The Role of Endothelial Barrier Function in the Fibrosis of Salivary Gland.

In the salivary glands, fibrosis occurs in many pathological conditions. Endothelial tight junction (TJ)-based barrier function plays a vital role in maintaining the homeostasis of the salivary glands. However, whether endothelial barrier function is changed and involved in the pathogenesis of glandular fibrosis is unknown. Here, by using a mouse model in which the main excretory duct of the submandibular gland (SMG) was ligated to induce inflammation and fibrosis, endothelial barrier function and TJ protein expression and distribution were examined. Both 4-kDa and 70-kDa fluorescence-labeled dextrans permeated more in the 1-, 3-, and 7-d ligated SMGs. Meanwhile, the mRNA level of claudin-5 was increased with an obvious redistribution from apicolateral membranes to lateral membranes and cytoplasm in the fibrotic glands. Notably, the TJ sealer AT1001 significantly attenuated the disrupted endothelial barrier function and thereby ameliorated the glandular fibrosis. Cytokine array detection showed that monocyte chemoattractant protein-1 (MCP-1) was highly enriched in the 3-d ligated SMGs, and MCP-1 directly impaired barrier function, increased claudin-5 expression, induced the relocalization of claudin-5, and activated p-ERK1/2 in cultured human endothelial cells. Furthermore, the upregulation and disorganization of claudin-5 as well as the elevation of MCP-1 and p-ERK1/2 signaling were also confirmed in fibrotic SMGs from patients with chronic sialadenitis and immunoglobulin G4-related sialadenitis. Altogether, our findings revealed that disrupted endothelial barrier function contributed to the progression of glandular fibrosis, and targeting endothelial TJs might be a promising approach to alleviate salivary gland fibrosis-related diseases.

[Effect of topical injection of cyclosporine A on saliva secretion and inflammation in the submandibular gland of non-obese diabetic mice].

OBJECTIVE: To investigate the effects of topical administration of cyclosporine A (CsA) on salivary secretion and inflammation of the submandibular glands in non-obese diabetic (NOD) mice. METHODS: Female NOD mice, 21 aged 14 weeks and 18 aged 21 weeks were selected and randomly divided into low-dose group, high-dose group and control group on average. CsA was injected into submandibular glands. One week later the saliva stimulated by pilocarpine was collected and measured. The submandibular glands were collected to make paraffin sections. The lymphocyte infiltration in submandi-bular gland was observed by microscope after hematoxylin-eosin (HE) staining. The number of lymphocyte infiltration foci was counted to calculate the focus sore and the ratio of lymphocyte infiltration area to total gland area was figured up by Leica image analysis system. The expressions of inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-13, IL-17F, IL22 and IL-23a in the submandibular glands of the NOD mice were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis in the submandibular gland was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The levels of serum creatinine (Scr), blood urea nitrogen (BUN), uric acid (UA), alanine aminotransferase (ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB) and γ-glutamyl transferase (GGT) were measured by automatic biochemical analyzer to evaluate liver and kidney functions. RESULTS: After topical injection of CsA in the submandibular gland, the stimulated salivary flow rate of the 14- and 21-week-old NOD mice significantly increased compared with the control group (P < 0.01 or P < 0.05), and the number and area of lymphocyte infiltration foci in the 14-week-old NOD mice low-dose group significantly decreased compared with the control group (P < 0.01). Low and high dose of CsA had similar effects on reducing inflammation and improving salivary secretion. The overall level of inflammatory cytokines in the submandibular gland did not decrease significantly. The number of cell apoptosis of submandibular gland in the NOD mice treated with CsA decreased compared with the control group, but there was no statistically significant difference. Topical injection of CsA had no adverse effect on liver and kidney function in the NOD mice. CONCLUSION: Topical injection of CsA can reduce lymphocyte infiltration in submandibular gland of NOD mice and improve salivary secretion.

Tricellulin Modulates Transport of Macromolecules in the Salivary Gland.

Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate-dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate-dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in Ildr1-/- mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function.

TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis.

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients' SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α-treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α-induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

[Role and mechanism of muscarinic acetylcholine receptor in the regulation of submandibular gland secretion].

