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Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Objectives The aim of this study was to investigate cardiac involvement detected by ECG in patients with idiopathic inflammatory myopathies (IIMs) and to evaluate possible associations between the autoantibody profile and ECG changes in these patients. Methods In a Scandinavian cross-sectional study, patients were included from two Danish centres and one Swedish centre. Resting 12-lead ECG was investigated in 261 patients with IIM compared with 102 patients with systemic sclerosis (SSc) and 48 healthy controls (HCs). ECG changes were correlated to clinical manifestations and myositis-specific and myositis-associated autoantibodies (MSAs and MAAs, respectively). Results Patients with IIM had a longer mean corrected QT (QTc) duration and more frequently presented with prolonged QTc (≥450 ms; P = 0.038) compared with HCs. A longer QTc duration was recorded in SSc compared with IIM [433 ms (s.d. 23) vs 426 (24); P = 0.011], yet there was no significant difference in the fraction with prolonged QTc (SSc: 22%, IIM: 16%; P = 0.19). In multivariable regression analyses, anti-Mi2 (P = 0.01, P = 0.035) and anti-Pl-7 (P = 0.045, P = 0.014) were associated with QTc duration and prolonged QTc in IIM. Elevated CRP was associated with prolonged QTc (P = 0.041). Conclusion The presence of QTc abnormalities was as common in patients with IIM as in patients with SSc, including prolonged QTc seen in almost one-fifth of the patients. Anti-Mi2, anti-Pl-7 and elevated CRP may serve as biomarkers for cardiac disease in IIM, but needs to be confirmed in a larger prospective study.

A co-culture assay with human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) was used to study whether selected angiogenesis inhibitors were able to inhibit differentiation and network formation of HUVECs in vitro. The effect of the inhibitors was determined by the morphology and the calculated percentage area covered by HUVECs. Neutralizing VEGF with avastin and polyclonal goat anti-VEGF antibody and inhibiting VEGFR2 with sorafenib and vatalanib resulted in the formation of HUVEC clusters of variable sizes as a result of inhibited EC differentiation. Furthermore, numerous inhibitors of the VEGF signaling pathways were tested for their effect on the growth and differentiation of HUVECs. The effects of these inhibitors did not reveal a cluster morphology, either individually or when combined to block VEGFR2 downstream pathways. Only the addition of N-methyl-p-bromolevamisole revealed a similar morphology as when targeting VEGF and VEGFR2, meaning it may have an inhibitory influence directly on VEGFR signaling. Additionally, several nuclear receptor ligands and miscellaneous compounds that might affect EC growth and differentiation were tested, but only dexamethasone gave rise to cluster formation similarly to VEGF-neutralizing compounds. These results point to a link between angiogenesis, HUVEC differentiation and glucocorticoid receptor activation.

