Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction.

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na+] and [Cl-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

TACI-Fc gene therapy improves autoimmune sialadenitis but not salivary gland function in non-obese diabetic mice.

OBJECTIVE: Patients with Sjögren's syndrome (SS) show aberrant expression of the B cell-related mediators, B cell-activating factor (BAFF), and a proliferation-inducing ligand (APRIL) in serum and salivary glands (SGs). We studied the biological effect of neutralizing these cytokines by local gene transfer of the common receptor transmembrane activator and CAML interactor (TACI) in an animal model of SS. MATERIAL AND METHODS: A recombinant serotype 2 adeno-associated virus (rAAV2) encoding TACI-Fc was constructed, and its efficacy was tested in the SGs of non-obese diabetic mice. Ten weeks later, SG inflammation was evaluated and serum and SG tissue were analyzed for inflammatory markers including immunoglobulins (Ig) and cytokines. RESULTS: AAV2-TACI-Fc gene therapy significantly reduced the number of inflammatory foci in the SG, owing to a decrease in IgD(+) cells and CD138(+) cells. Moreover, IgG and IgM levels, but not IgA levels, were reduced in the SG. Overall expression of mainly proinflammatory cytokines tended to be lower in AAV2-TACI-Fc-treated mice. Salivary flow was unaffected. CONCLUSION: Although local expression of soluble TACI-Fc reduced inflammation and immunoglobulin levels in the SG, further research will have to prove whether dual blockade of APRIL and BAFF by TACI-Fc can provide a satisfying treatment for the clinical symptoms of patients.

Temporal changes in salivary glands of non-obese diabetic mice as a model for Sjögren's syndrome.

OBJECTIVE: Non-obese diabetic (NOD) mice develop an autoimmune exocrinopathy that shows similarities with Sjögren's syndrome. They provide an experimental model to study the pathoetiogenesis of this disease. MATERIALS AND METHODS: Salivary gland (SG) function and salivary sodium content were measured in 8-, 12-, 16- and 20-week-old NOD and age-matched CB6 mice. In NOD mice, SG expression of phenotypic cell markers, B cell-stimulating and costimulatory molecules were evaluated. Cytokine levels were measured in serum and SG homogenates. RESULTS: Microscopically evident SG inflammation in NOD mice was preceded by expression of intercellular adhesion molecule 1 on epithelial cells in the presence of macrophages and relatively high levels of cytokines. Next, an influx consisting of mainly T, B, natural killer, plasma and dendritic cells was seen. Most cytokines, except for interleukin (IL)12/IL23p40 and B cell-activating factor, decreased or remained stable over time, while glandular function deteriorated from 16 weeks of age onward compared with CB6 mice. CONCLUSION: Sjögren's syndrome-like disease in NOD mice occurs in multiple stages; immunological and physiological abnormalities can be detected before focal inflammation appears and salivary output declines. Extrapolating this knowledge to human subjects could help in understanding the pathogenesis and aid the identification of potential therapeutic targets.

Deep sequencing of short RNAs reveals novel microRNAs in minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVES: Sjögren's syndrome is a complex autoimmune disease of the salivary gland with an unknown etiology, so a thorough characterization of the transcriptome would facilitate our understanding of the disease. We use ultradeep sequencing of small RNAs from patients with Sjögren's syndrome and healthy volunteers, primarily to identify and discover novel miRNA sequences that may play a role in the disease. METHODS: Total RNA was isolated from minor salivary glands of healthy volunteers and patients with either high or low salivary flow and sequenced on the SOLiD platform. Prediction of mature miRNAs from the sequenced reads was carried out using miRanalyzer, and expression was validated using Taqman qPCR assays. RESULTS: We validated the presence of six previously unidentified miRNA sequences in patient samples and in several cell lines. One of the validated novel miRNAs shows promise as a biomarker for salivary function. CONCLUSION: Sequencing small RNAs in the salivary gland is largely unprecedented, but here, we show the feasibility of discovering novel miRNAs and disease biomarkers by sequencing the transcriptome.

Helicobacter pylori and autoimmune diseases.

