Whole saliva and the diagnosis of Sjögren's syndrome: an evaluation of patients who complain of dry mouth and dry eyes. Part 1: Screening tests

Sjögren's Syndrome (SS) is a common autoimmune disorder characterised by generalised desiccation, exocrine hypofunction and serologic abnormalities, More than 90% of the patients are women. Objective : to determine if whole saliva could be used to diagnose this disease. Setting: The study was conducted at the School of Dental Medicine, SUNY, at Stony Brook. Patients : There were 49 subjects (48F; 1M), the mean age was 54 ± 13 years. In order to be admitted into the study, they had to complain of dry mouth and dry eyes. Tests : Whole saliva was collected by the spitting method. “Screening Tests'” were employed to measure the salivary flow rate, pH, buffer capacity; lactobacillus and yeast concentrations. Chemical tests were performed to determine protein, albumin, sodium and amylase activity. Lacrimal dryness was assessed by the Schirmer and Rose-Bengal methods. Results: Based on the sialometric findings, the patients were divided into 3 groups: Group 1: those with abnormally low resting (RFR) and stimulated (SFR) flow rates; Group 2: those with a low RFR but normal SFR; and Group 3: those with normal salivary flow rates. The group 1 patients were unique: their saliva demonstrated a low pH and buffer capacity, high lactobacillus and yeast concentrations, decreased protein output and amylase activity, and elevated albumin and sodium. Moreover, virtually all of them had abnormally low lacrimal flow rates. Conclusions : The findings suggested that whole saliva could be used to provisionally diagnose SS. Critical to this diagnosis was an abnormally low stimulated whole saliva flow rate. Other requisites included a low resting flow rate, the presence of dry mouth and dry eyes and evidence of lacrimal hypofunction. All of these attributes can easily be obtained by dentists in their clinics.     

Salivary gland hypofunction in elderly patients attending a xerostomia clinic

Objectives: To investigate the aetiological factors and the prevalence of salivary gland hypofunction (SGH) in patients complaining of xerostomia. Design Prospective, clinical study. Setting Xerostomia clinic in the Department of Oral Medicine at Liverpool University Dental Hospital. Subjects 100 consecutive patients, aged 60 years or older, referred for investigation of xerostomia. Interventions Patients were asked specific questions concerning their complaint of oral dryness and associated orofacial symptoms. A detailed medical history was recorded and patients underwent a systematic examination of the head, neck and oral structures. All patients underwent haematological, biochemical, immunological investigations, urinalysis and sialometry. Further investigations and referrals to other specialists were undertaken when appropriate. Main outcome measures The causes of xerostomia were established on the basis of clinical and laboratory findings and SGH was defined as an unstimulated whole salivary flow rate of <0.2ml/min, Results: The causes of xerostomia were identified as: Sjögren's Syndrome (40), iatrogenic (22), psychogenic (14), idiopathic (19), diabetes (1), candidosis (3) and alcohol (1). Sixty five percent of the patients studied had SGH. Conclusions This study has shown that 65% of patients whose presenting complaint was xerostomia had objective evidence of SGH. Several aetiological factors were identified, the most common of which was Sjögren's Syndrome. The possibility of associated systemic diseases should be considered when establishing the aetiology of SGH.     

Whole saliva and the diagnosis of Sjögren's Syndrome: an evaluation of patients who complain of dry mouth and dry eyes. Part 2: Immunologic findings

Sjögren's Syndrome (SS) is a multisystem, autoimmune disease characterised by generalised desiccation, exocrine hypofunction and serologic abnormalities. Last year we showed that the antibodies which are quasi-specific for diagnosis of SS, anti SS-A/Ro and anti SS-B/La, were present in the saliva of patients with this disease. Objective : To determine their presence in patients who complain of dry mouth and dry eyes. Setting: The study was conducted at the School of Dental Medicine, SUNY at Stony Brook. Patients : There were 49 patients (Mean Age= 54 ± 13 years). Tests : Serum was analysed for the SS and other antibodies with “Western Blot Autoantibody Strips”. Results: The findings showed that there was a strong correlation between the presence of the SS antibodies in the serum and the saliva. The SS antibodies were primarily found in the saliva of the patients whose resting and stimulated, whole saliva, flow rates were abnormally low. Antibodies to other autoimmune disease (Lupus, Seleroderma and Mixed Connective Tissue Disease) were also found in whole saliva. Conclusions : The findings in Part 1 of this study, of patients who had complained of dry mouth and dry eyes, suggest that those patients, who demonstrated low resting and stimulated flow rates and had lacrimal hypofunction, suffered from SS. The observation in this paper, that the whole saliva of these patients contains the SS antibodies, confirms this diagnosis. The data suggest that whole saliva can be used to establish the diagnosis of SS and other autoimmune diseases.     