Background Sporadic inclusion body myositis (sIBM) is a subgroup of the idiopathic inflammatory myopathies (IIMs) and is characterized by both degenerative and autoimmune features. Unlike other IIMs, myositis-specific autoantibodies had not been found in sIBM patients until recently. Objectives We aimed to establish the prevalence and clinical associations of anti-cN-1A in a large Danish cohort with connective tissue diseases (CTD). Methods In a cross-sectional study design, a total of 568 participants (183 IIMs (55 sIBM, 128 non-sIBM: dermatomyositis (DM), polymyositis (PM), immune-mediated necrotizing myopathy (IMNM)), 164 systemic lupus erythematosus (SLE), 121 systemic sclerosis (SSc), and 100 blood donors (BD)) were tested for the presence of: a) cN-1A autoantibodies using a commercial Anti-cN-1A ELISA and b) myositis specific and associated autoantibodies (anti-Jo-1, anti-PL-7, anti-PL-12, anti-OJ, anti-EJ, anti-SRP, anti-Mi-2α/β, anti-MDA5, anti-TIF1γ, anti-NPX2, anti-SAE1, anti-PM-Scl75, anti-PM-Scl100, anti-Ro52, anti-Ku) using a commercial lineblot kit. The patients were classified according to the ACR classification criteria for IIMs (2017), SLE (1997) and SSc (1980), respectively. Clinical features were compared between anti-cN-1A positive and anti-cN-1A negative sIBM patients using two-sample t-test and Mann-Whitney test for continuous normally and non-normally distributed data and Chi-square test for categorical data, as appropriate. Results: cN-1A antibodies were predominantly found in IIM patients. In the sIBM cohort, 24 patients (43.6%) were anti-cN-1A positive vs. 25 (19.5%) in the non-sIBM myositis cohort and 17 (10%) in the SLE cohort. None of the participants in the SSc or the BD cohorts had presence of anti-cN-1A. Anti-cN-1A positivity had a sensitivity of 43.6% and a specificity of 91.8% for sIBM. The positive and negative predictive values were 36.4% and 93.8%, respectively. There was no significant difference in gender, age at study entry, age at symptom onset, duration of symptoms or max creatine kinase (CK) levels during disease course between the anti-cN-1A positive and negative sIBM patients. Dysphagia was present in 19 (79%) of the anti-cN-1A positive and in 17 (55%) of the anti-cN-1A negative sIBM patients (P = 0.06). Conclusion Antibodies against cN-1A are the first and so far the only serological marker for sIBM. Our data showed that cN-1A autoantibodies are specific for sIBM and further corroborate the potential diagnostic role of cN-1A autoantibodies in this distinct subgroup of myositis. Disclosure of Interests: Louise Pyndt Diederichsen: None declared, Sine Sondergaard Korsholm: None declared, Line Vinderslev Iversen: None declared, Christoffer Tandrup Nielsen: None declared, Marie-Louise From Hermansen: None declared, Soren Jacobsen: None declared, Nanna Witting: None declared, Markus E. Krogager: None declared, Tina Friis Grant/research support from: Anti cN-1A ELISA kits and EUROLINE Autoimmune Inflammatory Myopathies 16 AG kits have been provided for a project free of charge from Euroimmun.

Background Idiopathic inflammatory myopathies (IIM) are rare devastating diseases characterized by progressive muscle weakness and muscle fatigue. IIM frequently affects other organs pointing to IIM as a systemic inflammatory disease. Cardiac involvement is associated with poor prognosis. The symptoms are often subclinical and therefore overlooked. QTc prolongation has been detected in patients with systemic sclerosis (SSc). Autoantibodies are important diagnostic tools to confirm IIM and are present in approx. 60% of IIM patients. Autoantibodies are increasingly being recognized as markers for specific organ involvement. A biomarker for cardiac involvement has yet to be elucidated. Objectives The aim is to generate new knowledge about cardiac involvement in IIM detected by electrocardiography (ECG) and to evaluate possible associations between autoantibodies and cardiac involvement detected on ECG in a cohort of IIM patients compared with ECG changes in a cohort of SSc patients. Methods In a Scandinavian cohort, 263 IIM patients (130 polymyositis patients, 77 dermatomyositis patients, and 56 inclusion body myositis patients) and 102 SSc patients were investigated by ECG and basic cardiovascular and disease specific assessments according to international guidelines. IIM patients were tested for myositis specific autoantibodies (MSAs; anti-Jo-1, anti-PL-7, anti-PL-12, anti-OJ, anti-EJ, anti-SRP, anti-Mi-2, anti-MDA5, anti-TIF1 γ, anti-NPX2, anti-SAE1, anti-HMGCR) and myositis associated autoantibodies (MAAs; anti-PM/Scl75, anti-PM/Scl100, anti-Ro52, anti-Ku, anti-cN1A). Results Twenty two IIM patients (8.49%) had abnormal QRS duration versus one SSc patient (1.06%) (P = 0.012). SSc patients had significantly longer QTc duration than IIM patients (QTc = 432.7 ms ± 22.2 and 426.4 ms ± 23.6, respectively) (P = 0.03). Multivariate regression analysis revealed that increased C-reactive protein (CRP) (P = 0.008), gender (P = 0.002), and hypertension (P = 0.007) were associated with QTc duration in IIM patients. Likewise, pulmonary arterial hypertension was associated with QTc duration (P In analysis of pooled data for IIM patients and SSc patients, factors associated with QTc duration were increased CRP (P = 0.005), gender (P = 0.001), and hypertension (P = 0.01). Conclusion Both IIM patients and SSc patients had ECG changes though no particular pattern was shown. The findings support our hypothesis on cardiac involvement in IIM patients. No significant association was found between presences of either myositis specific or myositis associated autoantibodies and ECG changes. This could be due to the relatively low number of each autoantibody. There is a need to conduct larger prospective studies to identify a possible autoantibody for cardiac involvement in IIM. Acknowledgement The authors would like to thank all participating patients and the study personnel at the including centres. Disclosure of interests Sine Sondergaard Korsholm: None declared, Maryam Dastmalchi: None declared, John Bonde Knudsen: None declared, axel Cosmus Diederichsen: None declared, ingrid E. Lundberg Grant/research support from: Dr. Lundberg has received honoraria from Bristol Myers Squibb and MedImmune and is currently receiving a research grant from Bristol Myers Squibb and from astra Zeneca., Consultant for: She is a scientific advisor for Bristol Myers Squibb, and aTyr, Daniel C. Andersson: None declared, Nanna Witting: None declared, Soren Jacobsen: None declared, Tina Friis Grant/research support from: anti cN-1A ELISA kits and EUROLINE autoimmune inflammatory Myopathies 16 aG kits have been provided for a project free of charge from Euroimmun., Markus E. Krogager: None declared, Louise Pyndt Diederichsen: None declared

