Monocyte secretory profiling in a clinical and MEFV genotype-characterized cohort of Danish familial Mediterranean fever patients: diagnostic potential of CCL1 and CXCL1.

OBJECTIVE: The autoinflammatory disease familial Mediterranean fever (FMF), characterized by recurrent attacks of sterile fever, serosal, and/or synovial inflammation, is caused by variants in the Mediterranean fever gene, MEFV, coding for the pyrin inflammasome sensor. The diagnosis of FMF is mainly based on clinical symptoms and confirmed by detection of disease-associated MEFV variants. However, the diagnosis is challenging among patients carrying variants of uncertain clinical significance (VUS). In this study, we aimed to identify potential FMF discriminatory diagnostic markers in a cohort of clinically characterized FMF patients. METHOD: We established a cohort of clinically and MEFV genotype-characterized FMF patients by enrolling patients from major Danish hospitals (n = 91). The secretory profile of pyrin inflammasome-activated monocytes from healthy donors (HDs) and MEFV-characterized FMF patients (n = 28) was assessed by analysing cell supernatants for a custom-designed panel of 23 cytokines, chemokines, and soluble tumour necrosis factor receptors associated with monocyte and macrophage function. RESULTS: MEFV genotypes in Danish FMF patients were associated with age at symptom onset (p < 0.05), FMF among relatives (p < 0.01), proportion of patients in colchicine treatment (p < 0.01), and treatment response (p < 0.05). Secretion of chemokines CCL1 and CXCL1 from pyrin-activated FMF monocytes was significantly decreased compared to HDs (p < 0.05), and could discriminate FMF patients with 'non-confirmatory' MEFV genotypes from HDs with 80.0% and 70.0% sensitivity for CCL1 and CXCL1, respectively (p < 0.05). CONCLUSION: Our data suggest that a functional diagnostic assay based on CCL1 or CXCL1 levels in pyrin-activated patient monocytes may contribute to FMF diagnosis in patients with VUS.

Characteristics of patients with familial Mediterranean fever in Denmark: a retrospective nationwide register-based cohort study.

Objectives: To investigate epidemiology, demography, and genetic and clinical characteristics of patients with familial Mediterranean fever (FMF) in Denmark. Method: In this population-based, cross-sectional cohort study, we identified FMF patients from discharge diagnoses using ICD-10 codes in the Danish National Patient Register, and linked data from the Danish Civil Registration System and laboratory databases for results of MEFV gene variant screening. Results: We identified 495 FMF patients (prevalence 1:11 680) with a median age of 29 years and a female ratio of 51%. The median age at diagnosis of FMF was 13 (IQR 7-22) years, with an estimated median diagnostic delay of 3 (IQR 0.7-6.9) years. The predominant ethnicities were Turkish (41.8%), Lebanese (15.8%), Syrian (6.5%), South-West Asian (7.9%), and South-East Asian (3.0%). The MEFV genotype distribution was 18.7% homozygous, 21.2% compound heterozygous, 32.0% heterozygous, 11.0% with complex alleles or unresolved zygosity, and 17.1% with no detected variants. M694V was the most prevalent variant in the overall cohort (32.5%). Homozygous or compound heterozygous MEFV exon 10 variants were associated with younger age at diagnosis (p < 0.001) and reduced number of hospital contacts before diagnosis (p = 0.008). The Charlson Comorbidity Index was ≥ 2 in 8.1% of patients. The prevalence of amyloidosis was 1.0%. Conclusions: FMF in Denmark is rare and patients are mainly of Eastern Mediterranean ethnicity. Diagnostic delay was long but patients with exon 10 MEFV variants were diagnosed at a younger age. Prolonged diagnostic delay is probably caused by lack of FMF awareness in the Danish healthcare system.

Muscle α-adrenergic responsiveness during exercise and ATP-induced vasodilation in chronic obstructive pulmonary disease patients.

