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1.  B cells in Sjögren's syndrome: indications for disturbed selection and differentiation in ectopic lymphoid tissue 
Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathological features. A hallmark of pSS is B-cell hyperactivity as manifested by the production of autoantibodies, hypergammaglobulinemia, formation of ectopic lymphoid structures within the inflamed tissues, and enhanced risk of B-cell lymphoma. Changes in the distribution of peripheral B-cell subsets and differences in post-recombination processes of immunoglobulin variable region (IgV) gene usage are also characteristic features of pSS. Comparison of B cells from the peripheral blood and salivary glands of patients with pSS with regard to their expression of the chemokine receptors CXCR4 and CXCR5, and their migratory capacity towards the corresponding ligands, CXCL12 and CXCL13, provide a mechanism for the prominent accumulation of CXCR4+CXCR5+ memory B cells in the inflamed glands. Glandular B cells expressing distinct features of IgV light and heavy chain rearrangements, (re)circulating B cells with increased mutations of cμ transcripts in both CD27- and CD27+ memory B-cell subsets, and enhanced frequencies of individual peripheral B cells containing IgV heavy chain transcripts of multiple isotypes indicate disordered selection and incomplete differentiation processes of B cells in the inflamed tissues in pSS. This may possibly be related to a lack of appropriate censoring mechanisms or different B-cell activation pathways within the ectopic lymphoid structures of the inflamed tissues. These findings add to our understanding of the pathogenesis of this autoimmune inflammatory disorder and may result in new therapeutic approaches.
PMCID: PMC2206371  PMID: 17697366
2.  Resistance to Rituximab Therapy and Local BAFF Overexpression in Sjögren’s Syndrome-Related Myoepithelial Sialadenitis and Low-Grade Parotid B-Cell Lymphoma 
B-cell expansion is a key feature of Sjögren’s syndrome (SS). Accordingly, several studies have reported the benefits of B-cell depletion with anti-CD20 monoclonal antibody (Rituximab) in the treatment of glandular and extraglandular manifestations of SS. Patients with SS are at increased risk of lymphoma development. B-lymphocyte stimulator (BAFF) is an essential cytokine for the control of B-cell maturation and survival, and high levels of BAFF were described in the serum and salivary glands of SS patients, strongly suggesting a crucial role in the proliferation of B cells in SS.
Patient and Methods
We describe the treatments employed, with particular regards to rituximab therapy, and the histopathologic and biologic studies, in particular BAFF levels in serum and in pathologic tissues before and after B-cell depletion therapy, and the characterization of the cultured epithelial cells obtained by the parotid gland MALT-lymphoma, in a case of a 51-year old woman with primary SS and mixed cryoglobulinaemia type II with features of systemic vasculitis, who developed a bilateral parotid MALT-type lymphoma. Rheumatoid factor (RF), cryoglobulins, BAFF levels were assessed monthly up to month +6, then at the end of follow-up (month +12), as well as peripheral blood CD19-positive B-cell level
A significant systemic effect of rituximab on B-cell biomarkers was documented, however, the cryoglobulinemic syndrome did not improve and the parotid enlargement did not decrease confirming the failure of B-cell depletion to affect the parotid lymphoma. BAFF levels decreased only under B-cell depletion associated with high-dose steroids. Tissue studies further documented the persistent overexpression of BAFF in the salivary gland pathologic tissue during the disease course.
Tissue and systemic overexpression of BAFF may have contributed to resistance to rituximab therapy, in MALT lymphoproliferation associated with SS. Thus, alternative treatment strategies should be then considered, possibly including BAFF-targeted approaches.
PMCID: PMC2577948  PMID: 19088870
3.  Bilateral multiple sialolithiasis of the parotid gland in a patient with Sjögren’s syndrome 
The presence of multiple calculi in the major salivary glands is an uncommon finding. Sjögren’s syndrome is a chronic autoimmune disease characterized by lymphocyte-mediated destruction of the exocrine glands. The case is presented of a 49-year-old female with Sjögren’s syndrome found to have bilateral multiple sialolithiasis in the parenchyma of the parotid glands. The patient presented with a right sided painful inflamed swelling of the parotid region. Even though she had been diagnosed with primary Sjögren’s syndrome 3 years prior to admission, she did not report any previous episode of sialadenitis. Full blood count showed leukocytosis (white blood cells = 14,900/106L) with neutrophilia (75%). Radiological assessment included ultrasound and computed tomography scan of the parotids which demonstrated intra-parenchymal multiple calculi of both parotid glands and obstruction of the right Stensen’s duct. The patient was treated with intravenous antibiotics and anti-inflammatory drugs. On the second day of hospitalisation, she reported spontaneous extrusion of a calculus during massage of the gland, with immediate relief of symptoms. In patients with Sjögren’s syndrome and radiological findings of calculi in the major salivary glands, close observation is mandatory for better control of recurrent sialadenitis and early recognition of mucosa-associated lymphoid tissue lymphomas.