Anti-Ro(52/60) and anti-La constitute the hallmark autoantibodies in primary Sjögren's syndrome, being present in 40-70% of sera. Several anti-Ro/La assays exist, but antibody detection appears to be assay-specific, thus the aim of this study was to compare several anti-Ro/La assays. In total, 96 sera from individuals with primary Sjögren's syndrome and 114 healthy controls were tested for anti-Ro 52/60 and anti-La in 17 immunoassays. Especially the immunoassays used for detection of anti-Ro 52 differed in their sensitivity (48-79%), while only small differences in sensitivities were observed for the anti-Ro 60 (69-77%) anti-La (39-44%) assays. Concordances of 65%, 79% and 73% for the anti-Ro 52, anti-Ro 60 and anti-La assays were found, respectively. The majority of the assays yielded high specificities, primarily ranging from 97 to 100%, except from a single anti-Ro 60 assay, which yielded a specificity of 79%. Occasionally, reactivity levels were increased in a few assays, indicating that false-positive results can be obtained when applying assays of reduced specificity. In general, the commercial assays appeared to perform better than the in-house analyses. When correcting the in-house assays for background reactivity, sensitivities were reduced by approximately 7%, 17%, and 19% for anti-Ro 52, anti-Ro 60 and anti-La assays, respectively, illustrating the pitfalls when applying immunoassays for detection of autoantibodies, which in theory may apply to commercial assays as well. Finally, increased total sensitivities were obtained when combining assays. These studies contribute to clarify the clinical utility of immunoassays for detection of autoantibodies of Ro 52, Ro 60 and La and illustrate that the most efficient strategy to maximize antibody sensitivity is to combine several assays.

One of the major targets of the autoimmune response in the rheumatic autoimmune diseases, Systemic Lupus Erythematosus and Sjogrens Syndrome, is the protein Ro60. Ro60 is known to associate with small misfolded RNAs, and is involved in RNA quality control and in enhancing cell survival during cellular stress, e.g. after ultaviolet irradiation. In this study, six monoclonal antibodies to Ro60 were analyzed in order to identify antigenic regions and the nature of these. Preliminary analyses revealed that two of the antibodies recognized continuous epitopes, while the remaining antibodies most likely recognized conformational epitopes. The continuous epitopes of Ro60 were characterised by modified immunoassays employing resin-bound peptides and free peptides. Peptide screenings located the epitopes to the N-terminus of Ro60, and further analyses indicated that the epitopes of the monoclonal antibodies TROVE2 and SSI-HYB 358-02 were located to amino acids 8-17 and 34-49, respectively. Moreover, charged amino acids were found to be especially important for antibody reactivity, although antibody reactivity of the monoclonal antibody TROVE2 primarily was found to be epitope backbone-dependent.

Autoantibody detection is a useful and obligatory tool for clinicians and researchers in diagnosing autoimmune diseases. Line blotting is a simple, sensitive, and flexible technique for fast semiquantitative detection of multiple antibodies. Line blotting enables the detection of antibodies on membrane strips coated with thin parallel lines of several purified, biochemically characterized antigens, which are fixed onto a synthetic support. Each strip can contain more than ten antigens, thus allowing simultaneous screening for multiple antibodies. Here, we describe the principle of line blotting and how the buffer composition can interfere with the results of autoantibody detection.

