at initiation sites of [Ca2+]i waves in polarized exocrine cells.

In the present work we examined localization and behavior of G protein-coupled receptors (GPCR) in polarized exocrine cells to address the questions of how luminal to basal Ca(2+) waves can be generated in a receptor-specific manner and whether quantal Ca(2+) release reflects partial release from a continuous pool or an all-or-none release from a compartmentalized pool. Immunolocalization revealed that expression of GPCRs in polarized cells is not uniform, with high levels of GPCR expression at or near the tight junctions. Measurement of phospholipase Cbeta activity and receptor-dependent recruitment and trapping of the box domain of RGS4 in GPCRs complexes indicated autonomous functioning of G(q)-coupled receptors in acinar cells. These findings explain the generation of receptor-specific Ca(2+) waves and why the waves are always initiated at the apical pole. The initiation site of Ca(2+) wave at the apical pole and the pattern of wave propagation were independent of inositol 1,4,5-trisphosphate concentration. Furthermore, a second Ca(2+) wave with the same initiation site and pattern was launched by inhibition of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps of cells continuously stimulated with sub-maximal agonist concentration. By contrast, rapid sequential application of sub-maximal and maximal agonist concentrations to the same cell triggered Ca(2+) waves with different initiation sites. These findings indicate that signaling specificity in pancreatic acinar cells is aided by polarized expression and autonomous functioning of GPCRs and that quantal Ca(2+) release is not due to a partial Ca(2+) release from a continuous pool, but rather, it is due to an all-or-none Ca(2+) release from a compartmentalized Ca(2+) pool.

Reduced oral wound healing in the NOD mouse model for type 1 autoimmune diabetes and its reversal by epidermal growth factor supplementation.

Using the NOD mouse, a model for type 1 diabetes, we examined how reduced concentrations of epidermal growth factor (EGF) in the saliva, after onset of type 1 diabetes, affect oral wound healing. Diabetic NOD/LtJ mice on insulin therapy, prediabetic NOD/LtJ, and age- and sex-matched BALB/cJ mice were given a cutaneous tongue punch and allowed to undergo normal healing. With diabetes onset and a reduction in saliva-derived growth factor levels, the rate of tongue wound healing was reduced compared with nondiabetic NOD/LtJ and healthy BALB/cJ mice. Addition of exogenous EGF to the drinking water did not accelerate the rate of healing in BALB/cJ or prediabetic NOD/LtJ; however, diabetic NOD/LtJ mice exhibited accelerated wound healing similar to healthy mice. These results demonstrate that loss of growth factors from saliva is associated with profoundly reduced oral wound healing, suggesting that therapeutic treatment with topical delivery may be beneficial to patients with type 1 diabetes and oral wound complications.

IL-4-dependent effector phase in autoimmune exocrinopathy as defined by the NOD.IL-4-gene knockout mouse model of Sjögren's syndrome.

NOD mice manifest many features of autoimmune exocrinopathy (Sjögren's syndrome), a disease generally characterized by a chronic, progressive immunological attack against the exocrine tissues of the salivary and lacrimal glands. Previous studies using the NOD congenic partner strain, NOD.Igmu(null), defined an important role for B lymphocytes in the development of xerostomia, implicating autoantibodies reactive with the acetylcholine muscarinic receptor (M3R) as the possible effector mechanism. In the present study, we have examined the impact of the cytokine, interleukin (IL)-4, on autoimmune exocrinopathy by using the IL-4 gene knockout (KO) NOD mouse strain, NOD.IL-4-/-. Despite manifesting the physiological aberrations and marked leukocytic infiltration of the salivary glands characteristic of autoimmune xerostomia in NOD mice, the NOD.IL-4-/- mice do not develop xerostomia. However, NOD.IL-4-/- mice that received adoptively transferred T lymphocytes derived from NOD.Igmu-/- mice progress to xerostomia, thereby reversing the defect. While progression or lack of progression to xerostomia correlated with the ability of the NOD.IL-4-/- mice to express detectable anti-M3R autoantibodies, the precise mechanism of how IL-4 influences the development of autoimmune xerostomia remains speculative.

Tumor necrosis factor-alpha and receptors for it in labial salivary glands in Sjögren's syndrome.