Sympathetic vasoconstriction is blunted in exercising muscle (functional sympatholysis) but becomes attenuated with age. We tested the hypothesis that functional sympatholysis is further impaired in chronic obstructive pulmonary disease (COPD) patients. We determined leg blood flow and calculated leg vascular conductance (LVC) during 1) femoral-arterial Tyramine infusion (evokes endogenous norepinephrine release, 1 µmol·min-1·kg leg mass-1), 2) one-legged knee extensor exercise with and without Tyramine infusion [10 W and 20% of maximal workload (WLmax)], 3) ATP (0.05 µmol·min-1·kg leg mass-1) and Tyramine infusion, and 4) incremental ATP infusions (0.05, 0.3, and 3.0 µmol·min-1·kg leg mass-1). We included 10 patients with moderate to severe COPD and 8 age-matched healthy control subjects. Overall, leg blood flow and LVC were lower in COPD patients during exercise ( P < 0.05). Tyramine reduced LVC in both groups at 10-W exercise (COPD: -3 ± 1 ml·min-1·mmHg-1 and controls: -3 ± 1 ml·min-1·mmHg-1, P < 0.05) and 20% WLmax (COPD: -4 ± 1 ml·min-1·mmHg-1 and controls: -3 ± 1 ml·min-1·mmHg-1, P < 0.05) with no difference between groups. Incremental ATP infusions induced dose-dependent vasodilation with no difference between groups, and, in addition, the vasoconstrictor response to Tyramine infused together with ATP was not different between groups (COPD: -0.03 ± 0.01 l·min-1·kg leg mass-1 vs. CONTROLS: -0.04 ± 0.01 l·min-1·kg leg mass-1, P > 0.05). Compared with age-matched healthy control subjects, the vasodilatory response to ATP is intact in COPD patients and their ability to blunt sympathetic vasoconstriction (functional sympatholysis) as evaluated by intra-arterial Tyramine during exercise or ATP infusion is maintained. NEW & NOTEWORTHY The ability to blunt sympathetic vasoconstriction in exercising muscle and ATP-induced dilation in chronic obstructive pulmonary disease patients remains unexplored. Chronic obstructive pulmonary disease patients demonstrated similar sympathetic vasoconstriction in response to intra-arterial Tyramine during exercise and ATP-induced vasodilation compared with age-matched healthy control subjects.

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients.

AIM: The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS: To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS: At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 µm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION: Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.

The effect of purinergic P2 receptor blockade on skeletal muscle exercise hyperemia in miniature swine.

PURPOSE: ATP could play an important role in skeletal muscle blood flow regulation by inducing vasodilation via purinergic P2 receptors. This study investigated the role of P2 receptors in exercise hyperemia in miniature swine. METHODS: We measured regional blood flow with radiolabeled-microsphere technique and systemic hemodynamics before and after arterial infusion of the P2 receptor antagonist reactive blue 2 during treadmill exercise (5.2 km/h, ~60 % VO2max) and arterial ATP infusion in female Yucatan miniature swine (~29 kg). RESULTS: Mean blood flow during exercise from the 16 sampled skeletal muscle tissues was 138 ± 18 mL/min/100 g (mean ± SEM), and it was reduced in 11 (~25 %) of the 16 sampled skeletal muscles after RB2 was infused. RB2 also lowered diaphragm blood flow and kidney blood flow, whereas lung tissue blood flow was increased (all P < 0.05). Infusion of RB2 increased arterial lactate concentration during exercise from 1.6 ± 0.5 to 3.4 ± 0.6 mmol/L and heart rate from 216 ± 12 to 230 ± 9 beats/min, whereas blood pressure was unaltered. Arterial ATP infusion caused a ~twofold increase in blood flow in 15 of the 16 sampled muscle tissues and this effect was abolished after RB2 infusion. CONCLUSIONS: These results indicate that P2 receptors play a role in regulating skeletal muscle blood flow during exercise in miniature swine.

Lifelong physical activity preserves functional sympatholysis and purinergic signalling in the ageing human leg.