Autoantibody detection is a useful and obligatory tool for clinicians and researchers in diagnosing autoimmune diseases. Line blotting is a simple, sensitive, and flexible technique for fast semiquantitative detection of multiple antibodies. Line blotting enables the detection of antibodies on membrane strips coated with thin parallel lines of several purified, biochemically characterized antigens, which are fixed onto a synthetic support. Each strip can contain more than ten antigens, thus allowing simultaneous screening for multiple antibodies. Here, we describe the principle of line blotting and how the buffer composition can interfere with the results of autoantibody detection.

Background Idiopathic inflammatory myopathies (IIMs) are characterised by progressive muscle weakness and muscle fatigue. IIMs frequently appear with other organ involvement, e.g. the heart. Cardiac affection is often subclinical but associated with poor prognosis which makes early detection critical. Presence of autoantibodies is common in IIMs and autoantibodies are important biomarkers to confirm the diagnosis of IIMs. Autoantibodies may even predict organ involvement, e.g. the common myositis specific autoantibody anti-Jo1, which is a marker of lung involvement. So far, an autoantibody predicting cardiac involvement has yet to be identified. Objectives The aim of this study was to identify and estimate presence of cardiac involvement detected by electrocardiography (ECG) and to evaluate possible associations between ECG changes and autoantibodies in a Scandinavian cohort of patients with IIMs. Methods In a Scandinavian cross-sectional study, 241 patients with polymyositis (PM), dermatomyositis (DM), or inclusion body myositis (IBM) and 48 healthy controls (HCs) were investigated by ECG, basic cardiovascular assessments, and autoantibody profile including myositis specific autoantibodies (MSAs) and myositis associated autoantibodies (MAAs). Results Compared to HCs, patients with IIMs more frequently had prolongation of QTc (p=0.037) and QRS (p=0.031). All patient groups had significantly longer QTc and QRS duration than HCs. In multivariate regression analysis of patients with IIMs, increased CRP (p=0.006) was associated to increased QTc. Pooled data for patients with IIMs and HCs showed an association between diagnosis of IIM and increased QTc duration (p Conclusions Patients with IIMs, no matter of clinical subgroup, had a higher occurrence of cardiac abnormalities detected by ECG than HCs. Increased CRP and presence of any MAA were associated with increased QTc and QRS duration, respectively. These results support our notion of possible associations between inflammation and autoimmunity and cardiac affection in patients with IIMs. There is now a pressing need to set up a larger prospective study to validate the present findings. Disclosure of Interest None declared

Natural, nonribosomal cyclotetrapeptides have traditionally been a rich source of inspiration for design of potent histone deacetylase (HDAC) inhibitors. We recently disclosed the total synthesis and full HDAC profiling of the naturally occurring azumamides ( J. Med. Chem. 2013 , 56 , 6512 ). In this work, we investigate the structural requirements for potent HDAC inhibition by macrocyclic peptides using the azumamides along with a series of unnatural analogues obtained through chemical synthesis. By solving solution NMR structures of selected macrocycles and combining these findings with molecular modeling, we pinpoint crucial enzyme-ligand interactions required for potent inhibition of HDAC3. Docking of additional natural products confirmed these features to be generally important. Combined with the structural conservation across HDACs 1-3, this suggests that while cyclotetrapeptides have provided potent and class-selective HDAC inhibitors, it will be challenging to distinguish between the three major class I deacetylases using these chemotypes.

