Immunohistochemical identification of antigen presenting cells in rat salivary glands

Mucosal dendritic cells affect immune responses through secretion of cytokines and exposure of na?ve B- and T-lymphocytes to foreign matter as antigen presenting cells (APCs). APC in oral tissues may play a role in the development of local and secretory immune responses [Crit. Rev. Oral Biol. Med. 7 (1996) 36]. Previous studies have shown that APC are present in the interstitial tissues of rat salivary glands [Arch. Oral Biol. 40 (1995) 1015]. This study sought to further define the distribution of APC in salivary glands. The major glands and ducts of male Sprague-Dawley and Wistar rats were fixed with 4% paraformaldehyde and prepared for immunofluorescence and pre- and post-embedding immunoelectron microscopy. Monoclonal antibodies to the dendritic cell marker Ia antigen (OX-6 antibody), monocyte lineage cytoplasmic antigen (ED-1), and resident tissue macrophage antigen (ED-2) were visualized with FITC-conjugated secondary antibodies for light microscopy and HRP- and gold-labelled secondary antibodies for electron microscopy. Light microscopy revealed numerous OX-6-positive cells with branching processes in the epithelium of striated and excretory ducts of both rat strains, as well as in the connective tissue stroma. ED-1-positive cells had a similar distribution but exhibited a more compact shape with fewer processes. ED-2-positive cells were found only in the connective tissue. Acinar and duct epithelial cells were unreactive. Electron microscopy confirmed that both OX-6-positive and ED-1-positive, non-epithelial cells were present within the duct epithelium. The presence of APC in the duct epithelium suggests that these ducts may be exposed to antigens, possibly by retrograde access from the oral cavity, and that APC located in the salivary gland epithelium may participate in local immune responses.     

Ultrastructural and biochemical analysis of the effects of alendronate on salivary glands of young rats

IntroductionThe bisphosphonates are drugs known by their antiresorptive potency and are widely prescribed for treating and preventing osteoporosis. In the past years the employment of this class of drugs had spread to other pathologies, and it is being prescribed to patients in a wide range of ages. Some adverse effects of bisphosphonate treatment have been highlighted recently, however, little is known about its potential side effects in salivary glands.MethodsNewborn rats received daily doses of 2.5 mg/kg/day of sodium alendronate during 30 days. On the thirtieth day the animals were stimulated with pilocarpine and their parotid and submandibular glands were collected, fixed and embedded for histological and ultrastructural analysis. Some glands were collected for analysis of protein content and amylase activity.ResultsAt light microscopy, the alendronate-treated animals presented an accumulation of secretion granules in their cytoplasm, which was confirmed by the ultrastructural examination. Biochemical analysis revealed an increase in total protein content and decreased amylase levels of both glands in the alendronate-treated animals in relation to the control.ConclusionBased on the current findings, alendronate is probably interfering in secretory pathways of parotid and submandibular glands.     

Characterization of cGMP-related phosphodiesterase isoenzymes in rat salivary glands

 Isolation and characterization of the cGMP-related phosphodiesterase (PDE) isoenzymes in rat salivary glands were investigated. Both cGMP- and cAMP-PDE activities were mainly present in the 100 000 g supernatant fractions from the parotid, submandibular, and sublingual glands. The results of inhibition studies and ion-exchange chromatography suggest that Ca²⁺/calmodulin markedly stimulates PDE1 in the parotid and sublingual glands, and slightly in the submandibular gland. PDE2 was detected only in the parotid gland. PDE3 was identified in the parotid and submandibular glands. PDE5 was detected in the submandibular and sublingual glands by using inhibition studies, ion-exchange chromatography, and Western blotting. Received: August 21, 2001 / Accepted: May 28, 2002     

Long- and short-term diabetes mellitus type 1 modify young and elder rat salivary glands morphology

ObjectiveIn this study we performed a temporal analysis of the effects of Diabetes Mellitus on morphology and laminin deposition in salivary glands of young (2 months-old) and aging (12 months-old) male Wistar rats, using immunohistochemistry.Materials and methodsThe animals were divided in control and diabetic (Streptozotocin induced) groups and euthanized after short and long-term diabetes induction.ResultsShort-term induction led to vacuolization of parotid acinar cells and increased laminin deposition in both animal ages. In young rats, no difference was observed between short or long-term diabetes regarding laminin deposition, but parotid acinar cells vacuolization was more discrete after long-term diabetes. A slight decrease of submandibular gland convoluted granular ducts was observed in young and elder diabetic animal ages. In diabetic aging rats was observed an increase of laminin content only in the parotid gland.ConclusionsThese results suggest that some Diabetes Mellitus effects on salivary glands are not progressive over time, possibly due to the existence of adaptive mechanisms in response to chronic hyperglycemia. They also show that the duration of the disease was more relevant to the morphological effects than the age, although it is known that aging per se affects salivary gland morphology and function.     

