Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren's syndrome.

BACKGROUND: The phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies. Primary Sjögren's syndrome (pSS) is a chronic immune-mediated inflammatory disease characterised by exocrine gland lymphocytic infiltration and B-cell hyperactivation which results in systemic manifestations, autoantibody production and loss of glandular function. Given the central role of B cells in pSS pathogenesis, we investigated PI3Kδ pathway activation in pSS and the functional consequences of blocking PI3Kδ in a murine model of focal sialoadenitis that mimics some features of pSS. METHODS AND RESULTS: Target validation assays showed significant expression of phosphorylated ribosomal protein S6 (pS6), a downstream mediator of the phosphatidylinositol 3-kinase delta (PI3Kδ) pathway, within pSS salivary glands. pS6 distribution was found to co-localise with T/B cell markers within pSS aggregates and the CD138+ plasma cells infiltrating the glands. In vivo blockade of PI3Kδ activity with seletalisib, a PI3Kδ-selective inhibitor, in a murine model of focal sialoadenitis decreased accumulation of lymphocytes and plasma cells within the glands of treated mice in the prophylactic and therapeutic regimes. Additionally, production of lymphoid chemokines and cytokines associated with ectopic lymphoneogenesis and, remarkably, saliva flow and autoantibody production, were significantly affected by treatment with seletalisib. CONCLUSION: These data demonstrate activation of PI3Kδ pathway within the glands of patients with pSS and its contribution to disease pathogenesis in a model of disease, supporting the exploration of the therapeutic potential of PI3Kδ pathway inhibition in this condition.

A Mouse Model for Dietary Xenosialitis: ANTIBODIES TO XENOGLYCAN CAN REDUCE FERTILITY.

Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah(-/-) mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fcγ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.

Inducible nitric oxide synthase increases secretion from inflamed salivary glands.

OBJECTIVE: Salivary gland secretion is dependent on cholinergic stimulation via autonomic nerves and calcium signalling in acinar cells. Secretory dysfunction associated with SS may be partly caused by the damaging effects of increased glandular concentrations of nitric oxide (NO) derived from up-regulation of inducible NO synthase (iNOS) that accompanies glandular inflammation. The present study examines the effects of increased iNOS expression on salivary gland secretory function. METHODS: The inflammogen lipopolysaccharide (LPS) was introduced intraductally into rat submandibular glands, and glandular responsiveness to cholinergic stimulation was determined. RESULTS: LPS provoked a rapid, long-lasting inflammation, increasing gland weight (by almost 20%) and inflammatory cell infiltration at 3 and 24 h. Immunoblotting of glandular homogenates indicated that iNOS expression was increased approximately 4-fold, and immunohistochemistry of frozen tissue sections showed increased iNOS expression in acinar cells. Salivary secretion from inflamed glands was significantly increased in response to low doses of methacholine and accompanied by increased acinar cell calcium signalling in vitro. Prior administration of the iNOS inhibitors, aminoguanidine or L-NIL [L-N6-(1-iminoethyl)-lysine dihydrochloride] abolished increased secretion and acinar cell calcium signalling. CONCLUSIONS: Up-regulation of glandular iNOS expression can increase cholinergically evoked salivary secretion and appears to offset any secretory hypofunction linked with glandular inflammation. It seems unlikely that increased glandular levels of NO are responsible for the secretory hypofunction that accompanies SS.

Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjögren's syndrome.