Ageing is associated with an impaired ability to modulate sympathetic vasoconstrictor activity (functional sympatholysis) and a reduced exercise hyperaemia. The purpose of this study was to investigate whether a physically active lifestyle can offset the impaired functional sympatholysis and exercise hyperaemia in the leg and whether ATP signalling is altered by ageing and physical activity. Leg haemodynamics, interstitial [ATP] and P2Y(2) receptor content was determined in eight young (23 ± 1 years), eight lifelong sedentary elderly (66 ± 2 years) and eight lifelong active elderly (62 ± 2 years) men at rest and during one-legged knee extensions (12 W and 45% maximal workload (WL(max))) and arterial infusion of ACh and ATP with and without tyramine. The vasodilatory response to ACh was lowest in the sedentary elderly, higher in active elderly (P < 0.05) and highest in the young men (P < 0.05), whereas ATP-induced vasodilatation was lower in the sedentary elderly (P < 0.05). During exercise (12 W), leg blood flow, vascular conductance and VO2 was lower and leg lactate release higher in the sedentary elderly compared to the young (P < 0.05), whereas there was no difference between the active elderly and young. Interstitial [ATP] during exercise and P2Y(2) receptor content were higher in the active elderly compared to the sedentary elderly (P < 0.05). Tyramine infusion lowered resting vascular conductance in all groups, but only in the sedentary elderly during exercise (P < 0.05). Tyramine did not alter the vasodilator response to ATP infusion in any of the three groups. Plasma [noradrenaline] increased more during tyramine infusion in both elderly groups compared to young (P < 0.05). A lifelong physically active lifestyle can maintain an intact functional sympatholysis during exercise and vasodilator response to ATP despite a reduction in endothelial nitric oxide function. A physically active lifestyle increases interstitial ATP levels and skeletal muscle P2Y(2) receptor content.

Vasodilator interactions in skeletal muscle blood flow regulation.

During exercise, oxygen delivery to skeletal muscle is elevated to meet the increased oxygen demand. The increase in blood flow to skeletal muscle is achieved by vasodilators formed locally in the muscle tissue, either on the intraluminal or on the extraluminal side of the blood vessels. A number of vasodilators have been shown to bring about this increase in blood flow and, importantly, interactions between these compounds seem to be essential for the precise regulation of blood flow. Two compounds stand out as central in these vasodilator interactions: nitric oxide (NO) and prostacyclin. These two vasodilators are both stimulated by several compounds, e.g. adenosine, ATP, acetylcholine and bradykinin, and are affected by mechanically induced signals, such as shear stress. NO and prostacyclin have also been shown to interact in a redundant manner where one system can take over when formation of the other is compromised. Although numerous studies have examined the role of single and multiple pharmacological inhibition of different vasodilator systems, and important vasodilators and interactions have been identified, a large part of the exercise hyperaemic response remains unexplained. It is plausible that this remaining hyperaemia may be explained by cAMP- and cGMP-independent smooth muscle relaxation, such as effects of endothelial derived hyperpolarization factors (EDHFs) or through metabolic modulation of sympathetic effects. The nature and role of EDHF as well as potential novel mechanisms in muscle blood flow regulation remain to be further explored to fully elucidate the regulation of exercise hyperaemia.

Contribution of intravascular versus interstitial purines and nitric oxide in the regulation of exercise hyperaemia in humans.

The regulation of blood flow to skeletal muscle involves a complex interaction between several locally formed vasodilators that are produced both in the skeletal muscle interstitium and intravascularly. The gas nitric oxide (NO) and the purines ATP and adenosine, are potent vasodilators that are formed by multiple cell types and released into the skeletal muscle interstitium and in plasma in response to muscle contraction. Cellular sources of ATP and NO in plasma are erythrocytes and endothelial cells, whereas interstitial sources are skeletal muscle cells and endothelial cells. Adenosine originates primarily from extracellular degradation of ATP. During exercise the concentrations of ATP and adenosine increase markedly in the interstitium with smaller increases occurring in plasma, and thus the interstitial concentration during exercise is severalfold higher than in plasma. The concentration of NO metabolites (NOx) in interstitium and plasma does not change during exercise and is similar in the two compartments. Adenosine and NO have been shown to contribute to exercise hyperaemia whereas the role of ATP remains unclear due to lack of specific purinergic receptor blockers. The relative role of intravascular versus interstitial vasodilators is not known but evidence suggests that both compartments are important. In cardiovascular disease, a reduced capacity to form adenosine in the muscle interstitium may be a contributing factor in increased peripheral vascular resistance.

Two weeks of muscle immobilization impairs functional sympatholysis but increases exercise hyperemia and the vasodilatory responsiveness to infused ATP.