PMCID: PMC2640017  PMID: 17601211
Parotid gland; Sialolithiasis; Sjögren’s syndrome
4.  Retrovirus in salivary glands from patients with Sjögren's syndrome. 
Journal of Clinical Pathology  1997;50(3):223-230.
AIMS: To investigate the possibility of an immune response to retroviral antigens or of detecting retrovirus in Sjögren's syndrome. METHODS: Retroviruses were sought in labial salivary glands and peripheral blood mononuclear cells from patients with Sjögren's syndrome by immunoblotting assay, immunohistochemical assay, polymerase chain reaction (PCR), reverse transcriptase (RT) activity assay, and transmission electron microscopy. RESULTS: Sera from five of 15 patients with Sjögren's syndrome (33%) reacted against p24 group specific antigen (gag) of human immunodeficiency virus (HIV). Labial salivary gland biopsy specimens from seven of the 15 patients with Sjögren's syndrome (47%) contained an epithelial cytoplasmic protein reactive with a monoclonal antibody to p24 of HIV. PCR was performed to detect HIV and human T lymphotropic virus type I (HTLV-I) genes from salivary gland tissues and peripheral blood mononuclear cells from patients with Sjögren's syndrome. Mn2+ dependent, Mg2+ independent RT activity was detected in the salivary gland tissues in three of 10 patients. A-type-like retroviral particles were observed in epithelial cells of salivary glands by transmission electron microscopy. Target genes for HIV and HTLV-I were not found in any of the salivary gland tissues or peripheral blood mononuclear cells from Sjögren's syndrome patients. CONCLUSIONS: The data suggest the presence of an unknown retrovirus similar to HIV in the salivary gland which might be involved in the pathogenesis of a subpopulation in Sjögren's syndrome.
PMCID: PMC499817  PMID: 9155673
5.  Human Parathyroid Hormone Is Secreted Primarily into the Bloodstream After Rat Parotid Gland Gene Transfer 
Human Gene Therapy  2010;22(1):84-92.
Hypoparathyroidism is a hormone deficiency syndrome that leads to low blood calcium levels and for which current replacement therapy is inadequate. Gene transfer to salivary glands leads to safe and abundant secretion of therapeutic protein into either saliva or the bloodstream. We previously reported the successful transduction of rat submandibular glands with an adenoviral vector encoding human parathyroid hormone (Ad.hPTH), but unfortunately most of the hPTH was secreted into saliva. Because submandibular and parotid glands are morphologically and functionally different, we hypothesized that hPTH sorting might be different in parotid glands. After 2 days, the pattern of hPTH secretion from transduced parotid glands of intact rats was reversed from that of transduced submandibular glands, that is, most transgenic hPTH was detected in serum (5 × 1010 viral particles per gland; the saliva-to-serum ratio of total hPTH secreted was 0.04). Vector copies were localized to the targeted parotid glands, with none detected in liver or spleen. Ad.hPTH next was administered to parotid glands of parathyroidectomized rats. Two days after delivery no hPTH was detectable in saliva, but high levels were found in serum, leading to normalization of serum calcium and a significant increase in the urinary phosphorus-to-creatinine ratio. This study demonstrates for the first time differential sorting of transgenic hPTH between submandibular and parotid glands, suggesting that hPTH may be a valuable model protein for understanding the molecular basis of transgenic secretory protein sorting in these exocrine glands. We also show the clinical potential of salivary gland hPTH gene therapy for patients with hypoparathyroidism.
Physiologically sufficient levels of calcium in the blood are important for a range of vital functions and hypocalcemia can lead to seizures, tetany, or heart failure. Parathyroid hormone (PTH) is central to maintaining adequate blood calcium concentration. In this study, Adriaansen et al. demonstrate that delivery of an adenoviral vector encoding human PTH to the parotid gland of hypocalcemic rats leads to a normalization of serum calcium levels.
PMCID: PMC3025188  PMID: 20977345
6.  Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjögren's syndrome. 
Journal of Clinical Investigation  1998;102(5):938-946.
Structures resembling germinal centers are seen in the salivary glands of patients with Sjögren's syndrome, but it is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal center response. Therefore, we cloned and sequenced rearranged Ig V genes expressed by B cells isolated from sections of labial salivary gland biopsies from two Sjögren's syndrome patients. Rearranged V genes from B cells within one cell cluster were polyclonal and most had few somatic mutations. Two adjacent clusters from another patient each contained one dominant B cell clone expressing hypermutated V genes. None of the rearranged V genes was found in both clusters, suggesting that cells are unable to migrate out into the surrounding tissue and seed new clusters. The ratios of replacement to silent mutations in the framework and complementarity determining regions suggest antigen selection of high-affinity mutants. These results show that an antigen-driven, germinal center-type B cell response is taking place within the salivary glands of Sjögren's syndrome patients. In view of the recent demonstration of a germinal center response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that germinal center- type responses can be induced in the nonlymphoid target tissues of a variety of autoimmune diseases.
