Marc R. Kok

Local adeno-associated virus-mediated interleukin 10 gene transfer has disease-modifying effects in a murine model of Sjögren's syndrome.

Female nonobese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are a widely used model of Sjögrens syndrome. We examined the feasibility of local salivary gland immunomodulatory gene delivery to alter these sequelae in NOD mice. We constructed recombinant adeno-associated virus (rAAV) vectors encoding either human interleukin 10 (rAAVhIL-10) or beta-galactosidase (rAAVLacZ, control vector). Mice received rAAVhIL-10 or rAAVLacZ by retrograde submandibular ductal instillation either at age 8 weeks (early, before onset of sialadenitis), or at 16 weeks (late, after onset of sialadenitis). As a systemic treatment control, separate mice received intramuscular delivery of rAAVhIL-10 at each time point. Both submandibular and intramuscular delivery of vector led to low circulating levels of hIL-10. After submandibular administration of rAAVhIL-10, salivary flow rates at 20 weeks for both the early and late treatment groups were significantly higher than for both rAAVLacZ-administered and untreated mice. Systemic delivery of rAAVhIL-10 led to improved salivary flow in the late treatment group. Inflammatory infiltrates in submandibular glands, however, were significantly reduced only in mice receiving rAAVhIL-10 locally in the salivary gland compared with mice receiving this vector intramuscularly, or rAAVLacZ or no treatment. In addition, after submandibular rAAVhIL-10 delivery, NOD mice exhibited significantly lower blood glucose, and higher serum insulin, levels than all other groups, indicating some systemic benefit of this treatment. These studies show that expression of hIL-10 by rAAV vectors can have disease-modifying effects in the salivary glands of NOD mice, and suggest that local immunomodulatory gene transfer may be useful for managing the salivary gland pathology in Sjögrens syndrome.

Pro-atherogenic lipid changes and decreased hepatic LDL receptor expression by tocilizumab in rheumatoid arthritis

OBJECTIVES Blocking the interleukin-6 pathway by tocilizumab (TCZ) has been associated with changes in the lipoprotein profile, which could adversely impact cardiovascular (CV) risk in patients with rheumatoid arthritis (RA). In the present study, we addressed the effect of TCZ on lipoproteins in both fasting and non-fasting state in RA patients and tested the effect of TCZ on LDL receptor (LDLr) expression in vitro. METHODS Twenty patients with active RA and an inadequate response to TNF blockers received monthly TCZ intravenously. On week 0, 1 and 6 blood was drawn before and after an oral fat load, the lipid profiles and HDL antioxidative capacity were measured. Effects of TCZ on LDLr expression in transfected HepG2 cells were subjected. RESULTS After 6 weeks of TCZ, total cholesterol increased by 22% (4.8 ± 0.9 to 5.9 ± 1.3 mmol/L; p < 0.001), LDLc by 22% (3.0 ± 0.6 to 3.6 ± 0.8 mmol/L; p < 0.001) and HDLc by 17% (1.4 ± 0.4 to 1.7 ± 0.7 mmol/L; p < 0.016). Fasting triglycerides (TG) increased by 48% (1.0 ± 0.4 to 1.4 ± 0.8 mmol/L; p = 0.011), whereas postprandial incremental area under the curve TG increased by 62% (p = 0.002). Lipid changes were unrelated to the change in disease activity or inflammatory markers. No difference in HDL antioxidative capacity was found. In vitro, LDLr expression in cultured liver cells was significantly decreased following TCZ incubation (P < 0.001). CONCLUSIONS TCZ adversely impacts on both LDLc as well as fasting and postprandial TG in patients with RA. The changes in hepatic LDLr expression following TCZ imply that adverse lipid changes may be a direct hepatic effect of TCZ. The net effect of TCZ on CV-morbidity has to be confirmed in future clinical trials.

Enhanced transduction of mouse salivary glands with AAV5-based vectors

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2β-galactosidase (βgal), rAAV4βgal, or rAAV5βgal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more βgal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.

Use of localised gene transfer to develop new treatment strategies for the salivary component of Sjögren’s syndrome

Effective treatment for Sjögren’s syndrome (SS) might be developed locally by introducing genes encoding cytokines, which are potentially anti-inflammatory, or by introducing a cDNA encoding a soluble form of a key cytokine receptor, which can act as an antagonist and decrease the availability of certain cytokines, such as soluble tumour necrosis factor α receptors. Currently, the preferred choice of viral vector for immunomodulatory gene transfer is recombinant adeno-associated virus. The use of gene transfer to help determine the pathophysiology and to alter the course of the SS-like disease in the NOD mouse model can ultimately lead to the development of new treatments for managing the salivary component in patients with SS.