Helicobacter pylori (H. pylori) is a widely prevalent microbe, with between 50 and 80% of the population infected worldwide. Clinically, infection with H. pylori is commonly associated with peptic ulcer disease, but many of those infected remain asymptomatic. H. pylori has evolved a number of means to affect the host immune response and has been implicated in many diseases mitigated by immune dysregulation, such as immune thrombocytopenic purpura (ITP), atrophic gastritis, and mucosa associated lymphoid tissue (MALT) lymphoma. Autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, are the result of a dysregulated host immune system which targets otherwise healthy tissues. The exact etiology of autoimmune diseases is unclear, but it has long been suggested that exposure to certain environmental agents, such as viral and bacterial infection or chemical exposures, in genetically susceptible individuals may be the catalyst for the initiation of autoimmune processes. Because of its prevalence and ability to affect human immune function, many researchers have hypothesized that H. pylori might contribute to the development of autoimmune diseases. In this article, we review the available literature regarding the role of chronic H. pylori infection in various autoimmune disease states.

Peptide-based ELISAs are not sensitive and specific enough to detect muscarinic receptor type 3 autoantibodies in serum from patients with Sjogren's syndrome.

OBJECTIVES: The detection of autoantibodies to the muscarinic receptor type 3 (M3R) in the serum of patients with Sjögrens syndrome (SS) by ELISA is controversial. A study was undertaken to test whether modification of M3R peptides could enhance the antigenicity and increase the detection of specific antibodies using an ELISA. METHODS: A series of controlled ELISAs was performed with serum from 71 patients with SS and 37 healthy volunteers (HV) on linear, citrullinated and/or cyclised and multi-antigenic peptides (MAP) of the three extracellular M3R loops to detect specific binding. RESULTS: Significant differences (p<0.05) in optical density (OD) between serum from patients and HV were detected for a cyclised loop 1-derived peptide and the negative control peptide. Furthermore, there were no statistically significant differences between the frequency of positive patients (defined as OD >2SDs above the mean of the HV) and HV on any of the peptides tested. CONCLUSIONS: Binding of serum from patients with SS to M3R-derived peptides does not differ from binding to a control peptide in an ELISA and no significant binding to M3R-derived peptides was found in the serum from individual patients compared with HV. These data suggest that peptide-based ELISAs are not sufficiently sensitive and/or specific to detect anti-MR3 autoantibodies.

Exosomes from human saliva as a source of microRNA biomarkers.

OBJECTIVE: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications. DESIGN: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays. RESULTS: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjögren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling. CONCLUSIONS: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies.

Cytokines in Sjögren's syndrome.

Cytokines play a central role in the regulation of immunity and are often found to be deregulated in autoimmune diseases. Sjögren's syndrome is a chronic autoimmune disease characterized by inflammation and loss of secretory function of the salivary and lachrymal glands. This review highlights the current knowledge of the expression and the function of pro- and anti-inflammatory cytokines both locally and systemically in Sjögren's syndrome patients. In the salivary glands, saliva and serum of these patients, many pro-inflammatory cytokines are upregulated. Concomitantly, most anti-inflammatory cytokines are not detectable or are expressed at low levels. Besides a role in inflammation, cytokines are also thought to be involved in salivary gland dysfunction by directly interfering with the epithelial cells in the glands. Future research on the role of novel cytokines in Sjögren's syndrome in combination with a better understanding of the effect of cytokines on exocrine dysfunction will aide the identification of the best therapeutic targets for Sjögren's syndrome.

Variant form of STAT4 is associated with primary Sjögren's syndrome.

Single nucleotide polymorphisms in the STAT4 gene have recently been shown to be associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Primary Sjögren's syndrome (pSS) is a related autoimmune disease thought to have a pathogenesis similar to these diseases. To test the hypothesis that the variant haplotype of STAT4 seen in RA and SLE is also associated with pSS, we genotyped rs7574865, the most strongly disease-associated SNP in the variant STAT4 haplotype, in 124 Caucasian pSS subjects and compared them to 1143 Caucasian controls. The disease-associated T allele was more common in chromosomes of the pSS patients (29.6%) than in controls (22.3%), leading to a P-value for association of 0.01. These results implicate polymorphisms in the STAT4 gene in the pathogenesis of pSS.