PMCID: PMC508959  PMID: 9727062
7.  Abnormalities of B cell phenotype, immunoglobulin gene expression and the emergence of autoimmunity in Sjögren's syndrome 
Arthritis Research  2002;4(6):360-371.
Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathologic features and the production of typical autoantibodies. In addition, characteristic changes in the distribution of peripheral B cell subsets and differences in use of immunoglobulin variable-region genes are also features of pSS. Comparison of B cells from the blood and parotid gland of patients with pSS with those of normal donors suggests that there is a depletion of memory B cells from the peripheral blood and an accumulation or retention of these antigen-experienced B cells in the parotids. Because disordered selection leads to considerable differences in the B cell repertoire in these patients, the delineation of its nature should provide important further clues to the pathogenesis of this autoimmune inflammatory disorder.
PMCID: PMC153845  PMID: 12453312
autoimmunity; B cells; IgV gene usage; lymphocytes; Sjögren's syndrome
8.  Primary T cell lymphoma of salivary gland: a report of a case and review of the literature 
Journal of Clinical Pathology  2002;55(1):61-63.
Lymphoma of the salivary gland accounts for 5% of cases of extranodal lymphoma and 10% of malignant salivary gland tumours. Most primary salivary gland lymphomas are B marginal zone lymphomas arising on a background of sialadenitis associated with autoimmune disorders such as Sjorgen's syndrome. Primary T cell lymphoma of the salivary gland is rare. This report describes a case of primary T cell lymphoma arising in the parotid gland of an elderly white man, which was notable for its striking resemblance to a B cell extranodal marginal zone lymphoma. Immunohistochemistry and gene rearrangement studies confirmed the clonal T cell nature of the tumour. There was no molecular evidence of Epstein-Barr virus (EBV) infection of neoplastic or surroundings cells. Only 14 cases of primary T cell lymphoma of the salivary glands have been recorded in the literature, most being from the Orient and having extremely variable prognosis. Those with a T/natural killer cell phenotype are associated with EBV infection. This case highlights the fact that T cell lymphoma in the salivary gland can mimic closely the morphological features of B cell extranodal marginal zone lymphoma.
PMCID: PMC1769572  PMID: 11825927
salivary gland; T cell lymphoma; Epstein Barr virus
9.  Sialometry and sialochemistry: a non-invasive approach for diagnosing Sjögren's syndrome 
Annals of the Rheumatic Diseases  2002;61(2):137-144.
Background: Analysis of salivary variables has frequently been proposed as a diagnostic tool for Sjögren's syndrome (SS). Because univocal salivary reference values are lacking, it is currently rather difficult to use sialometry and sialochemistry for diagnosing SS unless major changes have occurred in salivary secretion and composition.
Objective: To define reference values of several salivary variables, which offer a possible new and non-invasive means of diagnosing SS.
Methods: Cut off points were selected from receiver operating characteristic curves of gland-specific sialometrical and sialochemical variables, which have proved to be potentially relevant for diagnosing SS in a previous study—that is, sodium, chloride, and phosphate concentration in stimulated parotid and submandibular/sublingual (SM/SL) saliva, unstimulated and stimulated SM/SL flow rates, and lag phase of parotid secretion, respectively. By combining the most discriminating variables, two different diagnostic approaches for SS were applied in a group of 100 patients and subsequently evaluated in a second group of 20 patients. The first approach was to combine variables by applying their cut off points into sets of criteria for a positive diagnosis of SS. The second approach was to construct a logistic regression model that predicts the true state of a patient (SS or non-SS). From both approaches, the tests with highest likelihood ratio combined with the smallest number of rejected cases were selected for clinical use.
Results: The most accurate test combined the stimulated SM/SL flow rate and parotid sodium and chloride concentration as salivary variables for diagnosing SS; it had a sensitivity of 0.85 and a specificity of 0.96. The selected tests proved equally accurate in the second group of patients.
Conclusions: Because the proposed non-invasive diagnostic tools can be easily applied, do not need a laboratory other than for routine blood testing, and are very accurate, gland-specific sialometry and sialochemistry may eventually replace other, more invasive, diagnostic techniques for diagnosing SS.
PMCID: PMC1754002  PMID: 11796400
10.  Progression of salivary gland dysfunction in patients with Sjögren's syndrome 
Annals of the Rheumatic Diseases  2006;66(1):107-112.
Salivary gland dysfunction is one of the key manifestations of Sjögren's syndrome.
(1) To assess prospectively loss of function of individual salivary glands in patients with primary and secondary Sjögren's syndrome in relation to disease duration and use of immunomodulatory drugs. (2) To study changes in sialochemical and laboratory values and subjective complaints over time.
60 patients with Sjögren's syndrome were included in this study. Whole and gland‐specific saliva (parotid and submandibular/sublingual (SM/SL)), samples were collected at baseline and after a mean of 3.6 (SD 2.3) years of follow‐up. Disease duration was recorded for all patients.