Differential expression of salivary glycoproteins in aggressive and chronic periodontitis

Objectives

The aim of this study was to compare the pattern of secretion and the expression of mucin glycoprotein-2 (MG2) and lactoferrin in individuals with or without periodontitis.

Material and Methods

Five individuals with aggressive periodontitis (APG), 5 with generalized chronic periodontitis (CPG) and 5 without periodontitis (CG) were enrolled after informed consent. Non-stimulated and stimulated submandibular and sublingual saliva was collected and samples analyzed by Western blot probed with specific antibodies.

Results

Stimulated and non-stimulated salivary flow rates did not differ among groups. Western blot analysis revealed that stimulation led to: an increase in MG2 expression in all groups, and to lactoferrin expression in APG and CPG. In non-stimulated saliva, CG exhibited the highest expression of both glycoproteins. In stimulated saliva, CG exhibited the highest expression of MG2, whereas APG the highest of lactoferrin.

Conclusions

The pattern of secretion of MG2 and lactoferrin in health and disease is complex. Although the present study analyzed samples from a limited number of participants, the reduced expression of MG2 and lactoferrin in APG and CPG under non-stimulated condition, the predominant circumstance of salivary secretion during the day, suggests that these salivary constituents may play a role in the etiopathogenesis of these diseases.     

放疗对小型猪腮腺颌下腺舌下腺微血管损伤影响的实验研究

目的:观察放疗对小型猪腮腺、颌下腺、舌下腺的微血管损伤状况。方法:将6只实验用小型猪分为2组。2组动物进行放疗,将双侧腮腺、颌下腺、舌下腺加入放射野中,放疗组20Gy/每侧,对照组0Gy/每侧。放疗结束后4 h处死两组动物,取两组动物腮腺、颌下腺、舌下腺标本行HE染色与CD31免疫组化染色,观察放疗早期3种腺体微血管密度变化。结果:两组小型猪腮腺、颌下腺、舌下腺CD31阳性染色颗粒数有显著差异(P＜0.05);3个腺体间统计结果有显著性差异(P＜0.05);腮腺与颌下腺及舌下腺有显著性差异(P＜0.05),颌下腺与舌下腺无显著性差异。结论:放疗可导致小型猪腮腺、颌下腺、舌下腺微血管密度明显减低,且各腺体间微血管密度减低有差异,腮腺的损伤程度大于颌下腺和舌下腺。     

Imaging the major salivary glands

Advances in imaging have led to improved sensitivity in the diagnosis of diseases that involve the major salivary glands. Ultrasound (US), plain radiography and sialography, magnetic resonance imaging (MRI), computed tomography (CT), and nuclear scintigraphy/positron emission tomography (PET) all play a part, and imaging often assists in the planning of further management, operative or otherwise. We review the methods used for imaging the major salivary glands, and apply the indications for these methods to the principal pathological processes.     

Human salivary gland-specific daily variations in histatin concentrations determined by a novel quantitation technique

Histatins constitute a distinct family of human salivary antimicrobial peptides, of which histatins 1, 3 and 5 are the most abundant. To evaluate salivary gland-specific differences in histatin secretion, we used the recently developed histatin-zinc precipitation method to quantify histatins and to assess daily variations in secretions. Stimulated pure secretions from parotid glands (HPS) and submandibular/sublingual glands (SMSL) were collected from 10 different subjects at four different times of the day (9:35 a.m.; 12:40 p.m.; 2:50 p.m. and 5:00 p.m.). Zinc precipitation and subsequent reversed phase HPLC analysis were performed to determine concentrations of histatins 1, 3 and 5 with reference to purified histatin standards. Both HPS and SMSL secretions displayed daily variations in histatin concentrations. HPS values showed a maximum at mid-day and SMSL samples showed a maximum in the morning. Mean daily histatin concentrations were almost three fold higher in SMSL than in HPS. Mean histatin 1, 3 and 5 concentrations in HPS from 10 subjects ranged from 0.7 to 2.8, 0.6 to 4.3 and 1.0 to 4.3mg%, respectively. The corresponding means in SMSL were 2.8-12.2, 1.5-7.5 and 2.6-9.0mg%, respectively. Remarkably, although histatins constitute only 3-10% of total protein in these secretions, an almost perfect correlation between total protein and total histatin concentrations was observed for both glands. Despite a broad range in histatin concentrations between individuals, this study demonstrated a hitherto unidentified daily variation in histatin concentrations in HPS and SMSL secretions and a differential expression pattern which might have functional implications.     

