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1.  TACI-Fc gene therapy improves auto-immune sialadenitis but not salivary gland function in NOD mice 
Oral Diseases  2011;18(4):365-374.
Objective
Sjögren’s syndrome (SS) patients 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 (NOD) 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, due 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 pro-inflammatory 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.
doi:10.1111/j.1601-0825.2011.01885.x
PMCID: PMC3314152  PMID: 22212434
TACI-Fc; gene therapy; salivary gland; NOD mice; Sjögren’s syndrome
2.  Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis 
Annals of the Rheumatic Diseases  2005;65(3):294-300.
Background
Chemokine receptors and chemokines have a crucial role in leucocyte recruitment into inflamed tissue.
Objective
To examine the expression of an extensive number of chemokines and receptors in a unique bank of paired samples of synovial tissue (ST) and peripheral blood (PB) from patients with different forms of arthritis to assist in identifying suitable targets for therapeutic intervention.
Methods
Synovial biopsy specimens were obtained from 23 patients with rheumatoid arthritis (RA), 16 with osteoarthritis, and 8 with reactive arthritis. ST chemokine (CCL2/MCP‐1, CCL5/RANTES, CCL7/MCP‐3, CCL8/MCP‐2, CCL14/HCC‐1, CCL15/HCC‐2, CCL16/HCC‐4), chemokine receptor (CCR1, CCR2b, CCR5, CXCR4), and CD13 expression was analysed by immunohistochemistry and two colour immunofluorescence. Chemokine receptor expression (CCR1, CCR3, CCR5, CCR6, CCR7) on PB cells was studied by flow cytometry. Non‐parametric tests were used for statistical analysis.
Results
Abundant expression of CCR1, CXCR4, and CCR5 was found in all forms of arthritis, with a specific increase of CCL5 and CCL15 in RA. CCL7, CCL8, CCL14, CCL15, and CCL16 were detected for the first time in ST. The results for PB analysis were comparable among different arthritides. Interestingly, compared with healthy controls, significantly lower expression of CCR1 (p<0.005) and CCR5 (p<0.05) by PB monocytes in the patient groups was seen.
Discussion
A variety of chemokines and receptors might have an important role in several inflammatory joint disorders. Although other receptors are involved as well, migration of CCR1+ and CCR5+ cells towards the synovial compartment may play a part in the effector phase of various forms of arthritis.
doi:10.1136/ard.2005.037176
PMCID: PMC1798063  PMID: 16107514
arthritis; chemokines; pathogenesis; synovial tissue; chemokine receptors
3.  Local delivery of beta interferon using an adeno-associated virus type 5 effectively inhibits adjuvant arthritis in rats 
The Journal of General Virology  2007;88(Pt 6):1717-1721.
Beta interferon (IFN-β) is a cytokine with potent immunomodulatory properties and has been described as a promising therapeutic molecule for the treatment of rheumatoid arthritis (RA). IFN-β was previously overexpressed intra-articularly using an adenoviral vector in rats with adjuvant arthritis (AA) as a model of RA. This effect was powerful, albeit transient due to the vector chosen. Therefore, in the context of pre-clinical development, a delivery vector optimized for intra-articular gene transfer, recombinant adeno-associated virus type 5 (rAAV5), was selected. To exert an optimal effect, protein production should parallel the course of the disease. For this reason, the gene for IFN-β was placed under the control of an inflammation-responsive [nuclear factor (NF)-κB] promoter. After intra-articular injection of the rAAV5 constructs in rats with AA, local transcription of the transgene and production of the IFN-β protein was found, leading to a pronounced and sustained effect on paw swelling when the expression was under the control of the NF-κB-responsive promoter. Additionally, a significant beneficial effect was observed on proteoglycan depletion and erosions. Thus, intra-articular overexpression of IFN-β using a rAAV5 vector exhibits potential as an innovative therapy for the treatment of RA.
doi:10.1099/vir.0.82603-0
PMCID: PMC2884954  PMID: 17485531
4.  Reduction of arthritis following intra‐articular administration of an adeno‐associated virus serotype 5 expressing a disease‐inducible TNF‐blocking agent 
Annals of the Rheumatic Diseases  2007;66(9):1143-1150.