Proteomic analysis of saliva: a unique tool to distinguish primary Sjögren's syndrome from secondary Sjögren's syndrome and other sicca syndromes

Introduction

A growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS.

Methods

One hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network.

Results

A total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, α-amylases precursor, carbonic anhydrase VI, β-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities.

Conclusions

This study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.     

Issues in the epidemiological investigation of dry mouth

Despite decades of research, much remains unanswered about the epidemiology of dry mouth. This review aims to provide an overview of the condition's epidemiology and the issues to consider when planning an epidemiological study of dry mouth. The latter can be broadly grouped into: study design; sampling and statistical power considerations; the measurement of dry mouth; and the selection, nature and measurement of relevant exposure measures, including medications and potential confounding variables. Each of these is discussed, in order to provide guidance for prospective researchers based on experience with past research. Finally, an agenda for further epidemiological research into dry mouth is proposed.     

In vitro suppression of immune responses using monocyte-derived tolerogenic dendritic cells from patients with primary Sj?gren's syndrome

Introduction

Therapeutic vaccination with antigen-specific tolerogenic dendritic cells (tolDC) might become a future option of individualized therapy for patients with autoimmune diseases. In this study, we tested the possibility of generating monocyte-derived tolDC from patients with primary Sjögren''s syndrome (pSS). We analyzed phenotype, cytokine production and ability to suppress Ro/La-specific immune responses.

Methods

Monocyte-derived tolDC from patients with pSS were generated in the presence of dexamethasone, vitamin D3 and lipopolysaccharide (DexVD3 DC). The phenotype was analyzed by flow cytometry and the cytokine profile was investigated using a 25-plex Luminex assay and ELISA. The capacity to both stimulate Ro/La-specific T cells and suppress this response was evaluated by autologous mixed lymphocyte reaction (MLR).

Results

DC generated from patients with pSS had a similar phenotype and cytokine profile to those from healthy controls. DexVD3 DC from pSS patients induced little antigen-specific T cell proliferation, but DexVD3 DC-primed lymphocytes successfully suppressed Ro/La-specific T cell responses.

Conclusions

DexVD3 DC presenting Ro/La antigens might be a promising new therapeutic option for patients with pSS.     

Health-related quality of life in patients with primary Sjögren's syndrome and xerostomia: a comparative study

Objective: To compare the health status of groups of Primary Sjögren's and Xerostomia patients, using the Medical Outcomes Short Form 36 (SF‐36). The SF‐36 is a generic measure, divided into eight domains, used in the assessment of health‐related quality of life. Patients and methods : The SF‐36 was given to 2 groups: Group 1 comprised 43 patients diagnosed with Primary Sjögren's Syndrome (1SS) and an unstimulated whole salivary flow rate (UFR) of <0.1 ml/min). Group 2 (n = 40) reported Xerosiomia but had an UFR >0.2 ml/min. Sub groups of patients in Groups 1 and 2 were compared with community normative data, for the SF‐36 Results: There were trends to suggest lower SF36 scores for 1SS patients but there were no significant differences between the mean domain scores of Groups 1 and 2. 1SS and Xerostomia patients registered lower mean scores across all 8 domains, compared with normative community data. Conclusion: The SF‐36 was unable to detect significant differences between subjects with 1SS and Xerostomia but a larger sample size is required to confirm these findings. The results of this limited study suggest that a disease‐specific measure is required to assess the impact 1SS on health‐related Quality of life (QOL).     

Masticatory function in patients with xerostomia

The effects of reduced salivary output in patients suffering from xerostomia on masticatory function has not been previously studied. This study compares masticatory performance and kinematic activity of patients suffering from xerostomia with age-, sex-, and number of occluding pairs-matched healthy controls. Masticatory function was evaluated by assessment of chewing motion and muscle activity during chewing an artificial food (CutterSil®), chewing gum and swallowing a bolus of almond. Chewing motion was recorded with the Optotrak® computer system. Bilateral muscle activity of both masseter and anterior temporalis was recorded using surface electrodes. Results of this study revealed significant differences between patients and controls in their ability to process food and masticatory muscle activity. The majority of patients could not break down the artificial food, others had a larger median particle size than the controls. A significant difference was also observed in the number of chewing cycles required to swallow almonds, the patients required more than twice as many chews as the controls, P<0.001. The right masseter muscle displayed significantly less activity for the patient than the controls. These findings suggest that patients with xerostomia exhibit reduced ability to process food. The observed decline in masticatory performance is probably due to reduced activity of the muscles of mastication.     