Muscarinic acetylcholine receptors (mAChRs), including M1-M5 subtypes, are classic receptors in regulating water, ion, and solute transport in salivary gland. Our work focuses on the studies on the expression pattern and function of mAChR in the submandibular gland (SMG), and the underlying mechanism involved in the mAChR-regulated secretion, together with the effect of parasympathectomy on the salivary secretion. Microvascular autotransplantation of SMG into the temporal fossa provides a continuous and endogenous source of fluids, and is currently an effective method for treating severe keratoconjunctivitis sicca. By using RT-PCR, Western blotting, and immunofluorescence, our data demonstrated that the expression of M1 and M3 subtypes were decreased in latent period in rabbit SMG autotransplantation model, whereas carbachol stimulation promoted the salivary secretion, as well as M1 and M3 expressions. By contrast, mAChRs were hypersensitive in epiphora SMGs, whereas atropine gel and botulinum toxin A application significantly inhibited the hypersecretion in both animal models and patients. Furthermore, the possible intracellular signal molecules involved in the mAChR-modulated salivary secretion were explored. Activation of mAChR upregulated the expression of aquaporin 5 (AQP5), the main transporter that mediated water secretion through transcellular pathway, and led to AQP5 trafficking from lipid rafts to non-lipid microdomain. Extracellular signal-regulated kinase 1/2 (ERK1/2) was involved in the mAChR-regulated AQP5 content. mAChR activation also modulated the expression, distribution, and function of tight junction proteins, and increased paracellular permeability. ERK1/2/ß-arrestin2/clathrin/ubiquitin signaling pathway was responsible for the mAChR-regulated downregulation of tight junction molecule claudin-4. Cytoskeleton filamentous actin (F-actin) was also involved in the distribution and barrier function of epithelial tight junctions. Besides, endothelial tight junctions were opened by mAChR agonist-evoked salivation in the mice. Furthermore, parasympathetic denervation increased resting salivary secretion in the long terminrats and minipigs. Taken together, our work demonstrated that mAChR regulated saliva secretion via transcellular and paracellular pathways in SMG epithelium as well as tight junction opening in SMG endothelium. Modulation of mAChR might be a promising strategy to ameliorate SMG dysfunction.

[Subarachnoidal anesthesia in patients with concomitant diseases of the cardiovascular system].

Age-related changes limit blood circulatory reserves in vital organs, by increasing the risk of ischemic and hypoxic lesions. Patients from a peripheral vascular surgery department form a high cardiac risk group. Subarachnoidal anesthesia is the most optimal anesthetic support (if there are no absolute contraindications) during operations for lower extremity varicose veins. Thirty patients operated on for this condition were examined to solve the problem associated with the effectiveness and safety of this method of anesthesia and to define the pattern of hemodynamic rearrangement more precisely. All the patients had concomitant cardiovascular diseases, which forced them to be referred to as ASA Classes III-IV. The authors monitored basic hemodynamic parameters, such as systolic blood pressure (BP), diastolic BP, mean BP (BP(mean)), heart rate, and cardiac output, by using a noninvasive procedure by means of a computer equipped with a Doppler flowmetric transducer, and ST-segment changes by ECG). The values of total and specific peripheral vascular resistance and cardiac index were estimated by a calculating method. Analysis of hemodynamic changes revealed no significant differences between the groups of elderly and senile patients. On examination, all the patients were divided into 2 groups by the type of circulation: 1) those with eukinetic circulation and 2) those with hypokinetic circulation. Hyperkinetic circulation was not found in the study groups of patients. The patients with cardiac disease were observed to have increased vascular resistance, in those with hypokinetic circulation, this increase being more marked (p < 0.05). In addition, ST-segment had a more stable position in patients with hypokinetic circulation. There was a great scatter in ST-segment changes in patients with eukinetic circulation in the presence of decreased peripheral resistance under preganglionic sympathetic block. With the described changes, noteworthy was the stability of BP(mean) values that did not decreased below the critical ones. This fact permitted the authors to state that the compensatory reserves of the cardiovascular system were preserved under subarachnoidal blockade, which in turn suggests the effectiveness and safety of the used anesthetic procedure.

[Subarachnoid anesthesia in patients with high surgical risk]. / Subarakhnoidal'naia anesteziia u bol'nykh s vysokim operatsionnym riskom.

The hemodynamics was intraoperatively studied in 43 patients with multicentric atherosclerosis, ASA III-IV, operated on the peripheral vessels of legs with balanced anesthesia based on subarachnoid block. It was established in regional sympathetic block and in the course of the whole surgery that the parameter of AP were decreased by 20-25%, those of heart rate--by 15% and of TPVR--50%, whereas, the parameters of cardiac performance were stable. The authors discuss the specificity of hemodynamic restructuring under subarachnoid block in patients with at surgical risk due to vascular pathology.

[Immunological prognosis of the effectiveness of soybean diet]. / Immunologicheskii prognoz éffektivnosti soevogo pitaniia.