Background
In the context of preclinical development, we studied the potential of intra‐articular gene delivery using a recombinant adeno‐associated virus 5 (rAAV5) encoding a chimeric human tumour necrosis factorα (TNFα) soluble receptor I linked to a mouse immunoglobulin heavy chain Fc portion (TNF receptor I; TNFRI‐Ig).
Methods
Expression was under control of a nuclear factor kappa B (NFκB)‐responsive promoter and compared with a cytomegalovirus (CMV) promoter (rAAV5.NFκB‐TNFRI‐Ig and rAAV5.CMV‐TNFRI‐Ig, respectively).
Results
Fibroblast‐like synoviocytes transduced in vitro with rAAV5.NFκB‐TNFRI‐Ig were able to produce TNFRI‐Ig protein in response to several stimuli, and this was inhibited upon treatment with a specific NFκB blocking agent. A bioassay revealed that the synthesised TNFRI‐Ig was bioactive, showing a higher affinity for human than for rat TNFα. Transcription of the transgene and protein production were detectable in joints injected with both constructs. No dissemination of the vector was observed outside the joints. A significant reduction in paw swelling was seen in rats treated with rAAV5.NFκB‐TNFRI‐Ig. This clinical effect was accompanied by a decrease in pro‐inflammatory cytokine levels and an increase in IL10 expression in the synovium.
Conclusion
These results provide evidence that intra‐articular gene therapy using rAAV5 encoding TNFRI‐Ig may be a safe and feasible approach for the treatment of rheumatoid arthritis. The higher affinity for human TNFα suggests that in patients with rheumatoid arthritis the therapeutic effect might be even more pronounced than in rat adjuvant arthritis.
doi:10.1136/ard.2006.064519
PMCID: PMC1955149  PMID: 17363402
AAV; gene therapy; TNF receptor I; arthritis; gene transfer
5.  Detailed analysis of the cell infiltrate and the expression of mediators of synovial inflammation and joint destruction in the synovium of patients with psoriatic arthritis: implications for treatment 
Annals of the Rheumatic Diseases  2006;65(12):1551-1557.
Background
The synovial tissue is a primary target of many inflammatory arthropathies, including psoriatic arthritis (PsA). Identification of proinflammatory molecules in the synovium may help to identify potentially therapeutic targets.
Objective
To investigate extensively the features of cell infiltration and expression of mediators of inflammation and joint destruction in the synovium of patients with PsA compared with patients with rheumatoid arthritis matched for disease duration and use of drugs.
Methods
Multiple synovial tissue biopsy specimens were obtained by arthroscopy from an inflamed joint in 19 patients with PsA (eight oligoarthritis, 11 polyarthritis) and 24 patients with rheumatoid arthritis. Biopsy specimens were analysed by immunohistochemistry to detect T cells, plasma cells, fibroblast‐like synoviocytes, macrophages, proinflammatory cytokines, matrix metalloproteinases and tissue inhibitor metalloproteinase‐1, adhesion molecules and vascular markers. Stained sections were evaluated by digital image analysis.
Results
The synovial infiltrate of patients with PsA and rheumatoid arthritis was comparable with regard to numbers of fibroblast‐like synoviocytes and macrophages. T cell numbers were considerably lower in the synovium of patients with PsA. The number of plasma cells also tended to be lower in PsA. The expression of tumour necrosis factor alpha (TNFα), interleukin (IL) 1β, IL6 and IL18 was as high in PsA as in rheumatoid arthritis. The expression of matrix metalloproteinases, adhesion molecules and vascular markers was comparable for PsA and rheumatoid arthritis.
Conclusion
These data show increased proinflammatory cytokine expression in PsA synovium, comparable to results obtained for rheumatoid arthritis, and support the notion that, in addition to TNFα blockade, there may be a rationale for treatments directed at IL1β, IL6 and IL18.
doi:10.1136/ard.2005.050963
PMCID: PMC1798447  PMID: 16728461
6.  Experience with experimental biological treatment and local gene therapy in Sjögren's syndrome: implications for exocrine pathogenesis and treatment 
Annals of the Rheumatic Diseases  2006;65(11):1406-1413.