Immune responses following salivary gland administration of recombinant adeno‐associated virus serotype 2 vectors

Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long‐term secretion of the transgene product. Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications. Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno‐associated virus serotype 2 (rAAV2) vectors induce host immune responses. There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs.

Local expression of tumor necrosis factor-receptor 1:immunoglobulin G can induce salivary gland dysfunction in a murine model of Sjögren's syndrome

IntroductionTumor necrosis factor is a pleiotropic cytokine with potent immune regulatory functions. Although tumor necrosis factor inhibitors have demonstrated great utility in treating other autoimmune diseases, such as rheumatoid arthritis, there are conflicting results in Sjögrens syndrome. The aim of this study was to assess the effect of a locally expressed tumor necrosis factor inhibitor on the salivary gland function and histopathology in an animal model of Sjögrens syndrome.MethodsUsing in vivo adeno associated viral gene transfer, we have stably expressed soluble tumor necrosis factor-receptor 1-Fc fusion protein locally in the salivary glands in the Non Obese Diabetic model of Sjögrens syndrome. Pilocarpine stimulated saliva flow was measured to address the salivary gland function and salivary glands were analyzed for focus score and cytokine profiles. Additionally, cytokines and autoantibody levels were measured in plasma.ResultsLocal expression of tumor necrosis factor-receptor 1:immunoglobulin G fusion protein resulted in decreased saliva flow over time. While no change in lymphocytic infiltrates or autoantibody levels was detected, statistically significant increased levels of tumor growth factor-β1 and decreased levels of interleukin-5, interleukin-12p70 and interleukin -17 were detected in the salivary glands. In contrast, plasma levels showed significantly decreased levels of tumor growth factor-β1 and increased levels of interleukin-4, interferon-γ, interleukin-10 and interleukin-12p70.ConclusionsOur findings suggest that expression of tumor necrosis factor inhibitors in the salivary gland can have a negative effect on salivary gland function and that other cytokines should be explored as points for therapeutic intervention in Sjögrens syndrome.

Adult heart block is associated with disease activity in primary Sjögren's syndrome

Objectives: Congenital heart block occurring in the foetus and neonate may be associated with maternal anti‐SS‐A/anti‐SS‐B autoantibodies (anti‐SSA/anti‐SSB). The adult atrioventricular node is generally thought to be resistant to the damaging effects of anti‐SSA/anti‐SSB. However, case reports suggest that heart block developing in adult Sjögrens syndrome (SS) patients may be associated with these autoantibodies. Therefore, we investigated the relationship between serum antibodies and heart block in adult SS patients. Methods: We abstracted data from clinic patient records. Diagnosis of primary SS was based on American–European classification criteria. Electrocardiograms (EKGs), laboratory immunology parameters, lipid profiles, and focus scores from labial salivary gland biopsies were available for 51 SS patients. Fifteen patients had follow‐up EKGs. PR interval⩾200 ms was considered to be first‐degree heart block. Results: Five patients showed prolonged PR intervals; the presence of heart block was not related to the presence of anti‐SSA antibodies. However, significant differences between patients with prolonged and normal PR intervals were seen for mean focus scores (p<0.0001), anti‐cardiolipin immunoglobulin IgG (p = 0.0009), age (p = 0.01), IgG (p = 0.02), anti‐SSB antibodies (p = 0.02), and high density lipoprotein (HDL) cholesterol levels (p = 0.03). These parameters correlated with prolonged PR intervals. Conclusions: These results suggest an association between disease activity, the presence of anti‐SSB antibodies, and the occurrence of first‐degree heart block in adults with primary SS.

Taking advances from bench to bedside during the last decade.

The remarkable advances in understanding the pathogenesis and therapeutic options for rheumatoid arthritis over the last 10 years are a leap forward in the treatment of this disease. This has led to a shift in focus from established disease to early identification and treatment. Actualisation of treatment guidelines aiming for remission, and a vastly growing arsenal of new synthetic and biological agents have been major achievements. An area of ongoing research is the discovery and development of additional and improved biomarkers for (early) disease with the goal of designing a more personalised treatment regimen to prevent structural tissue damage. Developing valid tools to predict response and outcome for the individual patient remains, however, a great challenge. We will herein summarise some of the major achievements in diagnostic and therapeutic discoveries in rheumatoid arthritis of the past decade.

Remission of incapacitating acute cutaneous lupus erythematosus in a patient with systemic lupus erythematosus by B cell-depletive therapy.

