Influence of image artifacts on image-based computer simulations of the cardiac electrophysiology.

Myocardial infarct patients have an increased risk of scar-based ventricular tachycardia. Late gadolinium enhanced magnetic resonance (MR) imaging provides the geometric extent of myocardial infarct. Computational electrophysiological models based on such images can provide a personalized prediction of the patient's tachycardia risk. In this work, the effect of respiratory slice alignment image artifacts on image-based electrophysiological simulations is investigated in two series of models. For the first series, a clinical MR image is used in which slice translations are applied to artificially induce and correct for slice misalignment. For the second series, computer simulated MR images with and without slice misalignments are created using a mechanistic anatomical phantom of the torso. From those images, personalized models are created in which electrical stimuli are applied in an attempt to induce tachycardia. The response of slice-aligned and slice-misaligned models to different interval stimuli is used to assess tachycardia risk. The presented results indicate that slice misalignments affect image-based simulation outcomes. The extent to which the assessed risk is affected is found to depend upon the geometry of the infarct area. The number of unidirectional block tachycardias varied from 1 to 3 inducible patterns depending on slice misalignment severity and, along with it, the number of tachycardia inducing stimuli locations varied from 2 to 4 from 6 different locations. For tachycardias sustained by conducting channels through the scar core, no new patterns are induced by altering the slice alignment in the corresponding image. However, it affected the assessed risk as tachycardia inducing stimuli locations varied from 1 to 5 from the 6 stimuli locations. In addition, if the conducting channel is not maintained in the image due to slice misalignments, the channel-dependent tachycardia is not inducible anymore in the image-based model.

Human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA ) plays an important role in the removal of intravascular fibrin deposits and has several physiological roles and pathological activities in the brain. Its production by many other cell types suggests that t-PA has additional functions outside the vascular and central nervous system. Activity of t-PA is regulated at the level of its gene transcription, its mRNA stability and translation, its storage and regulated release, its interaction with cofactors that enhance its activity, its inhibition by inhibitors such as plasminogen activator inhibitor type 1 or neuroserpin, and its removal by clearance receptors. Gene transcription of t-PA is modulated by a large number of hormones, growth factors, cytokines or drugs and t-PA gene responses may be tissue-specific. The aim of this review is to summarise current knowledge on t-PA function and regulation of its pericellular activity, with an emphasis on regulation of its gene expression.

NF-κB is activated from endosomal compartments in antiphospholipid antibodies-treated human monocytes.

BACKGROUND: The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll-like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA. OBJECTIVE: To study the mechanisms of cell activation by aPLA, leading to pro-coagulant and pro-inflammatory responses. METHODS AND RESULTS: For this study, we used purified antibodies from the plasmas of 10 different patients with APS and healthy donors. We demonstrate that aPLA, but not control IgG, co-localizes with TLR2 and TLR1 or TLR6 on human monocytes. Blocking antibodies to TLR2, TLR1 or TLR6, but not to TLR4, decreased TNF and tissue factor (TF) responses to aPLA. Pharmacological and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in cell activation by aPLA. In addition, soluble aPLA induced NF-κB activation, while bead-immobilized aPLA beads, which cannot be internalized, were unable to activate NF-κB. Internalization of aPLA in monocytes and NF-κB activation were dependent on the presence of CD14. CONCLUSION: We show that TLR2 and its co-receptors, TLR1 and TLR6, contribute to the pathogenicity of aPLA, that aPLA are internalized via clathrin- and CD14-dependent endocytosis and that endocytosis is required for NF-κB activation. Our results contribute to a better understanding of the APS and provide a possible therapeutic approach.

Ex vivo pulsatile perfusion of human saphenous veins induces intimal hyperplasia and increased levels of the plasminogen activator inhibitor 1.

Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.

Retinoids and activation of PKC induce tissue-type plasminogen activator expression and storage in human astrocytes.