Sjögren's syndrome is an autoimmune exocrinopathy, mainly affecting the lacrimal and salivary glands, and resulting in ocular and oral dryness (keratoconjunctivitis sicca and xerostomia). The aetiology and pathogenesis are largely unknown, and only palliative treatment is currently available. Data obtained from experimental animal and human studies using biological agents or gene therapeutics can offer insight into the disease process of Sjögren's syndrome. This article reviews the current literature on these approaches and assesses the lessons learnt about the pathogenesis of Sjögren's syndrome.
doi:10.1136/ard.2006.052761
PMCID: PMC1798364  PMID: 16880196
7.  Pretreatment macrophage infiltration of the synovium predicts the clinical effect of both radiation synovectomy and intra‐articular glucocorticoids 
Annals of the Rheumatic Diseases  2006;65(10):1286-1292.
Objective
To explore whether pretreatment features of synovial tissue in patients with gonarthritis could predict the clinical effect of radiation synovectomy with yttrium‐90 (90Y) and glucocorticoids or with intra‐articular glucocorticoids alone.
Methods
A synovial biopsy was carried out blindly 2 weeks before treatment in 66 patients with persistent gonarthritis, who were randomised to treatment either with 90Y and triamcinolone or with placebo and triamcinolone. Immunohistochemistry was used to detect T cells, macrophages, B cells, plasma cells, fibroblast‐like synoviocytes, adhesion molecules and pro‐inflammatory cytokines. Stained sections were evaluated by digital image analysis. Individual patient improvement was expressed using a composite change index (CCI; range 0–12). Successful treatment was defined as CCI ⩾6 after 6 months.
Results
Patients with rheumatoid arthritis, psoriatic arthritis, undifferentiated arthritis and other causes of gonarthritis were included. The overall response rate was 47%. Clinical efficacy in both therapeutic groups was similar and not dependent on diagnosis. No significant differences were noted between baseline microscopic features of synovial tissue inflammation in patients with rheumatoid arthritis and in those with non‐rheumatoid arthritis (ie, all diagnoses other than rheumatoid arthritis). The number of macrophages in the synovial sublining was significantly higher in responders than in non‐responders (p = 0.002), independent of treatment group and diagnosis. The clinical effect was positively correlated with pretreatment total macrophage numbers (r = 0.28; p = 0.03), sublining macrophage numbers (r = 0.34; p = 0.005) and vascular cell adhesion molecule 1 expression (r = 0.25; p = 0.04).
Conclusion
The observations support the view that intra‐articular treatment either with 90Y and glucocorticoids or with glucocorticoids alone is especially successful in patients with marked synovial inflammation.
doi:10.1136/ard.2005.042333
PMCID: PMC1798328  PMID: 16627543
8.  Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjögren's syndrome 
Annals of the Rheumatic Diseases  2005;65(2):195-200.
Background
Sjögren's syndrome (SS), an autoimmune exocrinopathy mainly affecting lachrymal and salivary glands, results in ocular and oral dryness (keratoconjunctivitis sicca and xerostomia). The aetiology and pathogenesis are largely unknown; currently, only palliative treatment is available.
Objective
To determine whether gene transfer of vasoactive intestinal peptide (VIP), based on its immunomodulatory properties, might be useful in the management of SS.
Methods
A recombinant serotype 2 adeno‐associated virus encoding the human VIP transgene (rAAV2hVIP) was constructed and its efficacy tested in the female non‐obese diabetic (NOD) mouse model for SS after retrograde instillation in submandibular glands (SMGs). 1010 particles/gland of rAAV2hVIP or rAAV2LacZ (encoding β‐galactosidase; control vector) were administered at 8 weeks of age (before sialadenitis onset). Salivary flow rates were determined before vector delivery and at time of death (16 weeks). After death, saliva, serum, and SMGs were harvested. Salivary output, inflammatory infiltrates (focus scores), VIP protein expression, cytokine profile, and serum anti‐VIP antibodies were analysed.
