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Biomedical subjects

M G Humphreys-Beher

Publications and source records attributed to M G Humphreys-Beher.

At least 19 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Aging↗
PubMed2001

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.

Aging↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

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.

Aging↗
PubMed2000

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.

Aging↗
PubMed2000

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.

Adrenergic Uptake Inhibitors↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Adult↗
PubMed2000

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.

Alleles↗
PubMed1999

New concepts for the development of autoimmune exocrinopathy derived from studies with the NOD mouse model.

The non-obese diabetic (NOD) mouse is now recognized as an appropriate model to study autoimmune exocrinopathy prevalent in human Sjögren's syndrome patients. With increasing age, NOD mice undergo histopathological changes similar to human Sjögren's syndrome patients, but more importantly, exhibit the same clinical manifestation of declining exocrine tissue secretory function. Studies with the immunodeficient NOD-scid mouse have provided evidence for the temporal loss in the expression of several major salivary proteins and a decreased presence of acinar cells in salivary tissues. The diminished presence of acinar cells is accompanied by an increase in the enzymes associated with apoptosis in the absence of T- and B-lymphocytes. Despite these alterations, NOD-scid mice, unlike NOD mice, do not lose secretory function. Recent analyses of a second congenic NOD strain, the NOD.Igmnull, which lacks B-lymphocytes, indicate the histological presence of a T-cell infiltrate of the exocrine glands, increased caspace activity and induction of the biochemical alterations in protein expression observed in NOD and NOD-scid mice. NOD.Igmnull mice also do not lose secretory function, but can be manipulated to generate a reduced secretory response following the infusion of IgG fractions from autoimmune NOD mice or Sjögren's syndrome patients. These observations, in the absence of components of the adaptive arm of the immune system, have given rise to the concept that autoimmune exocrinopathy develops in two phases. The initial phase is lymphocyte independent and occurs as a consequence of an innate error in exocrine tissue homeostasis or differentiated function. The subsequent tissue specific immunological attack, generated in part by B-cell autoantibodies, is responsible for the loss of secretory function. Our preliminary observations in both NOD mice and Sjögren's syndrome patients is that antibody directed against the cell surface muscarinic/cholinergic receptors appears to play an important part in the onset of clinical disease.

Animals↗
PubMed1999

Elevated levels of human salivary epidermal growth factor after oral and juxtaoral surgery.

PURPOSE: Saliva provides a natural reservoir of growth factors whose purposes have remained elusive. Animal studies suggest that saliva-derived growth factors play a role in systemic and oral wound healing. In the current study, salivary concentrations of epidermal growth factor (EGF) were monitored in patients before and after oral and juxtaoral surgery. PATIENTS AND METHODS: Whole resting saliva was collected from a group of patients with parotid gland tumors requiring surgical resection. Another group of patients a history of periodontal disease requiring surgical intervention also provided whole salivary samples. Healthy age- and sex-matched persons served as controls. RESULTS: Salivary EGF levels were elevated in both groups of patients within 24 hours after surgery. In the periodontitis patients, a second smaller peak was assayed noted between 36 and 48 hours. After this, EGF concentrations returned to levels comparable to healthy controls in both experimental groups. CONCLUSIONS: Although the local cells have the ability to synthesize and secrete growth factors at a site of injury, these results suggest that surgery stimulates increased synthesis and secretion of growth factors in the saliva as well. This increased level of saliva-derived growth factor may also aid in promoting wound healing.

Adult↗
PubMed1999

An alternative perspective to the immune response in autoimmune exocrinopathy: induction of functional quiescence rather than destructive autoaggression.

Sjögren's syndrome is characterized by dryness of the eyes and the mouth due to mononuclear cell infiltration of the lacrimal and salivary glands. The aetiology is unknown but autoimmunity is considered to play a significant role in the pathogenesis. Recent studies have focused on the fact that tear and salivary flow involves an entire functional system that includes the mucosal surfaces with adnexes (the site of inflammation), efferent nerve signals sent to the midbrain (lacrimal and salivary response region), and afferent neural signals from the brain to the acinar/ductal epithelial structures in the gland. Mononuclear cell infiltration in exocrine glands can lead to glandular destruction, suggested to be mediated through apoptosis. However, the functional impairment of exocrine glands could be regulated by cytokines and/or antibodies against the muscarinic M3 receptor by inhibiting the neural stimulation of the residual glands. This review discusses the possibility that the pathogenesis of Sjögren's syndrome comprises aberrant immune-mediated neuro-hormonal events.

Animals↗
PubMed1999

Transforming and epidermal growth factors in degenerated intervertebral discs.

We studied the presence of anabolic growth factors in human herniated intervertebral discs (IVD) using a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Messenger RNA (mRNA) was isolated from the nucleus pulposus using oligo (dT)25 superparamagnetic beads and probing with gene-specific primers in RT-PCR. mRNA coding for TGF-alpha (3/10), EGF (0/10), TGF-beta1 (0/10) and TGF-beta3 (2/10) or the EGF receptor (EGF-R; 0/10) and TGF-beta type-II receptor (0/10) was found only occasionally. Beta-actin was always present and positive sample controls confirmed the validity of the RT-PCR assay. These RT-PCR findings were confirmed using immunohistochemical staining of EGF and TFG-beta, whereas TGF-alpha protein was always found associated with discocytes. We conclude that the nucleus pulposus of the herniated IVD is vulnerable to proteolytic degradation and depletion of proteoglycans due to the lack and/or low production of anabolic growth factors/receptors which could increase the local synthesis of the extracellular matrix.

Actins↗
PubMed1998

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren's syndrome.

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sjögren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren's syndrome.

Animals↗