An immunohistochemical study of the vasculature and stem cells and carbonic anhydrase IV in the ducts of irradiated and normal human submandibular salivary glands.

Therapeutic irradiation for cancers of the head and neck causes serious and irreversible damage to the salivary glands; the resulting adverse effects on salivary quality and quantity produce detrimental effects on teeth and oral mucosa. The salivary effects have been related mostly to loss of serous acini; damage to the ducts is relatively minor. Other radiation effects include fibrosis, adiposis and vascular damage. Stem cells in the salivary gland ducts have the potential to generate acinar cells in vitro and in vivo. I investigated the ducts and vasculature in irradiated and normal human submandibular glands using immunohistochemical localization of biomarkers of stem cells, duct function and blood vessels. Stem cell markers CK5 and Sca-1 labeled the cytoplasm of the basal and intercalated duct cells and all duct cells, respectively, in both normal and irradiated glands. CA IV, which participates in regulating salivary electrolytes and acid-base balance, labeled the cytoplasm of all ducts. CD34 labeling demonstrated more extensive vasculature in the irradiated glands than in the normal glands. My findings suggest that duct stem cells and at least one duct function persisted, and the vasculature was greater, despite moderate fibrosis, in the irradiated gland.

Follow-up study of veterans with white and red oral mucosal lesions at Veterans Affairs Dental Clinics.

OBJECTIVES: This analysis examined the clinical and histopathological characteristics of white and red oral mucosal lesions and patient lifestyle behaviors to understand how the lesions changed over 19-23 years, including among patients who developed oral and pharyngeal cancer. MATERIALS AND METHODS: Seventy-five individuals with red and/or white oral mucosal lesions with clinical diagnoses of smokeless tobacco lesions, leukoplakia, erythroplakia, lichen planus, ulcer, and virus-associated lesions were identified in six Veterans Affairs Medical Center Dental Clinics (VAMC) from 1996 to 2001. Biopsy results and patients' sociodemographic, medical, and tobacco/alcohol use characteristics were obtained. Study dentists used standardized forms to capture information about the lesions. Study participants were re-examined at intervals through January 2002. In 2020, a retrospective review of VAMC and public records ascertained whether participants developed oral cancer or died. RESULTS: The most common red or white oral mucosal lesions among the 75 study participants were leukoplakia (36.0%), smokeless tobacco lesions (26.7%), virus-associated lesions (18.7%), and lichen planus (16.0%). Lesions in 11% of participants with leukoplakia and one-third of participants with lichen planus persisted for 5 years or more. Dysplasia was present in four participants with leukoplakia. Seventeen percent of participants developed a new white or red oral mucosal lesion. Five patients (6.1%) developed oral or pharyngeal cancer, four among participants with leukoplakia (one with prior dysplasia) and one among participants with lichen planus. Four of the cancers developed 6-20 years after enrollment, and only one was at the original lesion site. CONCLUSIONS: The occurrence of oral and pharyngeal cancers in some study participants with white and red oral mucosal lesions many years after enrollment reinforces the need for patients, dentists, and health care systems to have better methods to identify and assess the malignant potential of oral lesions, monitor patients over time, and intercept high-risk oral lesions before they become cancerous.

Immunohistochemical profile of the anti-apoptosis, apoptosis and proliferation markers Bcl-2, caspase-3, p53, and Ki-67 in botryoid odontogenic cysts compared to lateral periodontal cysts and gingival cysts of the adult.

We investigated the differences in growth and rates of recurrence of the botryoid odontogenic cyst (BOC) and the less aggressive lateral periodontal cyst (LPC) and gingival cyst of the adult (GCA). We compared the immunohistochemical expression of selected biomarkers of apoptosis and proliferation and of regulators of their activity. Sections from archival paraffin blocks of 15 BOCs, six GCAs, six LPCs, and three odontogenic keratocysts (OKCs) were processed for immunohistochemical localization of Bcl-2, caspase-3, p53 and Ki-67. Labeled and unlabeled epithelial cells were counted and differences in the mean labeling index (LI) were evaluated statistically. The only significant differences in LI were for the anti-apoptotic marker, Bcl-2; the hierarchy was BOC > OKC > LPC > GCA. In two BOCs, 97% of the cells, and in all OKCs, all of the basal cells were labeled with Bcl-2. Otherwise, cells labeled with Bcl-2, p53 and caspase-3 were scattered among the basal and intermediate epithelial cell layers. Ki-67 labeled almost exclusively basal cells in the BOCs, LPCs and GCAs, and both basal and intermediate layer cells in the OKCs. Our findings suggest that while there was no significant difference in replicative potential of the GCAs, LPCs and BOCs, factors that influence apoptosis may be partially responsible for the more aggressive behavior of BOCs and OKCs.