BACKGROUND: Emerging data demonstrate important roles for tissue-type plasminogen activator (t-PA) in the central nervous system (CNS). In contrast to endothelial cells, little is known about the regulation of t-PA gene expression and secretion in astrocytes. OBJECTIVES: The aims of the present study were to investigate whether t-PA gene expression is regulated by retinoids and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in human astrocytes, and to study whether t-PA is stored and subject to regulated release from these cells, as with endothelial cells. METHODS: Native human astrocytes were treated with RA and/or PMA. mRNA was quantified by real-time RT-PCR and protein secretion determined by ELISA. Intracellular t-PA immunoreactivity in astrocytes was examined by immunocyto- and histochemistry. RESULTS: RA and/or PMA induced a time-dependent increase in t-PA mRNA and protein levels in astrocytes, reaching 10-fold after combined treatment. This was associated with increased amounts of t-PA storage in intracellular granular structures. Both forskolin and histamine induced regulated release of t-PA. The presence of t-PA in reactive astrocytes was confirmed in human brain tissue. CONCLUSIONS: These data show that RA and PKC activation induce a strong up-regulation of t-PA expression in astrocytes, and increased intracellular storage pools. Moreover, a regulated release of t-PA can be induced from these cells. This raises the possibility that astrocytes contribute to the regulation of extracellular t-PA levels in the CNS.

Tissue-type plasminogen activator has antiangiogenic properties without effect on tumor growth in a rat C6 glioma model.

Tissue-type plasminogen activator (tPA) plays a major role in the fibrinolytic system. According to several reports, tPA may also have antiangiogenic properties, especially in combination with a free sulfhydryl donor (FSD). In the rat C6 glioma model, in vitro and in vivo tPA synthesis by glioma cells is enhanced by differentiation therapy. To address the antiangiogenic potential of tPA in this model, tPA was overexpressed in glioma tumors by ex vivo transduction of C6 cells with a lentiviral vector encoding tPA. The transduced cells were subcutaneously implanted into nude mice. Gene transfer allowed for efficient synthesis of tPA by the C6 tumors. Although the treatment of tPA+ tumor-bearing animals with the FSD captopril generated angiostatin in situ and reduced endothelial vascularization of the tumors, it had no effect on tumor growth. Alternative mechanisms could account for this lack of effect and consequently have important implications for vascular the treatment of glioblastoma.

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

BACKGROUND: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. OBJECTIVE: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. METHODS: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. RESULTS: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on Matrigel(TM) and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. CONCLUSIONS: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.

Synovial inflammation does not change in the absence of effective treatment: implications for the use of synovial histopathology as biomarker in early phase clinical trials in rheumatoid arthritis.

OBJECTIVES: To determine the impact on synovial histopathology of changes in clinical disease activity in the absence of effective treatment. METHODS: Twelve patients with active RA not receiving effective treatment were studied over a 14 week period. Synovial biopsy specimens obtained at baseline and week 14 were analysed by histology and immunohistochemistry. RESULTS: Over the course of 14 weeks, there was a trend towards a decrease of the DAS28, with 7/12 patients being good or moderate DAS28 responders despite the absence of effective treatment. Patients' assessment of global disease activity and swollen joint count both decreased significantly. Histologically, there was a decrease of lining layer hyperplasia and lymphoid aggregates, a similar trend for vascularity, but there was no effect on global synovial infiltration. Accordingly, there was no decrease of the cellular infiltration with T lymphocytes (CD3, CD4, CD8), B lymphocytes (CD20), plasma cells (CD38), dendritic cells (CD1a, CD83), and even an increase of CD163+ sublining macrophages, with a similar trend for CD68+ sublining macrophages. The changes in DAS28 scores in these patients did not correlate with changes in histological variables, with the exception of an inverse correlation with plasma cells. Remarkably, even in the DAS28 responders, no significant changes in synovial inflammatory infiltration were noted. CONCLUSIONS: Despite variations in global disease activity, synovial inflammatory infiltration did not change significantly in the absence of effective treatment. The lack of a placebo effect on synovial markers of treatment response such as sublining macrophages can facilitate conclusive early phase trials with small numbers of patients with RA.

Involvement of neurotrophins and their receptors in spondyloarthritis synovitis: relation to inflammation and response to treatment.