Patients with Sjögren's syndrome with short disease duration had significantly higher stimulated flow rates at baseline than those with longer disease duration (p<0.05). When compared with healthy controls, the decrease in SM/SL flow rates at baseline was more prominent than that in parotid flow rates (p<0.05). Over time, there was a significant further decrease of stimulated flow rates, especially of the parotid gland, accompanied by increasing problems with swallowing dry food (p<0.05). The decrease was independent of the use of corticosteroids or disease‐modifying antirheumatic drugs (DMARDs). Sialochemical variables remained stable.
Early Sjögren's syndrome is characterised by a decreased salivary gland function (parotis>SM/SL), which shows a further decrease over time, regardless of the use of DMARDs or steroids. Patients with Sjögren's syndrome with longer disease duration are characterised by severely reduced secretions of both the parotid and SM/SL glands. These observations are relevant for identifying patients who would most likely benefit from intervention treatment.
PMCID: PMC1798390  PMID: 16728458
11.  Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland 
Thyroid  2013;23(8):1029-1036.
Physiologic iodide-uptake, mediated by the sodium/iodide symporter (NIS), in the salivary gland confers its susceptibility to radioactive iodine–induced damage following 131I treatment of thyroid cancer. Subsequent quality of life for thyroid cancer survivors can be decreased due to recurrent sialoadenitis and persistent xerostomia. NIS expression at the three principal salivary duct components in various pathological conditions was examined to better our understanding of NIS modulation in the salivary gland.
NIS expression was evaluated by immunohistochemistry in human salivary gland tissue microarrays constructed of normal, inflamed, and neoplastic salivary tissue cores. Cumulative 123I radioactivity reflecting the combination of NIS activity with clearance of saliva secretion in submandibular and parotid salivary glands was evaluated by single-photon emission computed tomography/computed tomography imaging 24 hours after 123I administration in 50 thyroid cancer patients.
NIS is highly expressed in the basolateral membranes of the majority of striated ducts, yet weakly expressed in few intercalated and excretory duct cells. The ratio of 123I accumulation between parotid and submandibular glands is 2.38±0.19. However, the corresponding ratio of 123I accumulation normalized by volume of interest is 1.19±0.06. The percentage of NIS-positive striated duct cells in submandibular salivary glands was statistically greater than in parotid salivary glands, suggesting a higher clearance rate of saliva secretion in submandibular salivary glands. NIS expression in striated ducts was heterogeneously decreased or absent in sialoadenitis. Most ductal salivary gland tumors did not express NIS. However, Warthin's tumors of striated duct origin exhibited consistent and intense NIS staining, corresponding with radioactive iodine uptake.
NIS expression is tightly modulated during the transition of intercalated to striated ducts and striated to excretory ducts in salivary ductal cells. NIS expression in salivary glands is decreased during inflammation and tumor formation. Further investigation may identify molecular targets and/or pharmacologic agents that allow selective inhibition of NIS expression/activity in salivary glands during radioactive iodine treatment.
PMCID: PMC3752512  PMID: 23441638
12.  Salivary gland expression of transforming growth factor β isoforms in Sjogren’s syndrome and benign lymphoepithelial lesions 
Molecular Pathology  2003;56(1):52-59.
Aim: Transforming growth factor β (TGF-β) is involved in the control of autoimmune reactions, cell proliferation, and the accumulation of lymphocytes within organs. The aim of this study was to determine the expression of TGF-β in salivary glands from patients with primary Sjogren’s syndrome (SS) and benign lymphoepithelial lesions (BLEL) with emphasis on ductal epithelium.
Methods: Immunoperoxidase staining for TGF-β isoforms and Ki67 antigen was performed on formalin fixed sections of labial glands from patients with primary SS (n = 15) and controls (n = 5) and parotid glands reported as BLEL (n = 5) or normal (n = 5). Ductal expression of TGF-β was quantified by absorbance measurements using image analysis. The specificity of staining was confirmed by peptide blocking studies.
Results: All TGF-β isoforms were detected within the cytoplasm of most lymphocytes, endothelial cells, and ducts in all specimens. Acinar expression was variable and weaker than that seen in ducts. Absorbance measurements revealed that the expression of all isoforms was greater in ducts within primary SS glands than in control glands. Ductal expression in control parotid glands was greater than that seen in BLEL glands, irrespective of the presence of adjacent lymphoid infiltrates. Comparisons between control specimens showed that ductal expression of all isoforms was highest in parotid glands, whereas no differences were detected between primary SS and BLEL glands. Ki67 positive lymphocytes and duct cells were mainly restricted to pathological specimens, with BLEL glands containing larger populations of positive cells than primary SS glands.
Conclusion: These results demonstrate complex and variable changes in ductal expression of TGF-β in primary SS and BLEL, which may be important in the control of lymphoid infiltration and the proliferation of lymphocytes and ductal epithelium.