Demonstration of ectopic germinal center-like structures (GC-LSs) in chronically inflamed tissues in patients with autoimmune disorders is a relatively common finding. However, to what extent ectopic lymphoid structures behave as true GC and are able to support class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig genes is still debated. In addition, no information is available on whether CSR and SHM can take place in the absence of GCs at extrafollicular sites in an ectopic lymphoid tissue. In this study, we show that in salivary glands (SGs) of Sjögren's syndrome (SS) activation-induced cytidine deaminase (AID), the enzyme responsible for CSR and SHM is invariably expressed within follicular dendritic cell (FDC) networks but is not detectable in SGs in the absence of ectopic GC-LSs, suggesting that FDC networks play an essential role in sustaining the Ag-driven B cell proliferation within SS-SGs. We also show that the recently described population of interfollicular large B cells selectively expresses AID outside ectopic GC in the T cell-rich areas of periductal aggregates. Finally, we report that AID retains its exclusive association with numerous, residual GCs in parotid SS-MALT lymphomas, whereas neoplastic marginal zone-like B cells are consistently AID negative. These results strongly support the notion that ectopic lymphoid structures in SS-SGs express the molecular machinery to support local autoantibody production and B cell expansion and may play a crucial role toward lymphomagenesis.

[Study of free-radical processes and antioxidant defense parameters during hirudotherapy of patients with diseases of salivary glands]. / Izuchenie pokazatelei svobodnoradikal'nykh protsessov i antioksidantnoi zashchity u lits s zabolevaniiami sliunnykh zhelez, poluchaiushchikh girudoterapiiu.

Hirudotherapy was used in the treatment of 39 patients (10 male and 29 female) aged 28-58 years with chronic sialadenitis and sialadenosis. Control group consisted of 15 normal subjects without diseases of salivary glands. Lipid peroxidation (LPO) and antioxidant defense (AOD) parameters were under study. Hirudotherapy led to improvement of the clinical status in the majority of patients with sialadenosis; the content of superoxide dismutase (SOD) normalized and ceruloplasmin (CP) level increased. The status of patients with sialadenitis also improved; catalase and glutathione peroxidase levels normalized and SOD and CP levels increased. The best therapeutic effect was attained in patients with sialadenitis. No appreciable improvement was observed in patients with chronic parenchymatous parotitis in the presence of Sjogren's syndrome.

Extracellular matrix molecules in chronic obstructive sialadenitis: an immunocytochemical and Western blot investigation.

The exact pathomechanism of inflammation progress and fibrosis in chronic sialadenitis is unknown. Connective tissue growth factor (CTGF), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of various fibrotic conditions. These factors are thought to be essential in the regulation of extracellular matrix turnover and the development of tissue fibrosis. In the present study, the expression of CTGF, MMP-2, -3, -9, -13 and TIMP-3 was examined in chronic obstructive sialadenitis. Tissue samples of 13 patients with chronic sialadenitis of the submandibular gland associated with sialolithiasis and 4 normal tissue samples of the submandibular gland were analyzed immunohistochemically and by Western blot analysis. An intense CTGF immunoreactivity was observed in the ductal system of inflamed salivary glands, whereas in normal glands no reactivity or a very low CTGF immunoreactivity was present. Immunohistochemical studies revealed a low to strong reactivity of MMP-2, -3, -9, -13, and TIMP-3 in the ductal system, in acinar cells and in lymphomonocytic infiltrates in normal and inflamed tissues. The expression of MMP-2, -3, -9, -13, and TIMP-3 was confirmed by Western blotting in all cases. Over-expression of CTGF in chronic obstructive sialadenitis suggests that this factor may play a role in glandular fibrosis. However, the physiological role of MMP-2, -3, -9, -13, and TIMP-3 in normal glands, as well as their possible role in inflammation progress and fibrosis in chronic obstructive sialadenitis, remains to be elucidated.

Alterations in nitric oxide synthase activity and expression in submandibular glands of NOD mice.

The non-obese diabetic (NOD) mouse model of autoimmune sialadenitis offers the possibility of studying the L-arginine/nitric oxide signaling pathway in salivary glands in basal and neurotransmitter-stimulated conditions and, thus, of analyzing the neural control of the secretory process in the target organ. The purpose of this study was to explore putative alterations in the activity and expression of nitric oxide synthase (NOS) in submandibular glands of NOD mice in relation to parotid glands and unrelated tissues. Here we report that NOD mice with incipient signs of secretory dysfunction presented a marked decrease in basal and vasoactive intestinal peptide (VIP)-stimulated NOS activity and a differential expression of NOS I in submandibular glands compared to control BALB/c mice. Similar alterations in NOS I were found in parotid glands but not in brain or spleen of NOD mice. No differences between NOD and controls appeared in NOS II and NOS III expression in any of the tissues studied.