OBJECTIVE: Modulation of TNF-alpha by neutralizing antibodies, soluble receptors and TNFR: Fc fusion proteins are being developed for the therapeutic modulation of immune inflammation. It is becoming increasingly important to understand the state and involvement of the TNF-alpha/TNFR system in various rheumatic diseases. Tumor necrosis factor-alpha (TNF-alpha) affects its target cells through binding to two different receptors, TNFR-p55 and TNFR-p75. Mitogenic, cytostatic and cytotoxic effects of TNF-alpha on various cells have been reported. In Sjögren's syndrome (SS) focal sialadenitis leads to salivary gland destruction and loss of function. Although TNF-alpha is one possible mediator in these processes, nothing is known about the spatial distribution of TNF-alpha in relation to its receptors/target cells in salivary gland tissue. METHODS: Labial salivary glands (LSG) were obtained from 16 SS patients and 13 healthy controls and stained using the immunohistochemical peroxidase-anti-peroxidase (PAP) method for TNF-alpha, TNFR-p55 and TNFR-p75. RESULTS: TNF-alpha, TNFR-p55 and TNFR-p75 staining was absent, weak or relatively inextensive in controls compared to SS patients. Infiltrating mononuclear inflammatory cells in SS patients displayed moderate to strong TNF-alpha and TNFR expression. In addition, resident vascular endothelial cells, ductal epithelial cells and fibroblasts co-expressed TNF-alpha and TNFR. In contrast, acinar end piece cells did not express TNF-alpha or TNFR-p75 although TNFR-p55 was expressed. CONCLUSION: The interrelated localization of TNF receptors and their ligand TNF-alpha in inflammatory and in endothelial cells suggests a proinflammatory role of TNF-alpha in SS. The expression of TNF-alpha and its receptors in fibroblasts and ductal cells may contribute to ductal hyperplasia and glandular fibrosis. However, in contrast to expectations, the cellular localization of the TNF-alpha/TNRF system argues against its role in acinar cell atrophy.

Elevated proapoptotic Bax and caspase 3 activation in the NOD.scid model of Sjögren's syndrome.

OBJECTIVE: Salivary gland epithelial cells in patients with Sjögren's syndrome (SS) and in NOD and NODscid mice express Fas and Fas ligand, and these cells die from apoptosis. To elucidate the intracellular molecular mechanisms responsible for this salivary gland epithelial cell apoptosis, expression of the Bcl-2 family of proteins (Bcl-2, Bcl-xL, Bax) and caspase (caspases 3 and 8) was studied in young (ages 8-10 weeks) and old (ages 17-28 weeks) NOD and NOD.scid mice. METHODS: Sections of frozen salivary gland tissue were obtained from NOD and NOD.scid mice and from the lip biopsy material of SS patients. Immunohistochemistry or Western blot analysis was performed to assess the apoptotic-associated proteins. RESULTS: Levels of Bax and caspase 3 were elevated in the epithelial cells of glands from old NOD mice, but not in those from young NOD mice. In contrast, epithelial cells from both young and old NOD.scid mice exhibited strong expression of Bax and caspase 3. Western blot analysis showed that the activated form of caspase 3 was increased 2-5-fold in the glands from old NOD, old NOD.scid, and young NOD.scid mice compared with those from young NOD mice. Caspase 3 was also significantly elevated (P < 0.01) in SS patients whose focus scores were grade 3 or 4. In the SS patients' biopsy tissue and in the mouse glands, cells with fragmented DNA were positive for caspase 3. CONCLUSION: These results demonstrate that salivary gland epithelial cells in NOD and NOD.scid mice overexpress the proapoptotic molecules Bax and caspase 3. Bax could be the gene responsible for initiation of caspase activation, epithelial cell destruction, and lymphocyte glandular localization in SS.

Differential absorption and distribution of epidermal growth factor and insulin-like growth factor in diabetic NOD mice.

Previous studies have shown that absorption of growth factors occurs through the gastrointestinal tract and the oral cavity. The non-obese diabetic (NOD) mouse, a model for spontaneous development of type 1 insulin-dependent diabetes (IDDM), was evaluated for the absorption and systemic distribution of growth factors. Radiolabeled epidermal growth factor (EGF) and insulin-like growth factor, type I (IGF-I), were administered by gavage into the stomach or by lozenge into the sublingual vasculature of either diabetic or nondiabetic mice. After a time-dependent uptake, the levels of absorption and distribution through the tissues were measured. A similar time course of EGF absorption following gavage administration was determined for NOD and C57BL/6 mice, with a maximum tissue distribution by 30-min post infusion. Diabetic NOD mice showed similar levels of IGF uptake and tissue distribution compared with nondiabetic NOD and normal healthy C57BL/6 mice, whether administered by gavage or sublingual lozenge. On the other hand, gavage uptake and tissue distribution of EGF was significantly higher in diabetic mice when compared to sublingual administration in nondiabetic NOD or C57BL/6 healthy control mice. These findings suggest that the overall potential uptake and distribution of saliva-derived growth factors in systemic wound-healing processes is retained with diabetes onset, and may offer a new avenue to treating this complication of diabetes.