PMCID: PMC1187290  PMID: 12560464
Sjogren’s syndrome; benign lymphoepithelial lesion; transforming growth factor β isoforms; salivary glands
13.  Parotid gland-recovery after radiotherapy in the head and neck region - 36 months follow-up of a prospective clinical study 
The aim of the present study was to evaluate the recovery potential of the parotid glands after using either 3D-conformal-radiotherapy (3D-CRT) or intensity-modulated radiotherapy (IMRT) by sparing one single parotid gland.
Between 06/2002 and 10/2008, 117 patients with head and neck cancer were included in this prospective, non-randomised clinical study. All patients were treated with curative intent. Salivary gland function was assessed by measuring stimulated salivary flow at the beginning, during and at the end of radiotherapy as well as 1, 6, 12, 24, and 36 months after treatment. Measurements were converted to flow rates and normalized relative to rates before treatment. Mean doses (Dmean) were calculated from dose-volume histograms based on computed tomographies of the parotid glands.
Patients were grouped according to the Dmean of the spared parotid gland having the lowest radiation exposure: Group I - Dmean < 26 Gy (n = 36), group II - Dmean 26-40 Gy (n = 45), and group III - Dmean > 40 Gy (n = 36). 15/117 (13%) patients received IMRT. By using IMRT as compared to 3D-CRT the Dmean of the spared parotid gland could be significantly reduced (Dmean IMRT vs. 3D-CRT: 21.7 vs. 34.4 Gy, p < 0.001). The relative salivary flow rates (RFSR) as a function of the mean parotid dose after 24 and 36 months was in group I 66% and 74%, in group II 56% and 49%, and in group III 31% and 24%, respectively. Multiple linear regression analyses revealed that the parotid gland dose and the tumor site were the independent determinants 12 and 36 months after the end of RT. Patients of group I and II parotid gland function did recover at 12, 24, and 36 months after the end of RT.
If a Dmean < 26 Gy for at least one parotid gland can be achieved then this is sufficient to reach complete recovery of pre-RT salivary flow rates. The radiation volume which depends on tumor site did significantly impact on the Dmean of the parotids, and thus on the saliva flow and recovery of parotid gland.
PMCID: PMC3201902  PMID: 21951317
head and neck cancer; irradiation; saliva; hyposalivation; parotid gland sparing; recovery
Cats injected into the parotid gland and testicle with a bacterial sterile filtrate of the salivary secretion of children in the active stage of parotitis or mumps can be made to develop a pathological condition having several points of resemblance to the condition present in mumps in human beings. After an incubation stage of from five to eight days definite changes have been noted in the temperature, blood leukocytes, and inoculated organs. The temperature rise begins within twenty-four hours of the inoculations and reaches a maximum in from seven to fourteen days. The febrile rise fluctuates between 1° and 2.5° C. The white blood cells begin to increase on the second day following the inoculation. The first change is a polymorphonuclear leukocytosis which precedes the glandular swellings. This initial rise is followed by a decline, after which the lymphocytes increase. The increase is confined to the small lymphocytes, which increase to from 7 to 10 per cent of their initial number. The inoculated glands become swollen and tender. The swelling and tenderness become apparent from the fifth to the ninth days and persist for a variable period. The parotid changes are less constant or less obvious than are the testicular. The latter are constant and endure from eight to twelve days. The rise of temperature and the leukocytosis precede the glandular swelling, but all the changes reach the maximum at about the same time, after which they decline gradually. What may be regarded as normal conditions are reestablished in four weeks or less. The intraparotid and intratesticular injections of extracts of normal parotid gland and testicles may cause a mild rise of temperature and leukocytosis of brief duration, but swelling and tenderness are absent. The white cells increased are the polymorphonuclears and not the lymphocytes. The intraparotid and intratesticular injections of filtrates of normal saliva may cause a mild rise of temperature of very brief duration, but leukocytosis, swelling, and tenderness do not appear. The histological changes in the parotid gland when present consist chiefly of edema of the interlobular connective tissue with mononuclear interstitial infiltration about the ducts and elsewhere. In cases of long duration the ducts may be dilated. But in some instances the swollen gland while showing congestion and edema in gross showed inconspicuous changes under the microscope. The histological changes in the testicle are of two kinds: inconstant changes of cellular invasion between the tubules and swelling or even multiplication of the interstitial cells, constant ones consisting of degeneration of the epithelium and interference with spermatogenesis, a condition to which we have applied the term "spermatorrhexis." The pathological conditions set up by the filtrate derived from the salivary secretion of cases of acute parotitis are intensified by successive transfers through a small series of cats of the extract and emulsion of the parotid gland and testicle previously inoculated. The pathological changes are also prevented or reduced when the extract or emulsion is previously incubated with a quantity of blood serum obtained from a cat which has survived inoculation. Normal serum, on the other hand, has no such inhibiting effect. The deduction from these experiments is to the effect that the salivary secretion in parotitis or mumps contains a filterable substance capable of setting up a series of definite pathological conditions when inoculated into the testicle and parotid glands of cats. Whether this active material is a microorganism and if so whether it is the specific microbic cause of parotitis or mumps remains to be ascertained.