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.

Tear lysozyme activity in frozen Schirmer strips and salivary gland biopsy as parameters of lacrimal gland function.

PURPOSE: The lysozyme concentration in human tears is an important parameter for tear gland function. The decline of lysozyme in tears reflects lacrimal gland destruction. In Sjögren's patients, lacrimal gland destruction parallels labial salivary gland destruction. The objective of this study was to determine whether human tear lysozyme that was frozen on Schirmer strips at -20 degrees C for several years maintained activity and whether there was a linear relation with inflammatory changes in labial salivary glands. METHODS: A total of 200 frozen Schirmer strips were processed. They were collected from 20 randomly selected patients each year of five consecutive years, all attending the UCSF Sjögren's Clinic. The tear lysozyme in the Schirmer strips was measured by a colorimetric assay. The average lysozyme concentration each year was calculated and compared. One third of the patients underwent labial salivary gland biopsy. The correlation was calculated between the tear lysozyme concentration and the lymphocytic focus scores in biopsy specimens. RESULTS: No significant difference of average lysozyme concentration in the Schirmer strips was found when the five different years of collection were compared. The linear relation between the tear lysozyme concentrations and the focus score in labial salivary gland biopsies showed a coefficient of r = -0.41. The linear relation between other diagnostic measurements, like Schirmer test, tear breakup time, or rose bengal staining pattern, and the focus score was lower. CONCLUSIONS: Human tear lysozyme in Schirmer strips can be stored at -20 degrees C for at least five years. There is little difference in lysozyme activity of frozen compared to unfrozen specimens. The lysozyme concentration in tears correlates better with the lymphocytic focus score in labial salivary gland biopsy than does clinical assessment and is therefore a parameter for the actual degree of tear gland destruction.

Lymphoepithelial cyst with crystalloid formation. Cytologic features of two cases.

BACKGROUND: The presence of amylase crystalloids (AC) in cystic lesions of the parotid gland is a rare occurrence and has been diagnosed to date as sialadenitis. We report the first two cases of parotid lymphoepithelial cyst (LC) containing this type of crystalloid. CASES: Case 1, a 56-year-old male, presented with a 3-cm parotid cyst. Fine needle aspiration (FNA) was performed on the mass. Smears showed numerous crystalloids identical to those described as crystallized amylase. Case 2, a 36-year-old female, had a 2-cm parotid mass. FNA smears exhibited the same features as did case 1. The two patients were treated with superficial parotidectomy, and an LC containing AC was diagnosed in both cases. CONCLUSION: When the above findings are present on FNA of parotid gland, the diagnosis of LC must be considered.

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sjögren's syndrome.

The enzyme aromatase is involved in the conversion of androgens to estrogens and in the modulation of various androgenic and estrogenic actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophysiology of Sjögren's syndrome. In the present study, aromatase was immunolocalized in 75 cases of inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sjögren's syndrome (19 cases), of chronic sialadenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sjögren's syndrome. Aromatase immunoreactivity was detected in myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts in 13/19 (68%) cases of primary Sjögren's syndrome. In contrast, aromatase expression was detected in only six of 34 (18%) cases of chronic sialadenitis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that increased aromatase expression in minor salivary glands with primary Sjögren's syndrome in premenopausal women may be involved in the biological features of primary Sjögren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.

Chronic submandibular sialadenitis: ultrastructure and phosphatase histochemistry.