OBJECTIVE: To investigate whether expression of the four members of the neurotrophin (NT) family and their four corresponding receptors is related to synovial inflammation in patients with spondyloarthritis (SpA). MATERIAL AND METHODS: Synovial fluid (SF) and serum NTs and their receptors were measured by ELISA. Immunohistochemistry was used for synovial tissue biopsy specimens from patients with SpA, rheumatoid arthritis, and osteoarthritis (OA). In SpA synovium, immunoreactivity of the receptors trkA and NGFRp75 was also assessed before and after 12 weeks of treatment with the monoclonal anti-tumour necrosis factor alpha antibody, infliximab. RESULTS: mRNA transcripts of all NTs and receptors were expressed in the inflamed synovium. At the protein level, brain derived neurotrophic factor and NT-3 were significantly higher in the SF of patients with SpA than in those with OA. In contrast, ELISA of serum samples showed that the highest member in SpA was NT-4. Immunohistochemistry demonstrated that the NT receptors trkA and NGFRp75 were highly expressed in the inflamed synovium of patients with SpA, correlating with vascularity and lymphoid aggregates, respectively. Additionally, immunoreactivity of both receptors was significantly decreased after infliximab treatment. CONCLUSIONS: NTs and their receptors are expressed in inflamed peripheral joints of patients with SpA. Their expression is not constitutive but related to inflammation and they may be involved in the local disease processes.

Anti-citrullinated peptide antibodies may occur in patients with psoriatic arthritis.

BACKGROUND: Anti-cyclic citrullinated peptide (anti-CCP) antibodies are considered highly specific markers of rheumatoid arthritis. Despite the high specificity of the test, anti-CCP antibodies have also been observed in psoriatic arthritis. OBJECTIVE: To determine the frequency of anti-CCP antibodies in psoriatic arthritis and to describe the clinical characteristics of such patients. METHODS: Serum samples from 192 patients with psoriatic arthritis were analysed for anti-CCP antibodies. A previously defined cut off point was applied at a specificity level of > or =98.5% (42 U/ml). Antibodies against pepA and pepB (two synthetic citrullinated peptides) were determined on samples containing anti-CCP antibodies by line immune assay. The swollen joint count and the numbers of affected joints (present or past) were recorded. Clinical features were noted and if available radiographs of hands and feet were scored for erosions. Rheumatoid factor was determined in all samples. RESULTS: Anti-CCP antibodies were found in 15 patients (7.8%); 13 of 15 anti-CCP2 positive samples were also positive for anti-pepA or pepB antibodies. The prevalence of anti-CCP antibodies was higher than expected in view of the highly specific cut off applied in the test. Detailed analysis of the clinical and radiological features makes it improbable that the high prevalence of anti-CCP antibodies resulted solely from concomitant psoriasis and rheumatoid arthritis or from misclassification. CONCLUSIONS: Anti-CCP antibodies may be present in patients with psoriatic arthritis. Although some of the present cohort could have had psoriasis with concomitant rheumatoid arthritis, a proportion at least had the typical characteristics of psoriatic arthritis as the primary diagnosis.

The effect of TNFalpha blockade on the antinuclear antibody profile in patients with chronic arthritis: biological and clinical implications.

Since the first proof of efficacy of TNFalpha blockade, both the number of patients treated worldwide and the number of indications for treatment with TNFalpha blockers have grown steadily. Surprisingly, the profound immunomodulation induced by anti-TNFalpha therapy is associated with a relatively low incidence of immune-related complications such as lupus-like syndromes and demyelinating disease. This contrasts sharply with the prominent induction of autoantibodies such as antinuclear antibodies (ANA) and anti-dsDNA antibodies during TNFalpha blockade. Although this phenomenon has been recognized for several years, the clinical and biological implications are not yet fully understood. In this review, recent studies analysing the effect of TNFalpha blockade (infliximab and etanercept) on the ANA profile in autoimmune arthritis will be discussed. Taken together, these reports indicate that the prominent ANA and anti-dsDNA autoantibody response is 1) not a pure class effect of TNFalpha blockers, 2) independent of the disease background, 3) largely restricted to the induction of short-term IgM anti-dsDNA antibodies, and 4) not associated with other serological or clinically relevant signs of lupus. Nevertheless, a careful follow-up of patients treated with TNFalpha blockers remains mandatory, including monitoring for lupus-like characteristics.