PMCID: PMC2125420  PMID: 19867993
15.  Secretory immunity with special reference to the oral cavity 
Journal of Oral Microbiology  2013;5:10.3402/jom.v5i0.20401.
The two principal antibody classes present in saliva are secretory IgA (SIgA) and IgG; the former is produced as dimeric IgA by local plasma cells (PCs) in the stroma of salivary glands and is transported through secretory epithelia by the polymeric Ig receptor (pIgR), also named membrane secretory component (SC). Most IgG in saliva is derived from the blood circulation by passive leakage mainly via gingival crevicular epithelium, although some may be locally produced in the gingiva or salivary glands. Gut-associated lymphoid tissue (GALT) and nasopharynx-associated lymphoid tissue (NALT) do not contribute equally to the pool of memory/effector B cells differentiating to mucosal PCs throughout the body. Thus, enteric immunostimulation may not be the best way to activate the production of salivary IgA antibodies although the level of specific SIgA in saliva may still reflect an intestinal immune response after enteric immunization. It remains unknown whether the IgA response in submandibular/sublingual glands is better related to B-cell induction in GALT than the parotid response. Such disparity is suggested by the levels of IgA in submandibular secretions of AIDS patients, paralleling their highly upregulated intestinal IgA system, while the parotid IgA level is decreased. Parotid SIgA could more consistently be linked to immune induction in palatine tonsils/adenoids (human NALT) and cervical lymph nodes, as supported by the homing molecule profile observed after immune induction at these sites. Several other variables influence the levels of antibodies in salivary secretions. These include difficulties with reproducibility and standardization of immunoassays, the impact of flow rate, acute or chronic stress, protein loss during sample handling, and uncontrolled admixture of serum-derived IgG and monomeric IgA. Despite these problems, saliva is an easily accessible biological fluid with interesting scientific and clinical potentials.
PMCID: PMC3595421  PMID: 23487566
IgA; IgG; mucosa-associated lymphoid tissue (MALT); gut-associated lymphoid tissue (GALT); nasopharynx-associated lymphoid tissue (NALT); salivary glands; crevicular fluid; polymeric Ig receptor (pIgR); secretory component (SC); mucosal vaccination
16.  Characterization of the primary structure of T cell receptor beta chains in cells infiltrating the salivary gland in the sicca syndrome of HIV-1 infection. Evidence of antigen-driven clonal selection suggested by restricted combinations of V beta J beta gene segment usage and shared somatically encoded amino acid residues. 
Journal of Clinical Investigation  1993;92(1):495-502.
Infection with HIV-1 occasionally results in a sicca syndrome, termed the diffuse infiltrative lymphocytosis syndrome, characterized by infiltration of the salivary glands with a predominance of CD8 T cells. This response is strongly associated with certain MHC class I and class II alleles. To define the salivary gland T cell receptor (TCR) repertoire, the primary structure of the TCR beta-chains was determined using in situ cDNA synthesis followed by the "anchored" polymerase chain reaction. The sequences of 59 beta-chains from five individuals with diffuse infiltrative lymphocytosis syndrome shared structural features suggesting antigenic clonal selection. Certain combinations of V beta J beta gene segments were selectively overrepresented in the repertoire sample, demonstrating a common restricted usage of certain V beta and J beta gene segments. The beta-chains derived from these overrepresented V beta J beta combinations revealed a preference for specific amino acids at position 97 in the third complementarity-determining region, a residue postulated to contact peptide antigen. Moreover, the nucleotides encoding this position were not germline in origin. TCR beta-chains in nonoverrepresented V beta J beta combinations did not exhibit preferential usage of selected somatically encoded residues. The pattern of TCR beta-chains expressed in the salivary gland of a control person with primary Sjögren's syndrome was considerably more heterogeneous and different from that found in diffuse infiltrative lymphocytosis syndrome.
PMCID: PMC293637  PMID: 8392093
17.  B-cell tolerance defects in the B6.Aec1/2 mouse model of Sjögren’s syndrome 
Journal of Clinical Immunology  2012;32(3):551-564.
Primary Sjögren’s syndrome (SjS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, B-cell clonal expansions and an increased risk of lymphoma. In order to understand the role of B cells in this disorder, the antibody repertoire and B-cell maturation were studied in a mouse model of SjS called B6.Aec1/2.
B6.Aec1/2 serum was analyzed for (auto)antibodies by ELISA and immunoprecipitation, B-cell development by flow cytometry, antibody gene rearrangements by CDR3 spectratyping and quantitative PCR. In order to test the functional consequences of the observed defects, B6.Aec1/2 mice were crossed with anti-dsDNA antibody heavy chain knock-in mice (B6.56R).
B6.Aec1/2 mice exhibit B-cell clonal expansions, have altered serum immunoglobulin levels and spontaneously produce multireactive autoantibodies. B6.Aec1/2 mice also have decreased numbers of bone marrow pre-B cells and decreased frequencies of kappa light chain gene deletion. These findings suggest that B6.Aec1/2 mice have a defective early B-cell tolerance checkpoint. B6.56R.Aec1/2 mice unexpectedly had lower anti-dsDNA antibody levels than B6.56R mice and less salivary gland infiltration than B6.Aec1/2 mice.