Eleven specimens of chronic submandibular sialadenitis were examined. A reduction in secretory material in acinar cells was seen with increasing atrophy until the acini resembled intercalary ducts. Myoepithelial cells and basement membrane were sometimes more conspicuous. Striated ducts showed a reduction of the folding of the plasma membranes in the basal part, and striated and excretory ducts showed a reduction in mitochondria. This possibly represents a functional atrophy secondary to reduced salivary flow. Very atrophic parenchyma largely consisted of simple cells. Phagosomes and apoptotic bodies were occasionally seen, and appear to be involved in the atrophy. Thiamine pyrophosphatase in the Golgi apparatus and acid phosphatase in the GERL were demonstrated in moderately atrophic parenchyma. This is similar to normal and indicates continuing synthetic activity. Acid phosphatase was demonstrated in lysosomes, which appear to be involved in the atrophy by their role in phagy. Alkaline phosphatase was occasionally demonstrated at luminal surfaces, and is likely to be involved in resorption of obstructed luminal contents. The changes are similar to those seen in experimentally obstructed glands and indicate that much of the parenchyma survives by adaptation to the altered environment which forms the basis for the successful results following conservative therapy.

Significance of vomiting for hyperamylasemia and sialadenosis in patients with eating disorders.

The authors investigated the significance of vomiting for hyperamylasemia and sialadenosis in patients with bulimia nervosa. Hyperamylasemia was found in 61% of the bulimics and in 20% of the restrictor anorectics but in no patients with binge-eating syndrome. In more than three fourths of the bulimics there was a close positive correlation between the frequency of vomiting and total serum amylase levels. Both frequency and type of vomiting seem to be relevant to the extent of salivary gland enlargement. The significance of vomiting for the etiopathology of hyperamylasemia and for the diagnosis of eating disorders will be discussed.

Approach to the diagnosis of sialadenosis using sialography.

Since parotid swelling is the most informative symptom of sialadenosis, we examined parotid swelling with sialography as a means of diagnosing sialadenosis. An X-ray was taken from a fixed position relative to the body, using a focus film distance (FFD) of 70 cm. To determine an "index" of parotid swelling, the distance between the submandibular bone ridge and the end of the main duct was measured on X-ray. After examination of 30 normal parotids, abnormal swelling was defined as an "index" exceeding 1.9 cm. Sixteen of 24 patients suspected of having sialadenosis showed swelling exceeding 1.9 cm. Six of 7 patients who were histologically diagnosed with sialadenosis showed swelling in excess of 1.9 cm. Our method is reproducible and recurrence of parotid swelling correlating with sialadenosis can be objectively demonstrated. Furthermore, serum amylase levels in patients who were diagnosed with sialadenosis were measured before and after sialography. After sialography serum amylase levels increased remarkably higher than those of normal subjects. Thus if a patient with underlying diseases has an "index" over 1.9 cm and his serum amylase level after sialography increases remarkably, a diagnosis of sialadenosis is likely.

Experimental models of acute and chronic sialoadenitis in the guinea pig.

Acute and chronic sialoadenitis were induced in ovalbumin-immunized guinea pigs by a single or repeated (once a day for 5 days) instillation of antigen into the parotid gland via the parotid duct. The acute sialoadenitis was characterized by infiltration of inflammatory polymorphonuclear leukocytes and the chronic one, by extensive tissue destruction together with infiltration of mononuclear leukocytes. In acute sialoadenitis, myeloperoxidase activity in the parotid gland, which was a marker of accumulation of neutrophils, was elevated, but in the chronic stage, it returned nearly to the control level. This observation is in accord with the histological findings that infiltrating cells in acute and chronic sialoadenitis were mainly polymorphonuclear and mononuclear leukocytes, respectively. Although cyclophosphamide suppressed the inflammation, both in acute and chronic sialoadenitis, indomethacin exerted its anti-inflammatory effect only in the acute stage. Our experimental models of acute and chronic sialoadenitis were easy to prepare, and had a high incidence. As the typical features of inflammatory development from acute to chronic phases were observed in these models, these models may be useful for studying the mechanism of the chronic course in immunologically induced inflammation and the effects of drugs on each phase and the chronic course of inflammation.