During 50 days a group of 23 volunteers received lunches prepared on the basis of textured proteins of soy beans. Each lunch included 50 g of proteins. Laboratory investigation was carried on dynamic values of lipid metabolism, cell-mediated, humoral and local immunity. A total of more than 30 tests were made. The analysis of immunological reactivity in volunteers has revealed the immunomodulatory activity of soy bean protein similar to the reference modulator with known quantitative estimation of effects on cell mediated, humoral and local immunity. Statistic analysis of preliminary individual laboratory results and plotting of prognostic curves allowed to develop an original method of volunteer selection for whom soy bean diet may bring positive or negative results.

[The local immunity reactions of swimmers on the Russian all-star team]. / Reaktsii mestnogo immuniteta u plovtsov sbornoi strany.

Dynamic observation of the national team of swimmers for a period of 3 years demonstrated that physical and psychosomal loadings of competitive sport led to the suppression of local immunity, depending on the level of competitive stress. In a certain number of sportsmen the phenomenon of the complete disappearance of some classes of immunoglobulins and normal antibodies from saliva was registered. The daily rhythms of immune reactions had no influence of the regularities of fluctuations in the levels of immunoglobulins and antibodies at different periods of sporting activities when these changes were most pronounced. Still the peaks of immunoglobulin levels were registered in the morning and those of normal antibodies, in the evening after physical strain. During the period of competitions at the time of a sharp drop in the content of immunoglobulins in saliva a rise in the level of anamnestic antibodies was observed. The dynamics of immune reactions in saliva did not essentially depend on the sex of swimmers. The results of these studies are indicative of the necessity of immunoprophylactic and immunocorrective measures in the group of immunological risk.

[Sorbed animal immunoglobulins--a reserve for increasing the output of therapeutic antitoxic and diagnostic sera]. / Sorbirovannye immunoglobuliny zhivotnykh--rezerv dlia uvelicheniia vypuska lechebnykh antitoksicheskikh i diagnosticheskikh syvorotok.

Conditions for the desorption of immunoglobulins from the surface of blood cells of animals used for the production of antitoxic therapeutic and diagnostic sera have been established. The possibility of obtaining, in principle, additional amounts of immunoglobulins and specific antibodies (in comparison with their initial content in sera) without increasing the number of producer animals has been demonstrated. The possibility of using desorbed immunoglobulins and specific antibodies in the production of therapeutic and diagnostic immunobiological preparations is discussed.

[Immunological criteria for the selection of servicemen for service under extreme conditions]. / Immunologicheskie kriterii otbora voennosluzhashchikh dlia sluzhby v ékstremal'nykh usloviiakh.

The paper deals with the results of the comparative immunological survey of three groups: group I (450 persons), a special unit "Quarantine" undergoing training for hostilities in Afghanistan; group II (800 persons), an armored regiment assigned for home service; and group III (578 persons), students of a vocational technical school. Among the members of all three groups disturbances in the systems of humoral and local immunity were detected, but the number of immunodeficiency cases in group I considerably exceeded that in groups II and III, which correlated with considerably greater morbidity figures registered in group I. On the basis of these data immunological risk groups were formed and immunocorrective measures were recommended.

[Quantitative changes of the blood serum immunoglobulins in patients with diabetes mellitus types I and II and microangiopathy detected by morphologic study]. / Kolichestvennye izmeneniia immunoglobulinov syvorotki krovi bol'nykh sakharnym diabetom I i II tipa s mikroangiopatiei, vyiavlennoi pri morfologicheskom issledovanii.

A study was made of the immunological mechanisms implicated in the evolution of diabetic microangiopathy. For this purpose in 270 patients with type I and II diabetes mellitus, the concentration of IgA, IgM and IgG was measured and compared with morphological alterations in skin biopsy specimens. The control group was made up of 30 normal persons (donors). The sections were stained with conventional methods, which made it possible to reveal by light microscopy the correlation between the intensity of diabetic microangiopathy and the rise of the IgG level.

[Experimental study of the immunostimulating properties of tomitsid]. / Eksperimental'noe izuchenie immunostimuliruiushchikh svoistv tomitsida.

Tomicide, a preparation with antibacterial properties, accumulates in the culture medium during the growth of one of streptococcal strains. The injection of the preparation into mice simultaneously with the antigen (sheep red blood cells) produces an adjuvant effect. Tomicide has been found capable of inducing the production of interferon detected in the serum of the animals. During experimental stress created by intensive exercise tomicide prevented a decrease in the normal (anamnestic) level of antibodies, produced an immunostimulating effect and, at the same time, enhanced the total physical endurance of the animals, manifested by an increased duration of swimming.