These data suggest that the early tolerance checkpoint defect in B6.Aec1/2 mice is not sufficient to promulgate disease in mice with pre-formed autoantibodies, such as B6.56R. Rather, B6.Aec1/2 mice may require a diverse B-cell repertoire for efficient T-B-cell collaboration and disease propagation. These findings imply that therapies aimed at reducing B-cell diversity or T-B interactions may be helpful in treating SjS.
PMCID: PMC3351565  PMID: 22350147
Sjögren’s syndrome; B cell; autoantibody; receptor editing
18.  Neutral endopeptidase (EC in labial salivary glands in healthy controls and in patients with Sjögren's syndrome. 
Annals of the Rheumatic Diseases  1996;55(8):513-519.
OBJECTIVE: Neuropeptides from nerve fibres can cause neurogenic inflammation. The potency of these peptides in vitro has led to the hypothesis that enzyme degradative systems are operative in vivo to limit their action. To consider this question neutral endopeptidase (NEP) in labial salivary glands in patients with Sjögren's syndrome was studied. METHODS: Synthesis of NEP mRNA in situ in labial salivary glands was studied using the reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical staining was used to localise the NEP enzyme protein and its neuropeptide substrates and fluorophotometry to measure the corresponding enzyme activities in saliva. RESULTS: NEP was found in nerve fibres and in perivascular, periductal, and periacinar axon terminal varicosities. Double labelling of PGP 9.5 and NEP confirmed this neuronal localisation of NEP. Although some fibroblast-like cells and occasional intravascular neutrophils were NEP positive, NEP mRNA was not found in labial salivary glands. Patients with Sjögren's syndrome and healthy controls did not have nerves containing NEP or neuropeptides (vasoactive intestinal peptide, substance P, or calcitonin gene related peptide (CGRP)) in lymphocyte foci. Salivary NEP activity was not decreased in patients compared with controls. CONCLUSION: NEP in labial salivary glands is almost totally of neuronal origin and plays a part in proteolytic modulation of neuropeptides in salivary glands and saliva. These regulatory interactions seem to be altered in focal lymphocyte accumulations in Sjögren's syndrome.
PMCID: PMC1010230  PMID: 8774178
19.  New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach 
PLoS ONE  2013;8(8):e71874.
Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin's tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin's tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin's tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.
PMCID: PMC3796443  PMID: 24205396
20.  Mammary Analogue Secretory Carcinoma (MASC) of salivary gland in four Mexican patients 
The Clinco-pathological, immunohistochemical and molecular findings of four cases of Mammary Analogue Secretory Carcinoma (MASC) of salivary glands found in Mexico are described. The cases were extracted from 253 salivary gland tumors from a single institution in Mexico City. The 85 candidates for initial selection were: low grade mucoepidermoid carcinoma (MEC) (N=70 ), acinic cell cancinoma (AciCC) (N=14), papillary cystadenocarcinoma (N=1), and adenocarcinoma NOS (N=0). Tumors with some histological features consistent with MASC (N= 17, 6.7%) were studied by immunohistochemistry for mammaglobin, STAT5, and S-100 protein and four cases were positive (1.5%), thus the diagnosis of MASC was established, and these were submitted for molecular studies for ETV6-NTRK3. Fusion gene was demonstrated in three cases, two had been erroneously diagnosed as poorly granulated AciCC, and one as low grade MEC with microcystic pattern. Female gender predominated (3:1); one occurred in the parotid, two in minor salivary glands and one in the submaxillary gland; infiltrating borders, atypical mitosis and lymph node metastases were seen in the parotideal tumor. Two patients with major salivary gland tumors are alive and well at 10 and 20 months respectively, the two patients with minor salivary gland tumors are lost. It can be concluded that is important to think in MASC in poorly granulated AciCC and low grade MEC with microcystic pattern. Immunohistochemisty studies confirm the diagnosis, preferentially supported by molecular studies. MASC may follow aggressive behavior or transform into a high grade neoplasm.
Key words:Acinic cell carcinoma, ETV6-NTRK3, Mammary Analogue Secretory Carcinoma, secretory breast carcinoma.
PMCID: PMC4320417  PMID: 25481229
21.  Lymphoepithelial carcinoma of the salivary gland: in situ detection of Epstein-Barr virus. 
Journal of Clinical Pathology  1995;48(11):1022-1027.