Immunohistochemical localization of amylase in sialoadenitis and salivary gland tumors.

Immunohistochemical demonstration of alpha-amylase has been made in sialoadenitis-involved tissue and salivary gland tumors, as well as in normal salivary glands. Immunoreactive alpha-amylase with trypsin pretreatment was confined to irregularly staining serous acinar cells in the parotid and submandibular glands, and to demilunes in sublingual glands. In obstructive adenitis, staining was irregular from high to negative in acini in early or intermediate stages. Dilated ductal segments contained cells positive for alpha-amylase in the early stage following obstruction. Pleomorphic adenomas were usually negative for alpha-amylase but in rare cases tumor epithelia stained variably positive; i.e., staining occurred throughout the cytoplasm or at the periphery or apical part of the tumor cells. Luminal cavities of tubular and duct-like structures contained alpha-amylase-positive material. Epithelia in Warthin's tumor were also negative in general; however, scattered single or grouped tumor cells containing alpha-amylase were found. Mucoepidermoid tumors were also negative, though slightly positive cells were found intermingled among the negative squamous and mucous tumor cells. Cystic lesions in mucoepidermoid tumor were sometimes positive in the wall cells together with material secreted into the lumen.

Immunohistochemical observations on carbonic anhydrase I and II in human salivary glands and submandibular obstructive adenitis.

Immunohistochemical identification of carbonic anhydrase I and II (CA-I, CA-II) was made in human major salivary glands and obstructive adenitis in submandibular glands. Normal salivary glands stained the strongest for CA-II in serious acinar cells and were negative in mucous cells. Moderate to strong staining for CA-I and CA-II was found in ductal segments. Submandibular glands with obstructive adenitis exhibited reduced CA-I activity in atrophic acinar cells, but not in ductal elements in the early and intermediate stages of the disorder. In the late stage of the obstructive lesion, CA staining in duct-like structures was moderate; however, almost degenerate ductal cells were negative for CA. During the progression of the degeneration in the obstructive lesion, the CA staining decreased dependent on acinar atrophy. Even after longstanding obstruction of the salivary gland, altered ductal epithelia may retain some of their functions.

Histochemical studies of obstructive adenitis in human submandibular salivary glands. I. Immunohistochemical demonstration of lactoferrin, lysozyme and carcinoembryonic antigen.

Immunohistochemical detection of lactoferrin (LF), lysozyme (LZ) and carcinoembryonic antigen (CEA) was made in obstructive adenitis of the submandibular glands. Atrophic and altered acinar cells in the early stage of the lesion stained strongly for LF, whereas they were unreactive or stained slightly for LZ. Ductal cells usually stained for LZ. Staining for CEA was strong and irregularly distributed in altered acinar cells. Duct-like structures and dilated ductal segments in the chronic stage were generally negative for LF, LZ and CEA. Secretory components in luminal cavities gave abundant staining for LF, LZ and CEA. Histocytes which infiltrated into the connective tissue in the later stage showed a positive LZ reaction.

[Phosphohexose isomerase activity in parotid and submandibular saliva as a parameter in the diagnosis of chronic sialadenitis]. / Phosphohexoseisomerase-Aktivität im Parotis- und Submandibularisspeichel als Parameter in der Diagnostik chronischer Sialadenitiden.

The phosphohexose isomerase (PHI) activity was measured in parotid and submandibular saliva of patients suffering from chronic sialadenitis and of healthy control persons. In control persons the mean PHI value determined in parotid saliva (10.93 +/- 1.17 U/l) was significantly lower compared to that measured in submandibular saliva (38.18 +/- 4.44 U/l). In chronic sialadenitis the salivary PHI activity was significantly increased and reached values up to 5440 U/l in parotid secretion and values up to 1620 U/l in submandibular secretion. The possible role of salivary PHI activity as a parameter for the diagnosis of chronic sialadenitis is discussed.