Biologic treatments for systemic rheumatic diseases.

Many rheumatologic disorders, most notably Sjögren's syndrome, are associated with dental complications and in some cases oral diseases may trigger or drive connective tissue disease. During the past three decades the treatment in rheumatology was revolutionized by the introduction of disease-modifying anti-rheumatic drugs. Advances in our understanding of the pathogenesis of rheumatic diseases have led to the discovery of critical mechanisms of inflammation and autoimmunity and the invention of new target-specific biologic agents. In this review, we will summarize the current state of biologic therapies in rheumatology and discuss the implications of these on oral health and disease.

Long-term effects of combination treatment with fludarabine and low-dose pulse cyclophosphamide in patients with lupus nephritis.

OBJECTIVES: To determine the safety and efficacy of a short course of fludarabine combined with cyclophoshamide in lupus nephritis. METHODS: A phase I/II open label pilot study. Thirteen patients with active proliferative lupus nephritis received monthly oral boluses of low-dose cyclophoshamide (0.5 gm/m(2) on day 1) and subcutaneous fludarabine (30 mg/m(2) on days 1-3) for 3-6 cycles. Concomitant prednisone was aggressively tapered from 0.5 mg/kg/day to a low-dose, alternate-day schedule. Patients were followed for at least 24 months after therapy. The primary outcome was the number of patients achieving renal remission defined as stable creatinine, proteinuria <1 gm/day and inactive urine sediment for at least 6 months. RESULTS: The study was terminated early because of bone marrow toxicity. Eleven patients who received at least three cycles were evaluated for efficacy. Ten patients improved markedly with seven patients achieving complete remission and three patients achieving partial remission. There were three serious haematological adverse events during the treatment with one death due to transfusion-associated graft vs host disease. Profound and prolonged CD4 (mean CD4: 98/microl at 7 months and 251/microl at 12 months) and CD20 lymphocytopenia was noted in most patients. Three patients developed Herpes zoster infections. CONCLUSIONS: A short course of low-dose fludarabine and cyclophoshamide can induce long-lasting remissions in patients with proliferative lupus nephritis, but severe myelosuppression limits its widespread use.

The Met66 allele of the functional Val66Met polymorphism in the brain-derived neurotrophic factor gene confers protection against neurocognitive dysfunction in systemic lupus erythematosus.

BACKGROUND: A common functional polymorphism of the brain-derived neurotrophic factor gene (BDNF Val66Met) was previously associated with diminished episodic memory performance in healthy people. As cognitive function is commonly impaired in patients with systemic lupus erythematosus (SLE), the association of the BDNF Val66Met with neurocognitive function was studied. OBJECTIVE: To study the association of the BDNF Val66Met with neurocognitive function in a cohort of patients with SLE. METHODS: Cognitive function was assessed in 59 patients with SLE with no previous or current central nervous system involvement. Cognitive tests were grouped into five domains (memory, attention/executive function, visuospatial skills, motor function and psychomotor speed) and used to obtain domain Z scores, reflecting the difference between averaged scores of performance on individual tests and published norms in each domain. Genotyping was carried out using a 5'-nuclease assay with 99.9% accuracy. Unpaired t test was used to assess the relationship between genotypes and cognitive function, whereas the effect of possible confounders was assessed in a multivariate analysis. RESULTS: Patients carrying the Met66 allele scored significantly higher on psychomotor, attention/executive and motor function tests, resulting in significantly higher domain Z scores for the psychomotor (p = 0.005) and motor (p = 0.002) domains. CONCLUSIONS: The BDNF Met66 allele was associated with better cognitive functioning in the psychomotor and motor domains, even after controlling for differences in ethnicity, sex, depression status and prednisone treatment. These data suggest that the BDNF Met66 allele confers protection against the decline of motor and psychomotor cognitive functions in patients with longstanding SLE.

Identification of parotid salivary biomarkers in Sjogren's syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis.