大鼠腮腺及颌下腺唾液的收集

目的:探讨大鼠腮腺及颌下腺唾液的收集方法。方法:采用微型Lashley吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集颌下腺唾液。毛果芸香碱刺激唾液分泌,假设唾液的比重为1.0 g/cm3,以唾液重量代表其体积,记录唾液流率。结果:大鼠腮腺唾液流率(9.9±1.4)μL/m in,颌下腺导管插管唾液流率(19.9±10.8)μL/m in。结论:可以采用微型吸盘法收集大鼠腮腺唾液,经口内颌下腺导管口直接插管法收集大鼠颌下腺唾液。     

45例舍格伦综合征伴发唾液腺肿物误诊病例分析

目的: 分析舍格伦综合征(Sjögren's syndrome,SS)伴发唾液腺肿物误诊病例的特点,为临床诊断提供参考。方法: 回顾分析45例接受腮腺下颌下腺肿物切除术并最终病理诊断为良性淋巴上皮病患者的相关临床资料,并与唾液腺肿瘤性疾病进行对比分析。采用 Microsoft Excel构建数据库并做统计分析。结果: 45例患者均为女性,24.4%的患者累及双侧唾液腺,少数患者有口干(11.1%)、眼干(4.4%)、易疲劳(6.7%)、关节疼痛(22.2%)等临床症状,近半数患者肿物质地中等(55.6%),活动度小(53.3%)。35例患者接受CT检查,13例(37.1%)提示自身免疫性疾病,15例接受B超检查,13例接受MRI检查,分别有8例(53.3%)和10例(76.9%)提示自身免疫性疾病。结论: 以唾液腺肿物为主诉就诊的中年女性,特别是肿物累及双侧的患者,需密切排除SS后再进行手术治疗。B超及MRI可作为CT以外的附加临床常规检查,用于唾液腺肿物的初期诊断。     

Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands

The constant ratio between saliva and plasma acetaminophen concentrations (S/P) during the elimination phase is assumed to result from the equilibrium established among the free-drug concentrations in the arterial blood, venous blood and saliva. Salivary secretion of acetaminophen is assumed to result from a passive diffusion of the drug to saliva from the blood that supplies the salivary glands. However, the constant S/P ratio during acetaminophen disposition and the finding that P-glycoprotein (P-gp), a protein recognized to pump substrates out of the cell, is expressed in duct cells of the submandibular glands questions the mechanisms involved in acetaminophen salivary secretion. Thus, we intended to evaluate the existence of a P-glycoprotein-mediated transport of acetaminophen in rat submandibular glands. Acetaminophen (30 mg/kg, i.v.) pharmacokinetics was assessed in controls and in rats pre-treated with erythromycin (100 mg/kg) as a P-glycoprotein inhibitor. Acetaminophen pharmacokinetic parameters were calculated from saliva and plasma levels considering a non-compartmental analysis. Mean plasma and salivary profiles of control and pre-treated animals were almost superimposable. No difference could be found in S/P ratios in control and erythromycin pre-treated animals (P > 0.05). Moreover, no statistical difference could be found in the kinetic parameters calculated from saliva or plasma drug level (P > 0.05). These observations indicate that acetaminophen salivary secretion in rat submandibular glands is not related to P-glycoprotein-mediated transport under the experimental conditions of the present work.     

Transglutaminase 2 expression in the salivary myoepithelial cells of mouse embryo

Earlier a strong transient expression of transglutaminase 2 (TGase 2) localized at the anchoring sites of muscle bundles in human embryo was observed. In this study, we report a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo by immunohistochemistry, RNA in situ hybridisation, and RT-PCR. From 35 submandibular glands of mouse embryos and postnatal mice, a consistent expression of TGase 2 in the myoepithelial cells via a stage-specific manner was identified by mono-clonal antibody to TGase 2 immunostaining. A similar expression pattern of TGase 2 in the myoepithelial cells was also observed by RNA in situ hybridisation analysis. The expression of TGase 2 in the salivary epithelium and mesenchyme during the prenatal 14.5-15.5 days was found minimally diffusely spread and became intensely focalised in the myoepithelial cells of salivary acini and ducts during the prenatal 16.5-18.5 days but thereafter gradually decreased until postnatal 7 days and remained weak in postnatal 3 weeks. Such transient rise and fall expressions of TGase 2 were also found with the sequential amount of RT-PCR products during the same period. The alpha-smooth muscle actin (alpha-SMA) as a positive control in the myoepithelial cells of mouse submandibular glands was consistently expressed during the prenatal and postnatal period. These results of transient expression of TGase 2 in the myoepithelial cells coincided with the formation of the dendritic basket structure in the periphery of acini and ducts, suggest a possible catalytic role of transglutaminase in a newly formed cellular matrixes during the cytodifferentiating stage of mouse prenatal and neonatal submandibular glands.     