An analysis of parotid salivary gland function with desipramine and age in female NIA Fischer 344 rats.

Cyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. Dry mouth is one of their major side effects. In this study we analyzed the effects of the long-term administration of the tricyclic antidepressant desipramine and the reversibility of this treatment following a 15-day washout period on different parameters in parotid gland function in aging rats. We hypothesized that glandular function would be decreased, and recovery delayed with age. Drug treatment affected body weight, glandular weight, DNA synthesis, and the concentration of soluble and structural membrane proteins. Surprisingly, parotid flow rate was increased with desipramine in all ages. While the concentration of secreted proteins was generally decreased with treatment, total proteins secreted were quite stable. SDS/PAGE analysis revealed prominent changes with desipramine. Amylase activity was depressed with treatment, but only low residual cellular enzyme activity was detected in the glandular supernatant. Therefore, a secretory impairment with desipramine was excluded. The content of the antimicrobial proteins peroxidase and lysozyme was increased with desipramine in all age groups. Most parameters measured revealed delayed recovery with age. These data indicate that the tricyclic antidepressant desipramine has profound effects on parotid gland function, accented with age.

Sjögren's syndrome: immunological response underlying the disease.

Sjögren's syndrome is a chronic autoimmnune disorder characterized primarily by the discomforts od dry eyes and dry mouth due to the progressive loss of exocrine gland function. Development of a number of animal models to study Sjögren's syndrome, especially the NOD mouse and its congenic partner strains, has permitted a systematic analysis of immunological and non-immunological factors that influence predisposition for development of the autoimmune response. These data are reviewed here.

Desipramine changes salivary gland function, oral microbiota, and oral health in rats.

Tricyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. Oral dryness is one of their major side-effects, leading in humans to increased oral disease and dysfunction of speech, chewing, swallowing and taste. We previously reported that the tricyclic antidepressant desipramine desensitizes beta-adrenergic signal transduction in salivary glands. In this study, we evaluated the effects of this treatment on parotid and submandibular gland function, oral microbiota, and oral health in rats. Total protein secretion and salivary alpha-amylase was not affected by treatment, while cellular alpha-amylase and the content of epidermal growth factor was depressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed increased secretion for proline-rich proteins and glycoproteins. Surprisingly, flow rates were temporarily increased. These alterations in salivary gland function may partially explain the observed changes in oral microbiota and the increased incidence of gingivitis. Under other nutritional conditions, desipramine might have more severe impacts on oral health.

An analysis of submandibular salivary gland function with desipramine and age in female NIA Fischer 344 rats.

Dry mouth is one of the major side effects of cyclic antidepressants, which are still a dominating group of psychotherapeutic drugs used in the treatment of depression. In this study we analyzed the effects of 28 day tricyclic antidepressant administration and the reversibility of this treatment following a 15 day washout period on different parameters in submandibular gland function in aging rats. We postulated that desipramine would decrease gland function, accented with age, and delay recovery in senescent animals. In contrast to body weight, desipramine had no effect on glandular wet weight. While glandular DNA synthesis was changed with age and treatment, the concentration of total membrane and soluble proteins was not affected. Flow rate was significantly changed with age, but desipramine increased salivary flow in the youngest animals only. Neither age nor treatment influenced salivary protein concentrations, but the total amount of proteins secreted, revealed perturbation with age. SDS- polyacrylamide gel analysis revealed changes in protein expression with treatment and age. Desipramine decreased epidermal growth factor (EGF) levels in all age groups, but increased the secretion of peroxidase and lysozyme. Analysis of total RNA showed a pronounced decrease with age. These data indicate that desipramine has profound effects on submandibular salivary gland function.

Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in nod mice.