AIM--To examine the role of Epstein-Barr virus (EBV) in lymphoepithelial carcinoma of the salivary gland in Hong Kong Chinese. METHODS--Ten cases of lymphoepithelial carcinoma of the salivary gland (eight parotid and two submandibular) were examined. In situ hybridisation was used to localise EBER RNA, immunohistochemical methods to detect expression of latent membrane protein 1 (LMP-1) in EBV positive tumours, and Southern blot analysis to examine the clonality of EBV in the two cases where frozen tissue was available. RESULTS--None of the cases had a history of Sjögren's syndrome or histological evidence of a benign lymphoepithelial lesion. The IgA antibody titre against EBV viral capsid antigen was elevated in four cases. All cases were EBV positive by in situ hybridisation, with a strong uniform positive signal in the epithelial cells, and all cases expressed LMP-1. Southern blot analysis revealed that the clonal episomal form of the virus was present. Two of the three female patients in this series also developed carcinoma of cervix. One of these carcinomas had histological features of a lymphoepithelioma-like carcinoma but was EBV negative. CONCLUSIONS--A consistent association between EBV and lymphoepithelial carcinoma of the salivary gland was found. The presence of the virus in a clonal episomal form, and the expression of LMP-1 viral oncoprotein is further evidence of the role of EBV in the oncogenesis of this tumour.
PMCID: PMC503007  PMID: 8543624
22.  Matriptase Deletion Initiates a Sjögren’s Syndrome-Like Disease in Mice 
PLoS ONE  2014;9(2):e82852.
The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model.
Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers.
Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren’s syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren’s syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers.
Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland dysfunction in the absence of inflammation while systemic deletion can induce a primary Sjögren’s syndrome like phenotype with autoimmunity and loss of gland function.
PMCID: PMC3923742  PMID: 24551030
23.  Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage 
Journal of dental research  2005;84(6):500-509.
Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.
PMCID: PMC1939692  PMID: 15914585
secretory granules; regulated secretion; amylase; parotid; saliva
24.  Systems Biology Analysis of Sjögren’s Syndrome and MALT Lymphoma Development in Parotid Glands 
Arthritis and rheumatism  2009;60(1):81-92.
To identify key target genes and activated signal pathways associated with the disease pathogenesis by conducting a systems analysis of parotid gland manifesting primary Sjögren’s syndrome (pSS) and pSS/mucosa-associated lymphoid tissue (pSS/MALT) lymphoma phenotypes.
A systems biologic approach was used to analyze parotid gland tissues obtained from non-pSS, pSS and pSS/MALT lymphoma patients. Concurrent expression microarray profiling and proteomic analysis were performed followed by weighted gene co-expression network analysis (WGCNA).
Gene co-expression modules related to pSS and pSS/MALT lymphoma are significantly enriched with genes known to be involved in immune/defense response, apoptosis, cell signaling, gene regulation, and oxidative stress. A detailed functional pathway analysis indicates that the pSS-associated modules are enriched with genes involved in proteasome degradation, apoptosis, signal peptides (MHC) class I, complement activation, cell growth and death, and integrin-mediated cell adhesion. The pSS/MALT-associated modules are enriched with genes involved in translation, ribosome, protease degradation, signal peptides (MHC) class I, G13 signaling pathway, complement activation, and Integrin-mediated cell adhesion. The combined analysis of gene expression and proteomics data implicates six highly connected hub genes for distinguishing pSS from non-pSS controls, and eight hub genes for distinguishing pSS/MALT lymphoma from pSS.
Systems biologic analysis of pSS and pSS/MALT parotid glands reveals pathways and molecular targets associated with the disease pathogenesis. The identified gene modules/pathways provide further insights into the molecular mechanisms of pSS and pSS/MALT lymphoma. The identified disease hub genes represent promising targets for therapeutic intervention, diagnosis, and prognosis.
PMCID: PMC2718690  PMID: 19116902
25.  Correspondence between salivary proteomic pattern and clinical course in primary Sjögren syndrome and non-Hodgkin's lymphoma: a case report 
In the last years human proteomic has represented a promising tool to promote the communication between basic and clinical science.
To explore the correspondence between salivary proteomic profile and clinical response, herein, we used a proteomic approach to analyse the whole saliva of a patient with primary Sjögren's Syndrome (pSS) and non-Hodgkin's-MALT type parotid lymphoma before, during and after a standard treatment with cyclophosphamide (CTX) and rituximab (RTX). To identify any discriminatory therapeutic salivary biomarker patient's whole saliva was collected at the baseline, after the fourth infusion of rituximab, and on remission and analysed combining two-dimensional electrophoresis (2DE) and MALDI-TOF/TOF mass spectrometry.
Proteomic results obtained from the comparison of salivary samples indicated several qualitative and quantitative modifications in the salivary expression of putative albumin, immunoglobulin J chain, Ig kappa chain C region, alpha-1-antitrypsin, haptoglobin and Ig alpha-1 chain C region.
This study suggests that clinical and functional changes of the salivary glands driven by autoimmune and lymphoproliferative processes might be reflected in patients' whole saliva proteins, shedding new light on the potential usefulness of salivary proteomic analysis in the identification of prognostic and therapeutic biomarkers for patients with pSS and non Hodgkin's lymphomas.
PMCID: PMC3223154  PMID: 22047044
primary Sjögren's Syndrome; B-cell non-Hodgkin's lymphoma; proteomic analysis

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