Rheumatoid factor, but not anti-cyclic citrullinated peptide antibodies, is modulated by infliximab treatment in rheumatoid arthritis.

OBJECTIVES: To analyse the effect of infliximab on IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies, and determine whether baseline autoantibody titres (IgM RF and anti-CCP antibodies) are associated with changes in acute phase reactants. PATIENTS AND METHODS: 62 patients with refractory RA were treated with infliximab combined with methotrexate. At baseline and week 30, serum samples were tested for IgM RF by two agglutination assays, and for anti-CCP antibodies by an ELISA. Percentage change in C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) was calculated. RESULTS: At baseline and week 30 RF titres were reduced significantly during infliximab treatment (p<0.001 and p = 0.038, respectively), whereas anti-CCP antibodies were unchanged (p = 0.240). Baseline IgM RF titres, but not anti-CCP antibodies, correlated inversely with changes in CRP and ESR during treatment. Patients with a marked decrease in acute phase reactants had lower IgM RF titres than those with a smaller decrease in CRP and ESR; no significant differences were found for anti-CCP antibodies. CONCLUSION: The differential effect of infliximab treatment on IgM RF and anti-CCP antibodies, and the different predictive value on changes in acute phase reactants during infliximab treatment support the existing evidence that RF and anti-CCP antibodies are independent autoantibody systems in RA.

Histological evidence that infliximab treatment leads to downregulation of inflammation and tissue remodelling of the synovial membrane in spondyloarthropathy.

OBJECTIVE: To confirm and extend the immunopathological evidence of effects of infliximab on the synovium in active spondyloarthropathy. METHODS: Synovial biopsies obtained in patients with spondyloarthropathy at baseline and week 12 were stained and scored by two independent observers. Two study populations were evaluated: I, a cohort of 10 patients treated with 5 mg/kg infliximab at week 0, 2, and 6, plus three placebo treated patients; and II, a pooled cohort of 20 patients fulfilling identical inclusion and exclusion criteria and treated with the same loading dose regimen. RESULTS: In study population I, treatment with infliximab induced reduction in synovial lining layer thickness (p = 0.015), endothelial activation (E-selectin, p = 0.034), and inflammatory cell infiltration with neutrophils (p = 0.041), macrophages (p = 0.034), and T cells (p = 0.026), but not with B cells and plasma cells; no such trends were observed in the placebo treated patients. Besides confirming the highly significant downregulation of inflammation, analysis of cohort II showed structural changes such as normalisation of lining layer thickness (p = 0.030), reduction in the number of blood vessels (p = 0.039), and downregulation of follicular organisation (p = 0.050). No differences in histopathological response were observed between spondyloarthropathy subtypes. CONCLUSIONS: Profound immunomodulatory changes in the synovium parallel the clinical benefit in patients with spondyloarthropathy treated with infliximab, independently of the subtype. The study provides histological evidence that TNF alpha blockade not only downregulates inflammation but also leads to tissue remodelling.

Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations.