Results
rAAV2hVIP significantly improved the salivary flow, increased SMG and serum expression of VIP, and reduced SMG cytokines interleukin (IL) 2, IL10, IL12 (p70), and tumour necrosis factor α, and serum RANTES, compared with the control vector. No difference in focus scores or apoptotic rates was found; neutralising antibodies were not detected.
Conclusions
Local delivery of rAAV2hVIP can have disease modifying and immunosuppressive effects in SMGs of the NOD mouse model of SS. The new strategy of employing VIP prophylactically may be useful for both understanding and managing the salivary component of SS.
doi:10.1136/ard.2005.038232
PMCID: PMC1798026  PMID: 15975969
vasoactive intestinal peptide; Sjögren's syndrome; gene transfer; adeno‐associated virus; autoimmune disease
9.  Monocyte migration to the synovium in rheumatoid arthritis patients treated with adalimumab 
Annals of the Rheumatic Diseases  2011;70(6):1160-1162.
Objectives
The mechanism of action of treatment with tumour necrosis factor (TNF) blockers in rheumatoid arthritis (RA) is still not completely understood. The aim of this study was to test if adalimumab treatment could affect the influx of monocytes into the synovium.
Methods
A novel technique was used to analyse the migration of labelled autologous monocytes before and 14 days after initiation of adalimumab treatment using scintigraphy. CD14 monocytes were isolated from patients with RA, using a positive selection procedure with magnetic-activated cell sorting, and labelled with technetium-99m-hexamethylpropylene-amino-oxime. Scintigraphic scans were made 1, 2 and 3 h after re-infusion.
Results
As early as 14 days after the start of treatment with adalimumab a significant decrease in disease activity score evaluated in 28 joints was shown. There was no significant decrease in the influx of monocytes into the joint at this time.
Conclusions
This study indicates that adalimumab treatment does not reduce the influx of monocytes into the synovium early after initiation of treatment. As previous studies showed a rapid decrease in macrophage infiltration after TNF-antibody therapy, which could not be explained by increased cell death, this points to an important role for enhanced efflux of inflammatory cells from the synovium.
doi:10.1136/ard.2010.141549
PMCID: PMC3086080  PMID: 21345816
15.  Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients 
Annals of the Rheumatic Diseases  2007;66(8):1008-1014.
Background
Rheumatoid arthritis (RA) is a heterogeneous disease with unknown cause.
Aim
To identify peripheral blood (PB) gene expression profiles that may distinguish RA subtypes.
Methods
Large‐scale expression profiling by cDNA microarrays was performed on PB from 35 patients and 15 healthy individuals. Differential gene expression was analysed by significance analysis of microarrays (SAM), followed by gene ontology analysis of the significant genes. Gene set enrichment analysis was applied to identify pathways relevant to disease.
Results
A substantially raised expression of a spectrum of genes involved in immune defence was found in the PB of patients with RA compared with healthy individuals. SAM analysis revealed a highly significant elevated expression of interferon (IFN) type I regulated genes in patients with RA compared with healthy individuals, which was confirmed by gene ontology and pathway analysis, suggesting that this pathway was activated systemically in RA. A quantitative analysis revealed that increased expression of IFN‐response genes was characteristic of approximately half of the patients (IFNhigh patients). Application of pathway analysis revealed that the IFNhigh group was largely different from the controls, with evidence for upregulated pathways involved in coagulation and complement cascades, and fatty acid metabolism, while the IFNlow group was similar to the controls.
Conclusion
The IFN type I signature defines a subgroup of patients with RA, with a distinct biomolecular phenotype, characterised by increased activity of the innate defence system, coagulation and complement cascades, and fatty acid metabolism.
doi:10.1136/ard.2006.063412
PMCID: PMC1954704  PMID: 17223656
16.  TWEAK and its receptor Fn14 in the synovium of patients with rheumatoid arthritis compared to psoriatic arthritis and its response to tumour necrosis factor blockade 
Annals of the Rheumatic Diseases  2009;69(1):301-304.
Objective:
To investigate the expression of tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor inducible 14 (Fn14) in the inflamed synovium of patients with arthritis, as TWEAK blockade has been observed to have a beneficial effect in an animal model of rheumatoid arthritis (RA).