To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell tracking for years, but knowledge regarding possible cytotoxic ultrastructural changes subsequent to iron-oxide particle labeling is limited. Hence, the purpose of this study was to label MSCs with dextran-coated ultrasmall super-paramagnetic iron-oxide (USPIO) particles conjugated with the transduction sequence of trans-activator of transcription (TAT) (IODEX-TAT) and evaluate the effect of labeling on ultrastructure, viability, phenotype and proliferative capacity of the cells.MSCs were labeled with 5 and 10 μg IODEX-TAT/10(5) cells for 2, 6 and 21 hours. IODEX-TAT uptake and cellular ultrastructure were determined by electron microscopy. Cell viability was determined by propidium iodide staining and cell proliferation capacity by 5-bromo-2-deoxyuridine (BrdU) incorporation. Maintenance of stem cell surface markers was determined by flow cytometry. Results. IODEX-TAT labeling for 2, 6 and 21 h did not influence cellular ultrastructure or viability. Moreover, neither stem cell surface markers nor cell proliferation capacity was affected by labeling with IODEX-TAT.Our results demonstrate that labeling of MSCs for 21 h with a clinically relevant dose of 10 μg IODEX-TAT/10(5) cells is feasible and does not affect MSC ultrastructure, viability, phenotype or proliferation capacity.

Optic neuritis (ON) is closely linked to multiple sclerosis (MS). It may, however, also be associated to a range of autoimmune or infectious diseases. The purpose of this study was to assess the differential diagnoses in patients with suspected ON. In this retrospective study, we reviewed the files of all patients referred to the Clinic of Optic Neuritis, Glostrup Hospital, University of Copenhagen, Denmark, between January 2000 and November 2011. All patients were referred by ophthalmologists with possible ON. Patients diagnosed with MS prior to referral were excluded from the study. A total of 643 patients were included in the study. Apart from ON, the most frequent diagnoses were tumors (n = 15), ischemic or hypertensive neuropathies (n = 13), and retinal or choroid disorders (n = 9). Six patients were diagnosed with neuromyelitis optica. Rarer causes of visual loss were infections (n = 5), giant cell arteritis (n = 4), sarcoidosis (n = 3), thyrotoxicosis (n = 2), and hereditary or toxic neuropathies (n = 2). Nine percent of patients referred to the Clinic of Optic Neuritis had symptoms caused by medical, neurosurgical or ophthalmic disorders, and 0.9 % of our patients had NMO. Though most of these conditions are rare, it is of importance to keep them in mind upon encountering patients with symptoms of ON.

Angiogenesis, the formation of new blood vessels from existing vessels is required for many physiological processes and for growth of solid tumors. Initiated by hypoxia, angiogenesis involves binding of angiogenic factors to endothelial cell (EC) receptors and activation of cellular signaling, differentiation, migration, proliferation, interconnection and canalization of ECs, remodeling of the extracellular matrix and stabilization of newly formed vessels. Experimentally, these processes can be studied by several in vitro and in vivo assays focusing on different steps in the process. In vitro, ECs form networks of capillary-like tubes when propagated for three days in coculture with fibroblasts. The tube formation is dependent on vascular endothelial growth factor (VEGF) and omission of VEGF from the culture medium results in the formation of clusters of undifferentiated ECs. Addition of angiogenesis inhibitors to the coculture system disrupts endothelial network formation and influences EC morphology in two distinct ways. Treatment with antibodies to VEGF, soluble VEGF receptor, the VEGF receptor tyrosine kinase inhibitor SU5614, protein tyrosine phosphatase inhibitor (PTPI) IV or levamisole results in the formation of EC clusters of variable size. This cluster morphology is a result of inhibited EC differentiation and levamisole can be inferred to influence and block VEGF signaling. Treatment with platelet factor 4, thrombospondin, rapamycin, suramin, TNP-470, salubrinal, PTPI I, PTPI II, clodronate, NSC87877 or non-steriodal anti-inflammatory drugs (NSAIDs) results in the formation of short cords of ECs, which suggests that these inhibitors have an influence on later steps in the angiogenic process, such as EC proliferation and migration. A humanized antibody to VEGF is one of a few angiogenesis inhibitors used clinically for treatment of cancer. Levamisole is approved for clinical treatment of cancer and is interesting with respect to anti-angiogenic activity in vivo since it inhibits ECs in vitro with a morphology resembling that obtained with antibodies to VEGF.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6–8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up ( p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 ( p < 0.001), angina attacks per week from 13.8 to 3.2 ( p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week ( p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life ( p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Aims . We evaluated the feasibility, safety and effi cacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this fi rst in man trial. Methods and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6 – 8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p 0.001), reduction in CCS Class (p 0.001), angina attacks (p 0.001) and nitroglycerin consumption (p 0.001), and improved Seattle Angina Questionnaire (SAQ) evaluations (p 0.001). For all parameters there was a tendency towards improved outcome with increasing numbers of cells injected. In the MRI substudy: ejection fraction (p 0.001), systolic wall thickness (p 0.03) and wall thickening (p 0.03) all improved. Conclusions. The study demonstrated that it was safe to treat patients with stable CAD with autologous culture expanded MSC. Moreover, MSC treated patients had signifi cant improvement in left ventricular function and exercise capacity, in addition to an improvement in clinical symptoms and SAQ evaluations.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.