BcellYdepleting therapy has been successfully used for rheumatoid arthritis and could be a novel strategy for numerous other autoimmune diseases. We report a patient with incapacitating lupus skin disease refractory to conventional therapy. Rituximab (anti-CD20) treatment resulted in a remarkable improvement of the skin lesions and quality of life. Systemic lupus erythematosus (SLE) was diagnosed in a 30-year-old woman at age 13 years based on arthritis, skin lesions, serositis, Raynaud phenomenon, antinuclear antibody, and antiYdouble-stranded DNA antibodies. Three years later, she had a biopsy-proven diagnosis of SLE nephritis. The kidney function remained normal while treated with azathioprine, hydroxychloroquine, and corticosteroids. During this treatment, at age 28 years, skin lesions on her hands, feet, face, neck, arms, chest, and abdomen worsened, and the dosage of prednisolone was increased to 60 mg without improvement. Clinically, the rash on the trunk, arms, hands, and fingers represented the disseminated maculopapular variant of acute cutaneous lupus erythematosus (ACLE). Annular-polycyclic or papulosquamous (psoriasiform) skin lesions as in subacute lupus erythematosus or scarring lesions as in discoid lupus erythematosus were not observed. Lesions of the hands were incapacitating as the patient could not use them normally because of pain upon touch. A skin biopsy confirmed the diagnosis of cutaneous lupus and showed granular depositions of IgG and complement along the basal membrane by direct immunofluorescence. Based on positive reports on the chimeric anti-CD20 monoclonal antibody rituximab for the treatment of SLE, we treated her with 2 intravenous infusions of rituximab (1000 mg) with an interval of 2 weeks, concomitant with methylprednisolone 100 mg and clemastine 2 mg as premedication. This had a dramatic positive effect on her skin lesions (Fig.). Three months after treatment, all skin lesions were in complete remission, and the prednisolone dosage could be tapered to 7.5 mg once daily. The antiYdouble-stranded DNA antibody levels, shown by Farr assay, decreased from 157 IE/mL before treatment to 38 IE/mL 10 months after treatment. The skin lesions relapsed 9 months later and again responded spectacularly after another course of rituximab. Skin manifestations represent the second most common symptoms of SLE and often require systemic treatment. Antimalarials are the drugs of first choice, but the effects are not always sufficient. Various immunosuppressive drugs have been used in resistant cases, including azathioprine, methotrexate, mycophenolate mofetil, and thalidomide, often combined with corticosteroids. Alleviating refractory and incapacitating skin lesions, if present, is often the most important issue for the patient. The efficacy of the B cellYdepleting antibody rituximab is illustrated by this case. Consistent with its known long-lasting pharmacokinetic effects, we observed clinical improvement of previously therapy-resistant skin disease for a period of 9 months after a single treatment course.

Influence of Disease Manifestations on Health-related Quality of Life in Early Psoriatic Arthritis

Objective. Psoriatic arthritis (PsA) is a multifaceted disease. Affecting joints, skin, entheses, and dactylitis, its effect on health-related quality of life (HRQOL) could be substantial. We aim to assess HRQOL in patients newly diagnosed with PsA and analyze its associations with disease manifestations. Methods. Data collected at time of diagnosis from patients with PsA included in the Dutch south-west Early Psoriatic Arthritis cohort (DEPAR) study were used. HRQOL was assessed using 8 domains of the Medical Outcomes Study Short Form-36 (SF-36) questionnaire. Patients were classified based on primary manifestation in arthritis subtypes (i.e., mono-, oligo-, or polyarthritis) and other subtypes (i.e., enthesitis, dactylitis, and axial disease). In all patients, presence of arthritis, enthesitis, dactylitis, psoriasis, and chronic inflammatory back pain was determined. Multivariable linear regression was used to determine associations of PsA manifestations with HRQOL. Results. Of 405 patients, primary manifestation was peripheral arthritis in 320 (78 monoarthritis, 151 oligoarthritis, and 91 polyarthritis), enthesitis in 37, axial disease in 9, and dactylitis in 39. Mean scores of SF-36 domains were lower than the Dutch reference population and similar across arthritis subtypes. A higher number of enthesitis locations and tender joints, and presence of chronic back pain, were independently associated with worse SF-36 scores. Psoriasis and dactylitis were not associated with worse scores. Conclusion. HRQOL was diminished in PsA at time of diagnosis compared to the Dutch reference population, and tender joints, enthesitis at clinical examination, and back pain as indicators of pain affected HRQOL.