Positive Association Between Neutrophil-Lymphocyte Ratio and Presence of Panoramically Imaged Carotid Atheromas Among Men.

PURPOSE: Heightened levels of systemic inflammation documented by increased neutrophil-to-lymphocyte ratios (NLRs) characterize a robust atherosclerosis processes evidenced by carotid and coronary artery plaques at ultrasound and angiography with associated strokes and myocardial infarctions (MIs). Therefore, this study investigated whether calcified carotid artery plaques (CCAPs) on panoramic images (PIs), known to herald future stroke and MI, are associated with increased NLRs. MATERIALS AND METHODS: Using a cross-sectional study design, electronic medical records and PIs of non-Hispanic white men at least 55 years old who were treated by the dental service (January 1, 2017 to December 31, 2017) were retrieved. Two groups of patients (n = 50 per group) with plaque (CCAP+) and without plaque (CCAP-) were constituted. The predictor variable was CCAP+ and the outcome variable was the NLR. A t test analyzed the differences in mean NLRs between groups. Other variables of interest, that is, atherogenic risk factors (hypertension, hyperlipidemia, and diabetes mellitus), were included in a logistic regression analysis to assess their influence on the association of CCAP with the NLR. Significance was set at .05 for all tests. RESULTS: The study group of 50 men with CCAP+ (mean age, 71.7 ± 7.47 yr) evidenced a mean NLR of 3.07 ± 1.43. The control group of 50 men with CCAP- (mean age, 69.8 ± 9.29 yr) evidenced a mean NLR of 2.13 ± 0.68. A t test analysis comparison showed a significant (P = .00007) difference (95% confidence interval, 0.49-1.39). Logistic regression failed to show any relevant relation of the NLR with the covariate and other variables of interest. CONCLUSION: There is a strong association between CCAP+ in older non-Hispanic white men and extent of systemic inflammation as evidenced by increased NLRs. These plaques are "risk factors or indicators" for future stroke and MI. Therefore, maxillofacial surgeons providing care for patients with CCAP+ should consider referring them for a comprehensive cerebrovascular and cardiovascular workup.

Microcalcifications in stone-obstructed human submandibular gland are associated with apoptosis and cell proliferation.

OBJECTIVE: Human submandibular gland (SMG) stones are associated with inflammation, fibrosis and microcalcifications in the surrounding tissues. However, there is little information about the accompanying cell injury-repair process, apoptosis, and cell proliferation. The purpose of this study was to investigate such an association and its clinical significance. DESIGN OF STUDY: Mid-gland paraffin sections of human SMGs ("stone glands") and normal SMGs ("non-stone glands") were subjected to stains for general histology (hematoxylin and eosin), fibrosis (Masson's trichrome), and calcification (alizarin red) and to immunohistochemistry for proliferative activity (Ki-67), and apoptosis (Caspase-3). Tissues were assessed for areas of inflammation, calcium deposition, and fibrosis, and for cycling and apoptotic cells. RESULTS: Acini were atrophic and proportionately fewer in lobules with fibrosis in stone glands. Additionally, stone glands had intraluminal calcifications (microliths) in scattered excretory and striated ducts and blood vessel walls. Areas of inflammation and fibrosis were small and uncommon, and calcifications were not seen in non-stone glands. Proliferating and apoptotic cells were common in the main duct of stone glands where ciliated and mucous cell hyperplasia and stratified squamous metaplasia had occurred, uncommon in the main duct of non-stone glands, and uncommon in all other parenchymal elements of both stone and non-stone glands. CONCLUSION: Stone obstruction in the main excretory ducts of SMG resulted in progressive depletion of acini from proximal to distal lobules via calcification, inflammation, fibrosis, and parenchymal cell atrophy, apoptosis and proliferation. Interlobular duct microliths contributed to this depletion by further provoking intralobular inflammation, fibrosis, and acinar atrophy.

Performance of self-reported measures for periodontitis in rheumatoid arthritis and osteoarthritis.