OBJECTIVES: To identify the most significant salivary biomarkers in Sjögren's syndrome (SS) using proteomic methods. METHODS: Parotid saliva from 20 non-SS subjects and 41 primary SS patients was analysed. Protein expression profiles for each sample were generated by surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry (SELDI-TOF-MS). Mean peak intensities of SS patients and non-SS subjects were compared by univariate analyses. Samples pooled by diagnosis (SS and non-SS) and labelled with different Cy dyes were compared by two-dimensional difference gel electrophoresis (2D-DIGE). Two protein levels that were most significantly different by SELDI-TOF-MS and 2D-DIGE were validated by enzyme-linked immunosorbent assay in individual samples. RESULTS: SELDI-TOF-MS of 10-200 kDa peaks revealed eight peaks with >2-fold changes in the SS group that differed from non-SS at P < 0.005. Peaks of 11.8, 12.0, 14.3, 80.6 and 83.7 kDa were increased, while 17.3, 25.4, and 35.4 kDa peaks were decreased in SS samples. 2D-DIGE identified significant increases of beta-2-microglobulin, lactoferrin, immunoglobulin (Ig) kappa light chain, polymeric Ig receptor, lysozyme C and cystatin C in all stages of SS. Two presumed proline-rich proteins, amylase and carbonic anhydrase VI, were reduced in the patient group. Three of these ten biomarkers have not been associated previously with SS. CONCLUSIONS: The salivary proteomic profile of SS is a mixture of increased inflammatory proteins and decreased acinar proteins when compared with non-SS. Future studies will test the ability of these biomarker levels, alone and in combination, to diagnose the salivary component of SS.

Membranous lupus nephritis.

Membranous lupus nephritis (MLN) represents about 20% of clinically significant renal disease in lupus. Few studies have addressed directly the pathogenesis of MLN. Our assumptions about the underlying mechanisms are based on the combination of extrapolations from idiopathic membranous nephritis (mainly from animal models) and proliferative lupus nephritis. Natural history studies of MLN suggest a relatively low rate of progression to end-stage renal disease but a high rate of significant comorbidities. Historical changes in the criteria for pathologic diagnosis and classification of membranous lupus nephropathy have precluded definitive descriptions of the natural history, prognosis and treatment of this disorder. Patients with membranous lupus nephropathy should be treated early with angiotensin antagonists to minimize proteinuria, as well as lifestyle changes and appropriate drugs to reduce attendant cardiovascular risk factors. In patients with protracted nephrotic syndrome, consideration should be given to immunosuppressive therapies, including corticosteroids, cyclosporine, mycophenolate and cyclophosphamide. Prospective controlled trials are clearly needed in order to establish solid clinical practice guidelines for use of these drugs and other experimental therapies currently under study in membranous lupus nephropathy.

Rationale for interleukin-6 blockade in systemic lupus erythematosus.

Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biological activities that plays an important role in immune regulation and inflammation. Among other actions, it induces terminal differentiation of B lymphocytes into antibody-forming cells and the differentiation of T cells into effector cells. IL-6 also has multiple potent proinflammatory effects. An association between IL-6 and lupus was demonstrated in murine models of SLE and blocking IL-6 improved lupus in all models tested. Data from several studies suggest that IL-6 plays a critical role in the B cell hyperactivity and immunopathology of human SLE, and may have a direct role in mediating tissue damage. Based on these data, we propose that blocking the effect of IL-6 in humans may improve lupus by interacting with the autoinflammatory process both systemically and locally.

Lymphocyte depletion with fludarabine in patients with psoriatic arthritis: clinical and immunological effects.