BACKGROUND: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies can be detected in rheumatoid arthritis (RA) sera. OBJECTIVE: To determine the diagnostic values of RF, anticitrullinated protein antibodies, and the shared epitope (SE), and their associations with radiological progression rates and extra-articular manifestations. METHODS: Population 1 consisted of sera from 315 patients, consecutively sent for detection of anticitrullinated protein antibodies, of which 264 were used to determine the sensitivity and specificity of RF and of antibodies against three synthetic citrullinated peptides: peptide A (pepA), peptide B (pepB), and CCP2. Population 2 consisted of sera from 180 longstanding RA patients and was used to determine associations of RA associated antibodies and the SE with radiological progression rates and extra-articular manifestations. Antibodies to pepA and pepB were detected by line immunoassay, and antibodies to CCP2 by ELISA. HLA Class II typing was performed by LiPA. RESULTS: In population 1, we defined adapted cut offs corresponding to a specificity of >/=98.5%. This yielded the following sensitivities: RF 12.8%; anti-pepA antibodies 63.6%; anti-pepB antibodies 54.2%; and anti-CCP2 antibodies 73.7%. In population 2, significant differences in radiological progression rates were found between positive and negative patients for different RA antibodies and the SE. RF, but not anticitrullinated protein antibodies or the SE, were more frequent in patients with extra-articular manifestations. CONCLUSION: A valid comparison of RA associated antibodies shows superior sensitivity of the anticitrullinated protein antibodies compared with RF. The presence of RA associated antibodies and the SE are indicative for poorer radiological outcome, and presence of extra-articular manifestations is associated with RF but not with anticitrullinated protein antibodies.

Aspirin inhibits endothelial cell activation induced by antiphospholipid antibodies.

BACKGROUND: Antiphospholipid antibodies (APLA) have been shown to activate endothelial cells (EC) in vitro, as documented by an increased expression of tissue factor as well as leukocyte adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. Currently, treatment of patients with the antiphospholipid syndrome includes aspirin, particularly for women with recurrent fetal loss. OBJECTIVE: The present study was undertaken to investigate whether aspirin interferes with EC activation induced by APLA in vitro. METHODS: IgG from 14 patients with APLA, and suffering from thrombotic complications and/or pregnancy morbidity, and control IgG were tested for their ability to modify the expression of VCAM-1 in human umbilical vein endothelial cells. VCAM-1 antigen was measured by flow cytometry and its mRNA by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Incubation of EC with IgG from most of the patients led to a higher VCAM-1 expression compared with incubation with control IgG. The effect of aspirin was studied for the eight IgG samples that induced a more than 50% increase in VCAM-1. Aspirin (10 mm) treatment of the cells significantly reduced the VCAM-1 response to these APLA. CONCLUSIONS: Our results indicate that besides its antiplatelet properties, aspirin exerts a protective effect towards APLA at the EC level by decreasing leukocyte adhesion molecule expression at the cell surface.

Effects of statins on adhesion molecule expression in endothelial cells.

BACKGROUND: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. OBJECTIVES: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. METHODS: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4-6 h activation with tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). RESULTS: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-alpha and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-alpha-mediated overexpression of E-selectin. CONCLUSIONS: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-alpha or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

Systematic safety follow up in a cohort of 107 patients with spondyloarthropathy treated with infliximab: a new perspective on the role of host defence in the pathogenesis of the disease?

BACKGROUND: Recent studies with infliximab indicate the therapeutic potential of tumour necrosis factor alpha blockade in spondyloarthropathy (SpA). Because defective host defence is implicated in the pathogenesis of SpA, the potential side effects of this treatment due to impact on the antimicrobial defence are a major concern. OBJECTIVE: To report systematically the adverse events seen in a large cohort of patients with SpA treated with infliximab, with special attention to bacterial infections. PATIENTS AND METHODS: 107 patients with SpA were treated with infliximab for a total of 191.5 patient years. All serious and/or treatment related adverse events were reported. RESULTS: Eight severe infections occurred, including two reactivations of tuberculosis and three retropharyngeal abscesses, and six minor infections with clear bacterial focus. One patient developed a spinocellular carcinoma of the skin. No cases of demyelinating disease or lupus-like syndrome were seen. Two patients had an infusion reaction, which, however, did not relapse during the next infusion. Finally, three patients with ankylosing spondylitis developed palmoplantar pustulosis. All patients recovered completely with adequate treatment, and infliximab treatment had to be stopped in only five patients with severe infections. CONCLUSIONS: Although the global safety of infliximab in SpA is good compared with previous reports in rheumatoid arthritis and Crohn's disease, the occurrence of infections such as tuberculosis and retropharyngeal abscesses highlights the importance of careful screening and follow up. Focal nasopharyngeal infections and infection related symptoms, possibly induced by streptococci, occurred frequently, suggesting an impairment of specific host defence mechanisms in SpA.