BACKGROUND: This study evaluates the performance of self-report against the reference standard of clinically defined periodontitis in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) after accounting for factors associated with periodontitis. METHODS: Six self-report periodontitis questions were evaluated in patients with RA and OA. Questions were validated against a reference standard of severe and moderate-to-severe periodontitis based on full-mouth examination. Multivariable logistic regression was used to evaluate the performance of: 1) self-report alone; 2) age, sex, education, and smoking status; and 3) a combination of the above. Model performance was assessed using the c-statistic. Convergent validity of self-reported "bone loss/deep pockets" and "loose teeth" was assessed; associations of self-report with RA disease characteristics were explored. RESULTS: Self-report performed similarly in RA and OA, with individual question specificity for periodontitis ≥ 68% and sensitivity from 9.8% to 45%. Question-only models yielded c-statistics of 0.66 to 0.72, whereas risk factor-only models yielded c-statistics of 0.74 to 0.79. The highest-performing models incorporated both self-report questions and periodontitis risk factors, with c-statistics ≥ 0.79. Greater radiographic alveolar bone loss was observed among participants reporting "bone loss/deep pockets" (P < 0.001) and "loose teeth" (P < 0.001). Among patients with RA, "loose teeth," but not other self-report items, was associated with rheumatoid factor positivity (P = 0.047) and higher disease activity (P < 0.001). CONCLUSIONS: Patient self-report, when combined with other risk factors, performs well in identifying periodontitis among patients with RA and OA. Self-report questions related to alveolar bone loss exhibit excellent convergent validity in these patient subsets.

Periodontitis and Porphyromonas gingivalis in patients with rheumatoid arthritis.

OBJECTIVE: To examine the degree to which shared risk factors explain the relationship of periodontitis (PD) to rheumatoid arthritis (RA) and to determine the associations of PD and Porphyromonas gingivalis with pathologic and clinical features of RA. METHODS: Patients with RA (n = 287) and patients with osteoarthritis as disease controls (n = 330) underwent a standardized periodontal examination. The HLA-DRB1 status of all participants was imputed using single-nucleotide polymorphisms from the extended major histocompatibility complex. Circulating anti-P gingivalis antibodies were measured using an enzyme-linked immunosorbent assay, and subgingival plaque was assessed for the presence of P gingivalis using polymerase chain reaction (PCR). Associations of PD with RA were examined using multivariable regression. RESULTS: Presence of PD was more common in patients with RA and patients with anti-citrullinated protein antibody (ACPA)-positive RA (n = 240; determined using the anti-cyclic citrullinated peptide 2 [anti-CCP-2] test) than in controls (35% and 37%, respectively, versus 26%; P = 0.022 and P = 0.006, respectively). There were no differences between RA patients and controls in the levels of anti-P gingivalis or the frequency of P gingivalis positivity by PCR. The anti-P gingivalis findings showed a weak, but statistically significant, association with the findings for both anti-CCP-2 (r = 0.14, P = 0.022) and rheumatoid factor (RF) (r = 0.19, P = 0.001). Presence of PD was associated with increased swollen joint counts (P = 0.004), greater disease activity according to the 28-joint Disease Activity Score using C-reactive protein level (P = 0.045), and higher total Sharp scores of radiographic damage (P = 0.015), as well as with the presence and levels of anti-CCP-2 (P = 0.011) and RF (P < 0.001). The expression levels of select ACPAs (including antibodies to citrullinated filaggrin) were higher in patients with subgingival P gingivalis and in those with higher levels of anti-P gingivalis antibodies, irrespective of smoking status. Associations of PD with established seropositive RA were independent of all covariates examined, including evidence of P gingivalis infection. CONCLUSION: Both PD and P gingivalis appear to shape the autoreactivity of RA. In addition, these results demonstrate an independent relationship between PD and established seropositive RA.

Microchimerism in salivary glands after blood- and marrow-derived stem cell transplantation.

Blood- and marrow-derived stem cells (BMDSCs) provide disease-ameliorating effects for cardiovascular and autoimmune diseases. Microchimerism from donor BMDSCs has been reported in several recipient tissues. We hypothesized that this finding suggests a potential use of BMDSCs in the treatment of salivary dysfunctions. We investigated the presence of Y chromosome-positive cells in salivary gland biopsies of 5 females who had received a marrow or blood stem cell transplant from male donors. One to 16 years after transplantation, all recipients exhibited scattered Y chromosome-positive cells in the acini, ducts, and stroma of their salivary glands (mean of 1.01%). Potentially, these cells can be markers of transplantation tolerance, contribute to neoplastic epithelial tissues, or engraft at sites of injury. In addition, transplantation of BMDSCs could be used for treatment of Sjögren's syndrome and salivary glands damaged by therapeutic irradiation for cancers of the head and neck.

Association of periodontitis with rheumatoid arthritis: a pilot study.