Altered autophagy and sympathetic innervation in salivary glands from high-fat diet mice

Objectiveto investigate the effects of a high fat diet (HFD) on salivary glands in vivo, in a mouse model. In particular, whether it will induce the appearance of fat cells in salivary glands, alterations related to autophagy, mTOR pathway and sympathetic innervation.Design27 adult female ICR mice were separated in six groups. Three groups fed with (HFD) containing 55% fat, for one, two and three month and another three groups fed with normal diet (2.7% of fat), for the same time periods. The submandibular glands and liver were dissected and part homogenized for protein analyses and part fixed in formalin for histological analyses.ResultsAfter three months the HFD fed mice total body weight fold change increased compared to controls. The Oil Red O staining showed no fat cells deposit in salivary gland however a large increase was observed in liver after three months of HFD. Adiponectin levels were significantly decreased in the HFD group after three months. The group fed with HFD for three months showed increased conversion of the LC3 autophagy marker in salivary gland. mTOR showed no activation regarding the time point studied. Tyrosine hydroxylase significantly decreased after two and three month of HFD.ConclusionHFD caused several changes after three months however the earliest change was noticed after two months regarding sympathetic innervation. This suggests neural alteration may drive other diet induced changes in salivary glands. These early changes may be the starting point for longer term alterations of salivary glands with alterations in diet.     

c-Met在小鼠颌下腺胚胎发育中的表达

目的探讨c-Met在小鼠颌下腺发育不同阶段的表达特点。方法制作ICR小鼠颌下腺不同发育阶段的冰冻组织切片,利用免疫组织化学方法对小鼠颌下腺自蕾状期至出生后两天不同发育阶段c-Met的表达情况进行了研究。结果 c-Met在小鼠颌下腺发育的蕾状期上皮中表达呈明显阳性,在假腺管期早期表达减弱,而在假腺管期晚期的上皮突起中表达增强。在微管期的分枝状上皮条索和顶端胚芽中呈不对称表达,周围细胞表达较强而中心细胞表达较弱。在终末分化期的腺泡、闰管的部分上皮细胞表达,在导管上皮中持续性强表达。结论 c-Met在小鼠颌下腺发育的细胞增殖、分支形态发生及腺泡和导管的形成中可能扮演重要角色。     

涎腺内镜治疗涎石病的回顾分析

目的 分析涎腺内镜在涎石病的诊断与治疗中的临床价值.方法 对52例(43例颌下腺、9例腮腺)涎石病行内镜探查及取石术.结果 34例颌下腺导管前段和(或)后段结石中,24例在内镜下直接取石;2例经手术及内镜取石;8例手术取石.8例颌下腺导管腺门处结石在内镜辅助下手术取石.9例腮腺结石中3例以抓篮取石;3例直接切开导管口取石;1例以抓篮套锁后于颊部切开取石.取石成功的49例随访1个月～2年无复发.结论 涎腺内镜可提高涎石病的诊断敏感性,且多数结石可在内镜辅助下取出.     

Increases in nerve growth factor immunoreactivity in the submandibular gland, but not in the parotid gland, of the rat following sympathetic denervation

The effect of sympathetic denervation on nerve growth factor (NGF) immunoreactivity in the submandibular and parotid glands of adult female rats using a two-site, enzyme-linked immunosorbent assay was investigated. Unilateral sympathetic denervation through the avulsion of the superior cervical ganglion resulted in elevated levels of total amounts (decreased from 40-20%) and concentrations (decreased from 45-32%) of NGF in the submandibular glands, but not in the parotid glands, on the operated side 7-28 days post-operatively. Sympathetic decentralisation had no effect. The present findings support the hypothesis that increased levels of NGF play an important part in the increased activity of the acetylcholine-synthesising enzyme, choline acetyltransferase, in the rat submandibular gland following sympathetic denervation.