Methods:
Synovial tissue (ST) biopsies were obtained from 6 early, methotrexate-naive patients with RA as well as 13 patients with RA and 16 patients with psoriatic arthritis (PsA) who were matched for treatment and disease duration. Serial ST samples were obtained from a separate cohort of 13 patients with RA before and after infliximab treatment. TWEAK and Fn14 expression was evaluated by immunohistochemistry and digital image analysis.
Results:
TWEAK and Fn14 were clearly expressed in ST of patients with RA and PsA. TWEAK expression was significantly higher in RA (sub)lining samples compared to PsA (p = 0.005 and p = 0.014, respectively), but Fn14 expression was comparable. Double immunofluorescence showed TWEAK and Fn14 expression on fibroblast-like synoviocytes and macrophages, but not T cells. Of interest, persistent TWEAK and Fn14 expression was found after anti-TNF therapy.
Conclusions:
TWEAK and Fn14 are abundantly expressed in the inflamed synovium of patients with RA and PsA. This raises the possibility that blocking TWEAK/Fn14 signalling could be of therapeutic benefit in inflammatory arthritis.
doi:10.1136/ard.2008.090548
PMCID: PMC2789939  PMID: 19147618
17.  Standardisation of synovial tissue infiltrate analysis: how far have we come? how much further do we need to go? 
Annals of the Rheumatic Diseases  2005;65(1):93-100.
Changes in cellular infiltrate and expression of cytokines, chemokines, and cell adhesion molecules as a result of therapeutic interventions in rheumatoid arthritis can be demonstrated in the synovial membrane. However, before synovial tissue analysis can be used as an outcome measure in such studies, standardisation of the site and method of synovial tissue acquisition, methods of tissue processing, and appropriate methods of detection and measurement of cell lineage specific markers and relevant biological proteins is needed.
doi:10.1136/ard.2005.036905
PMCID: PMC1797968  PMID: 15975970
immunohistochemistry; inflammatory arthritis; standardisation; synovial tissue
18.  Sustained changes in lipid profile and macrophage migration inhibitory factor levels after anti-tumour necrosis factor therapy in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2008;68(8):1316-1321.
Background:
Macrophage migration inhibitory factor (MIF) has recently emerged as an important cytokine possibly linking rheumatoid arthritis (RA) and atherogenesis. Because atherogenesis is accelerated in RA this study was conducted to investigate whether anti-tumour necrosis factor (TNF) therapy could lead to sustained downregulation of systemic MIF levels and improvement in lipid profiles.
Methods:
Fifty RA patients with active disease (disease activity score in 28 joints (DAS28) ⩾3.2), who started adalimumab therapy at 40 mg every other week, were included. At baseline, weeks 16 and 52 serum levels of MIF and lipids were assessed. In addition, the DAS28 and serum C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR) were determined.
Results:
After 16 weeks of adalimumab therapy, both DAS28 and MIF levels were significantly decreased (p<0.001 and p = 0.020, respectively). This was sustained up to week 52 (p<0.001 and p = 0.012, respectively). CRP levels and ESR were significantly reduced after 16 and 52 weeks of adalimumab therapy (p<0.001). High-density lipoprotein cholesterol levels increased at week 16 (p<0.001), but returned to baseline at week 52. Apolipoprotein (apo) A-I levels increased at week 16 (p<0.001) and remained stable (p = 0.005). This resulted in an improved apo B/A-I ratio.
Conclusions:
The results underline the sustained downregulation of MIF as a potential new mechanism by which anti-TNF therapy might reduce vascular inflammation, and as such perhaps cardiovascular morbidity in RA patients. This hypothesis is supported by an improved apo B/A-I ratio as well as reduced CRP levels in these patients.
doi:10.1136/ard.2007.086728
PMCID: PMC2703704  PMID: 18723565
19.  A prospective, randomised, placebo-controlled study to identify biomarkers associated with active treatment in psoriatic arthritis: effects of adalimumab treatment on synovial tissue 
Annals of the Rheumatic Diseases  2008;68(8):1303-1309.