Medication in elderly people: its influence on salivary pattern,signs and symptoms of dry mouth

doi:10.1111/j.1741‐2358.2009.00293.x
Medication in elderly people: its influence on salivary pattern, signs and symptoms of dry mouth Objective: To compare stimulated and non‐stimulated salivary flow, pH, buffering capacity and presence of signs and symptoms of hyposialie and xerostomia in elderly patients, with senile dementia using medication and healthy elderly subjects not using medication. Methods: Forty individuals (mean age: 68.5 years) were divided into two groups, according to the use (G1) or non‐use (G2) of medication and the presence (G1) or absence (G2) of senile dementia. Data with reference to the general health condition, use of medication and the patient’s complaints were collected during anamnesis. Clinical examination identified signs associated with hyposialie and xerostomia. Stimulated and non‐stimulated saliva flow, pH and buffering capacity were verified. Results: The stimulated saliva flow in both groups was below normal parameters. The drugs used by individuals in G1 showed xerostomic potential. Individuals with a higher consumption of xerostomic medication presented with dry and cracked lips. A significant negative relationship was found between drugs consumption and the buffering capacity (p < 0.001), and the resting saliva flow rate (p = 0.002). Conclusion: The use of medication increases the chance that an elderly person may present signs related to xerostomia and alterations in stimulated saliva flow and buffering capacity.     

Local delivery of AAV2-CTLA4IgG decreases sialadenitis and improves gland function in the C57BL/6.NOD-Aec1Aec2 mouse model of Sjögren's syndrome

Introduction

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a key negative costimulatory molecule that displays a wide range of anti-inflammatory properties and is currently approved to treat rheumatoid arthritis as a recombinant fusion protein (CTLA4IgG). To better understand the role of CTLA4IgG in primary Sjögren's syndrome (pSS), we generated a recombinant adeno-associated virus vector serotype 2 (AAV2) expressing a chimera of mouse CTLA-4 fused with a human immunoglobulin (AAV2-CTLA4IgG) and observed the effect of this molecule in C57BL/6.NOD-Aec1Aec2 mice, an animal model of pSS.

Methods

A recombinant adeno-associated virus-2 (AAV-2) vector was constructed encoding a CTLA4IgG fusion protein. The AAV2-CTLA4IgG vector and an AAV2 control vector encoding beta galactosidase (LacZ) were administered by retrograde cannulation of the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice. Protein expression was measured by ELISA and salivary glands were assessed for inflammation and activity.

Results

Recombinant CTLA4IgG blocked B7 expression on macrophages in vitro. In vivo, localized expression of CTLA4IgG in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice inhibited the loss of salivary gland activity and decreased T and B cell infiltration as well as dendritic cells and macrophages in the glands compared with control mice. In addition a decrease in several proinflammatory cytokines and an increase in transforming growth factor beta-1 (TGF-β1) expression were also observed.

Conclusions

These data suggest expression of CTLA4IgG in the salivary gland can decrease the inflammation and improve the xerostomia reported in these mice.     

Identification of autoantibody biomarkers for primary Sjögren's syndrome using protein microarrays

Sj?gren’s syndrome (SS) is a chronic, progressive autoimmune disease primarily affecting women. Diagnosis of SS requires an invasive salivary gland tissue biopsy and a long delay from the start of the symptoms to final diagnosis has been frequently observed. In this study,we aim to identify salivary autoantibody biomarkers for primary SS (pSS) using a protein microarray approach. Immune-response protoarrays were used to profile saliva autoantibodies from patients with pSS (n = 514), patients with systemic lupus erythematosus(SLE, n = 513), and healthy control subjects (n = 513). We identified 24 potential autoantibody biomarkers that can discriminate patients with pSS from both patients with SLE and healthy individuals. Four saliva autoantibody biomarkers, anti-transglutaminase, anti-histone, anti-SSA, and anti-SSB, were further tested in independent pSS (n = 534), SLE (n = 534), and healthy control (n = 534) subjects and all were successfully validated with ELISA. This study has demonstrated the potential of a high-throughput protein microarray approach for the discovery of autoantibody biomarkers. The identified saliva autoantibody biomarkers may lead to a clinical tool for simple, noninvasive detection of pSS at low cost.     

Lineage tree analysis of immunoglobulin variable-region gene mutations in autoimmune diseases: chronic activation, normal selection

Autoimmune diseases show high diversity in the affected organs, clinical manifestations and disease dynamics. Yet they all share common features, such as the ectopic germinal centers found in many affected tissues. Lineage trees depict the diversification, via somatic hypermutation (SHM), of immunoglobulin variable-region (IGV) genes. We previously developed an algorithm for quantifying the graphical properties of IGV gene lineage trees, allowing evaluation of the dynamical interplay between SHM and antigen-driven selection in different lymphoid tissues, species, and disease situations. Here, we apply this method to ectopic GC B cell clones from patients with Myasthenia Gravis, Rheumatoid Arthritis, and Sj?gren's Syndrome, using data scaling to minimize the effects of the large variability due to methodological differences between groups. Autoimmune trees were found to be significantly larger relative to normal controls. In contrast, comparison of the measurements for tree branching indicated that similar selection pressure operates on autoimmune and normal control clones.