YKL-40 is involved in remodelling and angiogenesis in non-cardiac inflammatory diseases. Aim was to quantitate plasma YKL-40 in patients with ST-elevation myocardial infarction (STEMI) or stable chronic coronary artery disease (CAD), and YKL-40 gene activation in human myocardium.We included 73 patients: I) 20 patients with STEMI; II) 28 patients with stable CAD; III) 15 CAD patients referred for coronary by-pass surgery. YKL-40 mRNA expression was measured in myocardium subtended by stenotic or occluded arteries and areas with no apparent disease; and IV) 10 age-matched healthy controls. Plasma YKL-40 was significantly increased in patients with STEMI (88 microg/l, median) and CAD (66 microg/l) compared to controls (16 microg/l, p0.01 for both). Plasma YKL-40 correlated with CRP at baseline in STEMI (r=0.53, p=0.02) and CAD patients (r=0.41, p=0.031).YKL-40 gene expression was similar in ischemic and non-ischemic myocardium.Plasma YKL-40 was significantly increased in patients with STEMI and stable CAD. Further studies will define the role of YKL-40 as a clinically useful marker for myocardial ischemia, remodelling and maybe prognosis.

Studies have shown that stem cell therapy could be a novel option for improving neovascularization and cardiac function in patients with ischemic heart disease. Human mesenchymal stromal cells (MSC) have generated wide interest in the clinical setting because of their ability to regenerate tissue. The aim of the study was to test whether freezing and storage of human BM mononuclear cells (BM-MNC) and ex vivo-expanded MSC influenced their phenotypic and functional characteristics as well as proliferation capacity.MNC were isolated from BM and divided into two portions: one part was immediately cultured (MSC P0) whereas the second part was frozen for a week before cultivation and analysis (F-MSC P1). Confluent MSC (P0) were harvested and divided: one was analyzed as MSC P1 and the other was frozen for a week before further cultivation and analysis as F-MSC P2.MSC P1, F-MSC P1 and F-MSC P2 had similar proliferation capacities and demonstrated almost identical expression levels of markers characteristic for MSC. The capacity to form endothelial vascular structures was independent of freezing.The proliferation and differentiation capacity as well as the cellular characteristics were identical in cultivated MSC derived from freshly isolated BM-MNC and MSC derived after freezing and storage of either freshly isolated BM-MNC or ex vivo-cultivated MSC. This highlights the potential clinical use of MSC in patients with cardiac and degenerative diseases, as it would be possible to inject MSC obtained from the same BM aspiration at different time points.

Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.

Histone deacetylase (HDAC) inhibitors have attracted considerable attention due to their promise as therapeutic agents. Most HDAC inhibitors adhere to a general “ cap-linker-Zn 2+ -binding group ” architecture but recent studies have indicated that potent inhibition may be achieved without a Zn 2+ - coordinating moiety. Herein, we describe the synthesis of an azumamide analogue lacking its native Zn 2+ -binding group and evaluation of its inhibitory activity against recombinant human HDAC1 – 11. Furthermore, kinetic investigation of the inhibitory mechanism of both parent natural product and synthetic analogue against HDAC3-NCoR2 is reported as well as their activity against Burkitt's lymphoma cell proliferation.