BACKGROUND: Similarities exist in the epidemiology and immunopathogenesis of periodontitis and rheumatoid arthritis (RA), but the associations between their respective disease activities and severities are less well documented. We evaluated the prevalence and severity of periodontitis in United States (U.S.) veterans with RA and their relationship to RA disease activity and severity. METHODS: Patients with RA from an outpatient rheumatology clinic were eligible, and patients with osteoarthritis (OA) served as controls. Dentists, masked to the rheumatologic diagnoses, performed periodontal probing and examined dental panoramic radiographs to assess the presence and severity of periodontitis. Associations of periodontitis with RA were examined using multivariate regression, whereas the association of periodontitis with disease-severity measures in RA was examined using the chi(2) test. RESULTS: Sixty-nine patients with RA (57 males and 12 females) and 35 patients with OA (30 males and five females) were studied. Moderate to severe periodontitis was more prevalent in patients with RA (51%) than controls (26%) (P = 0.03), an association independent of age, race, smoking, diabetes mellitus, and gender. Patients with RA who were seropositive for rheumatoid factor (RF) were more likely to have moderate to severe periodontitis (59%) than patients who were RF negative (15%) (P = 0.02). Likewise, patients with RA who were positive for the anti-cyclic citrullinated peptide (CCP) antibodies were more likely to have moderate to severe periodontitis (56%) than patients who were anti-CCP negative (22%) (P = 0.01). There were no associations of periodontitis status with other measures of RA disease activity or severity. CONCLUSIONS: In a cohort of U.S. veterans, periodontitis was more common and severe in patients with RA compared to patients with OA. Although unrelated to disease activity, the presence of periodontitis in patients with RA was associated with seropositivity for RF and the anti-CCP antibody, which was highly relevant given the associations of these autoantibodies with poor outcomes and disease pathogenesis in RA.

Isolation of intact nuclei of high purity from mouse liver.

Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600 g to remove gross debris and then at moderate centrifugal force (MCF, 16,000 g) or high centrifugal force (HCF, 70,000 g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Self-awareness of premalignant oral lesions among veterans attending six U.S. Veterans Affairs Medical Centers.

Early identification is key to reducing the morbidity and mortality of oropharyngeal cancer. This study identified factors associated with self-awareness among patients newly diagnosed with a premalignant oral lesion. Data describing sociodemographics, medical/dental histories, tobacco/alcohol use and oral health were obtained by questionnaire and clinical examination of 73 veterans at six U.S. Veterans Affairs Medical Centers. Lesion types included homogenous and non-homogenous leukoplakia, smokeless tobacco lesion (STL), papilloma, lichen planus and erythroplakia. Prior to diagnosis, 29 subjects (39.7%) were unaware of their lesion. In bivariate analyses, lesion self-awareness was associated with anatomic location, multifocal/generalized appearance, pain, oral sores, and cigar use (p<0.05). Awareness varied with lesion diagnosis and was more likely with STL and less likely with homogenous leukoplakia (p<0.05). In multivariate analyses, awareness was predicted by the presence of a lesion on easily visible mucosa (adjusted odds ratio, OR=11.2) and a history of mouth sores (OR= 11.2). These findings identified marked variations in patient self-awareness of oral premalignant conditions.

Orofacial and gastrointestinal hyperplasia and neoplasia in smad4+/- and elf+/-/smad4+/- mutant mice.

BACKGROUND: Smad4 is vital to the roles of Smads 2 and 3 in transforming growth factor-beta (TGF)-beta signal transduction, and inactivated Smad4 is common to human gastrointestinal cancers. The embryonic liver fodrin (ELF) is a beta-spectrin that facilitates the nuclear translocation of activated Smad4. METHODS: Smad4+/- mice, known to develop gastrointestinal cancer, were crossbred with elf+/- mice. The smad4+/- and smad4+/-/elf+/- offspring were autopsied as abnormalities developed. RESULTS: In addition to polyps and adenocarcinomas of the stomach and duodenum, the smad4+/- mice developed squamous cell carcinomas of the skin, oral mucosa and forestomach, benign neoplasms of connective tissue and lacrimal gland, and a lymphoma. The smad4+/-/elf+/- mice developed extensive hyperplasia and neoplasia of the gastric mucosa. CONCLUSION: These findings indicate that investigating interactions among smad4, elf, and other genes involved in TGF-beta signaling should be useful in further delineating the processes of neoplasia in a wide variety of tissues.

Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells. Role in apical Ca2+ influx.

Receptor-coupled [Ca2+]i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca2+-signaling proteins. However, localization of Ca2+ channels that are regulated by such Ca2+-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca2+ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and phospholipase Cbeta1 and -beta2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cbetas, Galpha(q/11), inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical 45Ca2+ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical 45Ca2+ uptake and transepithelial 45Ca2+ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca2+]i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca2+ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca2+] and secretion.