Objective:
To determine which of the changes in synovial tissue correlates best with clinical response associated with effective therapy (adalimumab) to facilitate the planning of future studies with therapeutic agents for psoriatic arthritis (PsA).
Methods:
A total of 24 patients with active PsA were randomised to receive adalimumab (n = 12) or placebo (n = 12) for 4 weeks. Synovial biopsies were obtained before and after 4 weeks of treatment. Immunohistochemical analysis was performed to characterise the cell infiltrate, expression of cytokines and matrix metalloproteinases (MMPs) and vascularity. Sections were analysed by digital image analysis. Statistical analysis was performed using covariance analysis.
Results:
The mean Disease Activity Score in 28 joints (DAS28) after 4 weeks was 1.92 units lower (95% confidence interval (CI) 1.07 to 2.77) after adalimumab therapy compared with placebo. Paired pretreatment and post-treatment synovial samples were available from 19 patients. Many cell types were reduced after adalimumab treatment compared to placebo. After applying a ranked analysis of covariance (ANCOVA) model to correct for baseline imbalances, a significant effect of treatment was observed on CD3-positive cells: there was a median reduction of 248 cells/mm2 after adalimumab versus placebo treatment (p = 0.035). In addition, the expression of MMP13 was significantly reduced after active treatment: the integrated optical density (IOD)/mm2 was 18 190 lower after adalimumab treatment as compared to placebo (p = 0.033).
Conclusion:
Adalimumab therapy in PsA is associated with a marked reduction in T cell infiltration and MMP13 expression in synovial tissue, suggesting that these parameters could be used as biomarkers that are sensitive to change after active treatment in small proof of concept studies in PsA.
doi:10.1136/ard.2008.091389
PMCID: PMC2703703  PMID: 18647851
21.  Bone mineral density in rheumatoid arthritis patients 1 year after adalimumab therapy: arrest of bone loss 
Annals of the Rheumatic Diseases  2008;68(3):373-376.
Objective:
To explore the effects of anti-tumour necrosis factor (TNF)α antibody therapy on bone mineral density (BMD) of the lumbar spine and femur neck in patients with rheumatoid arthritis (RA).
Methods:
A total of 50 patients with active RA (DAS28⩾3.2) who started adalimumab (40 mg subcutaneously/2 weeks) were included in an open label prospective study. All patients used stable methotrexate and were allowed to use prednisone (⩽10 mg/day). The BMD of the lumbar spine and femur neck was measured before and 1 year after start of treatment.
Results:
Disease activity at baseline (28-joint Disease Activity Score (DAS28)) and disease duration were inversely correlated with femoral neck BMD and lumbar spine BMD (p<0.05). Mean BMD of lumbar spine and femur neck remained unchanged after 1 year of adalimumab therapy (+0.3% and +0.3%, respectively). Of interest, a beneficial effect of prednisone on change in femur neck BMD was observed with a relative increase with prednisone use (+2.5%) compared to no concomitant prednisone use (−0.7%), (p = 0.015).
Conclusion:
In contrast to the progressive bone loss observed after conventional disease-modifying antirheumatic drug therapy, TNF blockade may result in an arrest of general bone loss. Consistent with previous observations, the data also suggest that the net effect of low-dose corticosteroids on BMD in RA may be beneficial, possibly resulting from their anti-inflammatory effects.
doi:10.1136/ard.2008.091611
PMCID: PMC2945478  PMID: 18408246
22.  The clinical response to infliximab in rheumatoid arthritis is in part dependent on pretreatment tumour necrosis factor α expression in the synovium 
Annals of the Rheumatic Diseases  2007;67(8):1139-1144.
Objective:
To determine whether the heterogeneous clinical response to tumour necrosis factor (TNF)α blocking therapy in rheumatoid arthritis (RA) can be predicted by TNFα expression in the synovium before initiation of treatment.