Objectives. Pulmonary disease is a rare complication in JDM, described in only a few studies. This longterm follow-up study aimed to (i) describe pulmonary involvement in a national cohort of JDM patients estimated by conventional spirometry, (ii) compare pulmonary impairment with overall JDM outcome, and (iii) identify possible associations between pulmonary impairment and myositis-specific autoantibodies (MSAs). Methods. Fifty-one JDM patients performed conventional spirometry in a cross-sectional follow-up study. The scores of the Myositis Damage Index (MDI), Myositis Damage by visual analogue scale (MYODAMVAS) and physician’s global damage assessment were used to estimate JDM outcome. ANAs, MSAs and myositis-associated autoantibodies were analysed in all patients. Results. Forty-two patients (82%) (mean follow-up time 14.3 years) had normal lung function. Four patients (8%) were diagnosed with JDM-related restrictive interstitial lung disease. No patients reported pulmonary symptoms. Patients with restrictive pulmonary function had increased long-term damage estimated by MDI (P = 0.008), MYODAM-VAS (P = 0.04), global assessment (P = 0.03) and number of organ systems involved (P = 0.009). We found significant correlation between the restrictive pulmonary function test and damage by the MDI (r = 0.43, P = 0.003), MYODAM-VAS (r = 0.44, P = 0.002), and global damage assessment (r = 0.43, P = 0.003). No association was found between the restrictive pulmonary function test and autoantibodies. Conclusion. In a long-term follow-up study of JDM patients, the majority of patients demonstrated normal lung function. However, restrictive pulmonary impairment was identified in 8% of patients, indicating a need for repetitive pulmonary follow-up in JDM patients. Restrictive pulmonary involvement was associated with increased long-term JDM damage.

Background.Regenerative therapy is an emerging treatment modality. To determine migration and retention of implanted cells, it is crucial to develop noninvasive tracking methods. The aim was to determineex vivomagnetic resonance imaging (MRI) detection limits of ultrasmall superparamagnetic iron-oxide (USPIO) labeled mesenchymal stromal cells (MSCs).Materials and Methods.248 gel-phantoms were constructed and scanned on a 1.5T MRI-scanner. Phantoms contained human MSCs preincubated with USPIO nanoparticles for 2, 6, or 21 hours using 5 or 10 μg USPIO/105MSCs. In addition, porcine hearts were scanned after injection of USPIO labeled MSCs.Results.Using 21 h incubation time and 10 μg USPIO/105MSCs, labeled cells were clearly separated from unlabeled cells on MRI using 250.000 (P<0.001), 500.000 (P=0.007), and 1.000.000 MSCs (P=0.008). At lower incubation times and doses, neither labeled nor unlabeled cells could be separated. In porcine hearts labeled, but not unlabeled, MSCs were identified on MRI.Conclusions.As few as 250.000 MSCs can be detected on MRI using 21 h incubation time and 10 μg USPIO/105MSCs. At lower incubation times and doses, several million cells are needed for MRI detection. USPIO labeled cells can be visualized by MRI in porcine myocardial tissue.

It has been questioned whether bone marrow-derived mesenchymal stromal cells (MSCs) from patients with ischemic heart disease are suitable for use in regenerative stem cell therapy. We compared MSCs from patients with chronic coronary artery disease (CAD) and MSCs from young healthy donors with respect to phenotype, proliferation and endothelial differentiation capacity.MSCs from 16 young healthy donors and 15 elderly CAD patients were isolated, expanded by ex-vivo cultivation for two cell passages and characterized by flow cytometry, real time PCR and angiogenesis assay.MSCs from healthy donors and CAD patients expressed the same surface markers and had similar proliferation capacity. In both groups VEGF-stimulation significantly increased the expression of the endothelial genes thrombospondin 1, Tie-2 and von Willebrand Factor and induced the capacity to form ring structures on extracellular matrix.MSCs from young healthy donors and CAD patients proliferate equally well, express the same surface markers and increase in endothelial gene expression and ring structure formation capacity in the angiogenesis assay upon VEGF-stimulation. MSCs from CAD patients do not seem to be inferior to MSCs from young healthy donors thus indicating that autologous MSCs may be suitable for cell therapy in CAD patients.

Despite progress in percutaneous coronary intervention, bypass surgery and drug therapy, rates of mortality and morbidity after acute coronary syndrome are high due to ventricular remodeling and heart failure. Mesenchymal stromal cells (MSCs) from adult bone marrow or adipose tissue are considered potential candidates for therapeutic regenerative treatment in cardiovascular disease. Recent animal studies have demonstrated that MSCs can induce neovascularization and improve myocardial function in postinfarction myocardial ischemic hearts. This review will focus on the present preclinical and clinical knowledge about the use of mononuclear cells and MSCs for cardiac regenerative medicine, the source of MSCs for clinical use and problems to consider when conducting clinical MSC therapy.