OBJECTIVE: Antibodies directed against general and specific target-organ autoantigens are present in the sera of human patients and animal models with autoimmune disease. The relevance of these autoantibodies to the disease process remains ambiguous in most cases. In autoimmune exocrinopathy (Sjögren's syndrome), autoantibodies to the intracellular nuclear proteins SSA/Ro and SSB/La, as well as the cell surface muscarinic cholinergic receptor (M3) are observed. To evaluate the potential role of these factors in the loss of secretory function of exocrine tissues, a panel of monoclonal and polyclonal antibodies was developed for passive transfer into the NOD animal model. METHODS: Monoclonal antibodies to mouse SSB/La, rat M3 receptor, and a rabbit polyclonal antiparotid secretory protein antibody were obtained for this study. These antibody reagents were subsequently infused into NOD-scid mice. Saliva flow rates were subsequently monitored over a 72-hour period. Submandibular gland lysates were examined by Western blotting for alteration of the distribution of the water channel protein aquaporin (AQP). RESULTS: Evaluation of the secretory response indicated that only antibodies directed toward the extracellular domains of the M3 receptor were capable of mediating the exocrine dysfunction aspect of the clinical pathology of the autoimmune disease. In vitro stimulation with a muscarinic agonist of submandibular gland cells isolated from mice treated with anti-M3 antibody, but not saline or the isotype control, failed to translocate AQP to the plasma membrane. CONCLUSION: These findings define a clear role for the humoral immune response and the targeting of the cell surface M3 signal transduction receptor as primary events in the development of clinical symptoms of autoimmune exocrinopathy. Furthermore, the anti-M3 receptor activity may negatively affect the secretory response through perturbation of normal signal transduction events, leading to translocation of the epithelial cell water channel.

Salivary EGF levels reduced in diabetic patients.

Oral problems such as periodontitis are recognized major complications associated with diabetes. Salivary derived growth factors, including epidermal growth factor (EGF), are thought to play a role in helping maintain levels of oral health, promoting wound healing, and maintaining mucosal integrity. In the present study, salivary levels of EGF in diabetic vs. healthy control patients was evaluated. Twenty-one diabetic patients participated in this study. Age, race, sex and smoking histories were matched with 21 systematically healthy nondiabetic patients. Three milliliters of unstimulated resting whole saliva was collected from each patient at 6 h intervals up to 42 h and whole saliva protein concentrations were determined for each sample. EGF concentrations for each sample were quantitated spectrophometrically utilizing an immunoassay. Diabetic patients had greater salivary protein concentrations over 42 h of collection with a mean of 1.502+/-0.09 vs. 1.242+/-0.05 mg/ml for healthy control patients. The EGF concentration was significantly lower (p<0.05) for the diabetic patients compared to control patients, whether expressed relative to 1 ml volume of saliva (873.43+/-106.5 vs. 1101.09+/-116.8 pg/ml) or 1 mg whole saliva protein (629.18+/-92.6 vs. 931.20+/-124.6 pg/mg saliva protein). This study suggests that reduced levels of salivary EGF in diabetic patients may contribute to the development of oral and systemic complications of diabetes, which may have future clinical applications.

Alleles from chromosomes 1 and 3 of NOD mice combine to influence Sjögren's syndrome-like autoimmune exocrinopathy.

OBJECTIVE: NOD mice exhibit at least 2 overlapping autoimmune diseases: autoimmune endocrinopathy (Type I, insulin dependent diabetes) and autoimmune exocrinopathy (Sjogren's syndrome, SS). To date, 18 chromosomal regions have been identified that contribute to development of diabetes in NOD mice; however, genetic mapping of similar susceptibility loci for autoimmune exocrinopathy is just beginning. We investigated if these 2 autoimmune diseases share a genetic predisposition. METHODS: Congenic partner strains of NOD and C57BL/6 mice containing defined genetic intervals influencing susceptibility to diabetes (Idd) were screened for histological and biochemical markers for their effect on the development of SS-like disease. Saliva flow rates, protein concentration, amylase activity, and cysteine protease activity were evaluated. RESULTS: In contrast to the nonsusceptible parental C57BL/6 strain, C57BL/6.NOD Idd5 congenic partner strain, containing a genetic region derived from chromosome 1 of the NOD mouse, exhibited pathophysiological characteristics of autoimmune exocrinopathy. Replacement of individual diabetes susceptibility intervals Idd3, Idd5, Idd13, Idd1, and Idd9, as well as a combination of the Idd3, Idd10, and Idd17 intervals, with resistance alleles had little effect on development of autoimmune exocrinopathy. Conversely, NOD mice, in which the chromosome regions containing both Idd5 and Idd3 have been replaced by intervals derived from C57BL mice, exhibit a reduced pathophysiology associated with SS-like autoimmune exocrinopathy. CONCLUSION: Alleles on chromosomes 1 (Idd5) and 3 (Idd3) in combination appear to greatly influence susceptibility and resistance to development of autoimmune exocrinopathy. The association with certain Idd, but not other Idd loci, indicate that genetic overlap is present but probably not inclusive.