Methods:
Prior to initiation of infliximab treatment, arthroscopic synovial tissue biopsies were obtained from 143 patients with active RA. At week 16, clinical response was evaluated using the 28-joint Disease Activity Score (DAS28). Immunohistochemistry was used to analyse the cell infiltrate as well as the expression of various cytokines, adhesion molecules and growth factors. Stained sections were evaluated by digital image analysis. Student t tests were used to compare responders (decrease in DAS28 ⩾1.2) with non-responders (decrease in DAS28 <1.2) and multivariable regression was used to identify the independent predictors of clinical response.
Results:
Synovial tissue analysis confirmed our hypothesis that the baseline level of TNFα expression is a significant predictor of response to TNFα blocking therapy. TNFα expression in the intimal lining layer and synovial sublining were significantly higher in responders than in non-responders (p = 0.047 and p = 0.008, respectively). The numbers of macrophages, macrophage subsets and T cells (all able to produce TNFα) were also significantly higher in responders than in non-responders. The expression of interleukin (IL)1β, IL6, IL18, IL10, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was not associated with response to anti-TNFα treatment.
Conclusion:
The effects of TNFα blockade are in part dependent on synovial TNFα expression and infiltration by TNFα producing inflammatory cells. Clinical response cannot be predicted completely, indicating involvement of other as yet unknown mechanisms.
doi:10.1136/ard.2007.080440
PMCID: PMC2564801  PMID: 18055470
23.  Synovial tissue response to rituximab: mechanism of action and identification of biomarkers of response 
Annals of the Rheumatic Diseases  2007;67(7):917-925.
Objective:
To investigate the synovial tissue in patients with rheumatoid arthritis (RA) treated with rituximab and to identify possible predictors of clinical response.
Methods:
A total of 24 patients with RA underwent synovial biopsy before, 4 and 16 weeks after initiation of rituximab treatment (without peri-infusional corticosteroids to prevent bias). Immunohistochemical analysis was performed and stained sections were analysed by digital image analysis. Linear regression analysis was used to identify predictors of clinical response.
Results:
The 28-joint Disease Activity Score (DAS28) was unaltered at 4 weeks, but significantly reduced at 16 and 24 weeks. Serum levels of IgM-rheumatoid factor (RF) decreased significantly at 24 weeks and anti-citrullinated peptide antibody (ACPA) levels at 36 weeks. Peripheral blood B cells were depleted at 4 weeks and started to return at 24 weeks. Synovial B cells were significantly decreased at 4 weeks, but were not completely depleted in all patients; there was a further reduction at 16 weeks in some patients. We found a significant decrease in macrophages at 4 weeks, which was more pronounced at 16 weeks. At that timepoint, T cells were also significantly decreased. The reduction of plasma cells predicted clinical improvement at 24 weeks.
Conclusions:
The results support the view that B cells orchestrate local cellular infiltration. The kinetics of the serological as well as the tissue response in clinical responders are consistent with the notion that rituximab exerts its effects in part by an indirect effect on plasma cells associated with autoantibody production, which could help explain the delayed response after rituximab treatment.
Trial registration number: ISRCTN05568900.
doi:10.1136/ard.2007.080960
PMCID: PMC2564787  PMID: 17965121
24.  Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years 
PLoS Pathogens  2012;8(9):e1002889.
CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.
Author Summary
Several viruses have found ways to evade the human immune system and cause latent infections. Examples include HIV and herpes-viruses. Most humans carry these herpes-viruses. The human immune system mounts continuous responses against these viruses to prevent them from causing disease. If this balance is disturbed, these viruses can cause extensive pathology. We do not know how the immune response against these viruses evolves over time. Understanding this response might help to understand why the immune system does not clear these viruses and might help in preventive and therapeutic strategies. Here we used a new technology that allowed us to track virus specific immune cells (CD8+ T cells) over time in a quantitative manner. When we used this technology to study the evolution of latent responses against herpes-viruses (from infection until 5 years later) we found that immune responses were very rigid and did not evolve over time. Collectively our data shows that – for these herpes-viruses – the initial immune response is maintained despite the fact that this does not result in clearance of the virus. Therefore, if a virus survives the initial response, it will not be cleared in the future. This is an important consideration in understanding latent infection and for vaccination-design.
doi:10.1371/journal.ppat.1002889
PMCID: PMC3460621  PMID: 23028307

Results 1-25 (29)