Udgivelsesdato: 2008 OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. MATERIAL AND METHODS: Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. RESULTS: FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. CONCLUSIONS: We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.

Background— Granulocyte-colony stimulating factor (G-CSF) after myocardial infarction does not affect systolic function when compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejection fraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells. Methods and Results— We included 78 patients (62 men; 56±8 years) with ST-elevation myocardial infarction treated with primary percutaneous intervention 9 G-CSF mobilized CD34 + cells, compared with 3×10 9 cells in placebo patients ( P 11 mesenchymal stem cells, compared with 2.0×10 11 in the placebo group ( P + cells/leukocyte increased during G-CSF treatment (from 0.3±0.2 to 1.1±0.9 ×10 −3 , P −3 , P =0.01 when compared with placebo). An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found (regression coefficient −6.8, P =0.004), however none of the mesenchymal cell subtypes analyzed, were independent predictors of systolic recovery. Conclusions— The dissociated pattern for circulating CD34 + and mesenchymal stem cells could be attributable to reduced mesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cells to the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recovery may be of clinical importance and should be explored further.

The synthetic anthelmintic compound Levamisole has previously been used in cancer treatment as an adjuvant in combination with 5-fluorouracil. Its mode of action remains unclear, but an immune-stimulatory effect has been suggested. Here, we show that Levamisole inhibits angiogenesis in vitro and tumor growth in vivo. In vitro, Levamisole specifically inhibits proliferation and differentiation of endothelial cells propagated in co-culture with fibroblasts. In vivo, Levamisole inhibits the growth in nude mice of a transplanted human tumor. The use of nude mice as tumor hosts permits the discrimination between the angiogenesis inhibitory effect of Levamisole and its assumed immune-stimulatory effect. Our findings support a possible therapeutic effect of angiogenesis inhibitors in the treatment of cancer and call for further investigations of the mechanism(s) underlying the anti-angiogenic effect of Levamisole.

The oxidation of spermidine or homospermidine with bovine serum amine oxidase (BSAO) was monitored in situ, using proton nuclear magnetic resonance spectroscopy in water with 10% D(2)O. NMR assignments were performed by spin decoupling and COSY spectra or by comparison with data from synthetic aminoaldehydes. The results represent the first in situ characterisation of the highly reactive aminoaldehydes and showed oxidation at the N(1) amino group of spermidine and homospermidine. Comparison of homospermidine with a variety of substrates revealed that among straight chain di- and polyamines both an aminopropyl group and two primary amino groups separated by seven (norspermidine) or eight (spermidine) carbon atoms were required for optimal substrate ability. However, highest activity was seen with the substrate N-(4-aminobutyl)hexahydropyrimidine, showing that the substrate channel of BSAO has a dual substrate preference, with moderately bulky substituents at the distal end of a diamine contributing equally well as an alkyl amino group. Cytotoxic investigations of a variety of substrates for BSAO, confirmed previous results, that cytotoxicity is primarily linked to polyamines encompassing the aminopropyl moiety. No acrolein was observed at any time during the oxidation showing that it reacts very fast with available amino groups forming a variety of derivatives.

Porcine calcitonin has been purified by the method of Tenenhouse et al. followed by gel filtration. Forty-five MRC units were injected i.m. or i.v. into seven hypercalcaemic and five normocalcaemic patients. Four control subjects received the vehicle for calcitonin. The serum concentrations and the urinary excretion rates of calcium, magnesium, phosphate, sodium, potassium, and creatinine were measured at short intervals for three days. The hormone was injected on the second day. Complexed and ionized calcium, alkaline phosphatase, and protein in serum were determined. The calcium turnover and bone formation rate were determined by the technique of Heaney and Whedon using 47Ca. There was a significant hypocalcaemic effect in all patients, most pronounced in the hypercalcaemic group. However, the effect seemed to be better correlated to the bone formation rate than to the degree of hypercalcaemia. A significant decrease in serum magnesium concentration was observed in the hypercalcaemic group. It was difficult to evaluate the effect on serum phosphate because of large diurnal fluctuations. An increased urinary sodium excretion rate after calcitonin administration was registered.