Salivary epidermal growth factor levels decrease in patients receiving radiation therapy to the head and neck.

OBJECTIVE: The objective of this study was to assess changes in salivary epidermal growth factor (EGF) in patients receiving radiation therapy to the head and neck and to determine whether salivary EGF levels correlate with the severity of radiation-induced oral mucositis. STUDY DESIGN: Thirteen patients and 18 control subjects were enrolled in the study. Saliva was collected before, during (weekly), and after radiation therapy. Salivary total protein (TP) and EGF concentrations were measured and correlated with the severity of oral mucositis. The variability in normalized EGF (ngEGF/mgTP) values and mucositis scores were analyzed with analysis of covariance, and the adjusted correlation coefficient was calculated. RESULTS: EGF levels decreased (P =.004), whereas TP levels increased over time (P =.039). A strong correlation was seen with decreasing normalized EGF values and more severe mucositis (P =. 0001). CONCLUSION: A strong negative correlation between normalized EGF and mucositis severity suggests a possible role for EGF in the progression of radiation-induced mucosal breakdown.

Aberrant proteolytic digestion of biglycan and decorin by saliva and exocrine gland lysates from the NOD mouse model for autoimmune exocrinopathy.

OBJECTIVE: The protein components of the extracellular matrix (ECM) are responsible for driving tissue morphogenesis, the development of differentiated function, and the sequestration of biologically active molecules such as growth factors in close proximity to tissue and organ cells. Recent reports indicate that saliva and exocrine tissue lysates from Sjögren's syndrome patients and the non-obese diabetic (NOD) mouse model for autoimmune exocrinopathy demonstrate elevated levels of specific enzymes that degrade the ECM, especially the matrix metalloproteinases (MMPs). To determine if elevated levels of MMPs could be important in exocrine tissue destruction, we examined proteolytic activity against two ECM proteoglycans, decorin and biglycan. METHODS: Purified recombinant human core protein for decorin or biglycan was incubated with saliva or gland lysates from either control BALB/c or NOD mice. Degraded proteoglycan products were estimated by Western blotting analysis using anti-decorin or anti-biglycan monospecific polyclonal antibodies. The levels of TGF beta protein were measured by ELISA. RESULTS: Proteolytic activity for decorin and biglycan was not observed in the saliva and salivary gland lysates collected from C57BL/6 or BALB/c mice used as normal controls. In contrast, both proteoglycans were degraded by saliva and exocrine gland lysates from NOD mice and the congenic partner strains NOD-scid and NOD.B10.H2b. This proteolytic activity for proteoglycans was inhibited by the MMP inhibitors, EDTA, GM6001 and 1,10-phenanthroline. Protein steady state levels for TGF beta were increased in the saliva and gland lysates from 20-week old NOD strains, as compared to BALB/c mice and NOD treated with the MMP inhibitor GM6001. With the inhibition of MMP activity, TGF beta levels declined in saliva and gland lysates. CONCLUSION: Proteolytic degradation of the ECM molecules decorin and biglycan is elevated in the exocrine tissues of the NOD mouse model for Sjögren's syndrome. Furthermore, the proteolysis of small leucine-rich proteoglycans correlates with the presence of elevated levels of TGF beta in gland lysates and saliva.

Desipramine induced changes in salivary proteins, cultivable oral microbiota and gingival health in aging female NIA Fischer 344 rats.

Cyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. One of their major side effect is salivary gland dysfunction (oral dryness, xerostomia), leading in humans to increased oral disease and dysfunction of speech, chewing, swallowing and taste. The purpose of this study was to assess the effects of the long-term administration of the tricyclic antidepressant desipramine and the reversibility of this treatment following a 15 d washout period on specific salivary proteins, composition of oral microbiota, and oral health (gingivitis) of aging female F344 rats. Total salivary proteins showed decreased concentrations with age and desipramine. Similar SDS/PAGE protein profiles appeared in all phases but in different relative amounts with age and treatment. While certain proteins maintained steady levels (lactoferrin) or decreased with age and treatment (amylase), the synthesis of proline-rich proteins, high molecular weight mucin-type glycoproteins, and lysozyme was induced with desipramine and age. The oral microbiota was significantly changed with age and the administration of the antidepressant. The incidence of gingivitis with desipramine was highest in the oldest animals, For the different parameters measured, recovery was delayed with age. These data indicate, that desipramine has profound effects on salivary protein secretion. This may partially explain the changes in microbiota and the increased incidence of gingivitis.