OBJECTIVE: To obtain preliminary information on the safety and efficacy of fludarabine in PsA and analyse its immunomodulatory effects in peripheral blood and synovial tissue. METHODS: 15 patients with active PsA who did not respond to DMARDs were randomly allocated to receive fludarabine every four weeks or placebo. Primary outcomes were the proportion of patients who met the ACR20 and the psoriatic arthritis response criteria (PsARC) at 16 weeks. Secondary outcomes were changes in tender or swollen joint counts and scores of the psoriasis area and severity index (PASI). Phenotypic analysis of peripheral blood mononuclear cells (PBMC), synovial immunohistochemistry, and functional analysis of PBMC were used to determine the immunomodulatory effects of fludarabine. RESULTS: At 16 weeks the ACR20 criteria were met by 3/7 (43%) fludarabine treated v 0/8 placebo treated patients (p=0.08); the PsARC was achieved by 4/7 (57%) fludarabine treated v 2/8 (25%) placebo treated patients; and 3/7 (43%) fludarabine treated v 0/7 placebo treated patients had > or =20% improvement in the PASI. Marked peripheral lymphopenia involving naive (CD4(+) CD45RA(+)) and memory (CD4(+) CD45RO(+)) T cells, CD8(+) T cells, and B cells was seen in fludarabine treated patients. CONCLUSIONS: In PsA fludarabine induces significant peripheral, but modest, synovial lymphopenia, and a trend towards improved clinical response.

Renal flares are common in patients with severe proliferative lupus nephritis treated with pulse immunosuppressive therapy: long-term followup of a cohort of 145 patients participating in randomized controlled studies.

OBJECTIVE: Immunosuppressive agents have become the standard of therapy for proliferative lupus nephritis, but some patients may relapse after discontinuing treatment. We reviewed the cases of renal flares in a cohort of patients who participated in 2 randomized controlled clinical trials at the National Institutes of Health and explored the prevalence, outcome, and predictive factors of renal flares. METHODS: Data were obtained on 145 patients treated with pulse cyclophosphamide, pulse methylprednisolone, or the combination of both. Patients had not received immunosuppressive therapy for at least 6 months and had experienced complete or partial response according to defined criteria. Renal flares were classified as either proteinuric or nephritic based on changes in urinary protein and sediment. Most patients who experienced a flare received additional immunosuppressive therapy. RESULTS: Seventy-three patients had a complete response, and 19 had partial response/stabilization. Forty-one of these patients (45%) experienced renal flares (nephritic in 33, proteinuric in 8) after a mean followup of 117 months; 31 of them received additional immunosuppressive therapy. The median time to renal flare was 36 months in the complete responders and 18 months in the partial responders. Eleven of the 41 patients (27%) progressed to end-stage renal disease (ESRD); 9 had nephritic flares (all severe except for 1) and 2 had proteinuric flares (1 in each responder group). Compared with patients who had a complete response, those with a partial response were more likely to experience a flare, to have a severe nephritic flare, or to progress to ESRD. Low C4 at the time of response and African American ethnicity were significant independent risk factors for renal flare, by multivariate Cox proportional hazards analysis. CONCLUSION: Nephritic flares are common in patients with proliferative lupus nephritis, even in those with a complete response to therapy, but they do not necessarily result in loss of renal function if treated with additional immunosuppressive agents. Renal flares are an important feature of the natural history of lupus nephritis and provide an opportunity for additional preventive strategies, as well as measures of efficacy in future therapeutic trials.

IL-10 improves skin disease and modulates endothelial activation and leukocyte effector function in patients with psoriatic arthritis.

Psoriatic arthritis (PsA) provides an ideal disease model in which to investigate the bioactivities of potentially therapeutic cytokines at multiple sites of tissue inflammation. We investigated the effects of IL-10, an antiinflammatory cytokine, given s.c. for 28 days in a double-blind, placebo-controlled study in PsA patients. Synovial/skin biopsies, peripheral blood leukocytes, articular magnetic resonance images, and clinical disease activity scores were obtained sequentially. Modest, but significant clinical improvement in skin, but not articular disease activity scores with only minor adverse effects was observed. Type 1, but not type 2 T cell cytokine production in vitro was suppressed in human rIL-10 compared with placebo recipients. Similarly, monokine production in vitro was reduced, whereas serum soluble TNFRII levels were elevated, indicating suppression of monocyte function. Decreased T cell and macrophage infiltration in synovial tissues was accompanied by reduced P-selectin expression. Moreover, suppressed synovial enhancement on magnetic resonance imaging and reduced alpha(v)beta(3) integrin expression on von Willebrand factor(+) vessels were observed. Together these data demonstrate that a short course of IL-10 modulates immune responses in vivo via diverse effects on endothelial activation, and leukocyte recruitment and effector function. Such biological changes may result in clinically meaningful improvement in disease activity.