During exercise, contracting muscles can override sympathetic vasoconstrictor activity (functional sympatholysis). ATP and adenosine have been proposed to play a role in skeletal muscle blood flow regulation. However, little is known about the role of muscle training status on functional sympatholysis and ATP- and adenosine-induced vasodilation. Eight male subjects (22 ± 2 yr, Vo(2max): 49 ± 2 ml O(2)·min(-1)·kg(-1)) were studied before and after 5 wk of one-legged knee-extensor training (3-4 times/wk) and 2 wk of immobilization of the other leg. Leg hemodynamics were measured at rest, during exercise (24 ± 4 watts), and during arterial ATP (0.94 ± 0.03 µmol/min) and adenosine (5.61 ± 0.03 µmol/min) infusion with and without coinfusion of tyramine (11.11 µmol/min). During exercise, leg blood flow (LBF) was lower in the trained leg (2.5 ± 0.1 l/min) compared with the control leg (2.6 ± 0.2 l/min; P < 0.05), and it was higher in the immobilized leg (2.9 ± 0.2 l/min; P < 0.05). Tyramine infusion lowers LBF similarly at rest, but, when tyramine was infused during exercise, LBF was blunted in the immobilized leg (2.5 ± 0.2 l/min; P < 0.05), whereas it was unchanged in the control and trained leg. Mean arterial pressure was lower during exercise with the trained leg compared with the immobilized leg (P < 0.05), and leg vascular conductance was similar. During ATP infusion, the LBF response was higher after immobilization (3.9 ± 0.3 and 4.5 ± 0.6 l/min in the control and immobilized leg, respectively; P < 0.05), whereas it did not change after training. When tyramine was coinfused with ATP, LBF was reduced in the immobilized leg (P < 0.05) but remained similar in the control and trained leg. Training increased skeletal muscle P2Y2 receptor content (P < 0.05), whereas it did not change with immobilization. These results suggest that muscle inactivity impairs functional sympatholysis and that the magnitude of hyperemia and blood pressure response to exercise is dependent on the training status of the muscle. Immobilization also increases the vasodilatory response to infused ATP.

Peripheral vasodilatation determines cardiac output in exercising humans: insight from atrial pacing.

In dogs, manipulation of heart rate has no effect on the exercise-induced increase in cardiac output. Whether these findings apply to humans remain uncertain, because of the large differences in cardiovascular anatomy and regulation. To investigate the role of heart rate and peripheral vasodilatation in the regulation of cardiac output during steady-state exercise, we measured central and peripheral haemodynamics in 10 healthy male subjects, with and without atrial pacing (100­150 beats min(−1)) during: (i) resting conditions, (ii) one-legged knee extensor exercise (24 W) and (iii) femoral arterial ATP infusion at rest. Exercise and ATP infusion increased cardiac output, leg blood flow and vascular conductance (P < 0.05), whereas cerebral perfusion remained unchanged. During atrial pacing increasing heart rate by up to 54 beats min(−1), cardiac output did not change in any of the three conditions, because of a parallel decrease in stroke volume (P < 0.01). Atrial pacing increased mean arterial pressure (MAP) at rest and during ATP infusion (P < 0.05), whereas MAP remained unchanged during exercise. Atrial pacing lowered central venous pressure (P < 0.05) and pulmonary capillary wedge pressure (P < 0.05) in all conditions, whereas it did not affect pulmonary mean arterial pressure. Atrial pacing lowered the left ventricular contractility index (dP/dt) (P < 0.05) in all conditions and plasma noradrenaline levels at rest (P < 0.05), but not during exercise and ATP infusion. These results demonstrate that the elevated cardiac output during steady-state exercise is regulated by the increase in skeletal muscle blood flow and venous return to the heart, whereas the increase in heart rate appears to be secondary to the regulation of cardiac output.

Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease.

BACKGROUND: Previous studies have demonstrated considerable variation in the antiplatelet effect of aspirin. OBJECTIVES: To investigate the impact of platelet turnover on the antiplatelet effect of aspirin in patients with stable coronary artery disease (CAD) and to identify determinants of platelet turnover. METHODS: Platelet turnover was evaluated by measurements of immature platelets and thrombopoietin in 177 stable CAD patients on aspirin monotherapy, including 85 type 2 diabetics and 92 non-diabetics. Whole blood platelet aggregation was determined using the VerifyNow(®) Aspirin test and multiple electrode aggregometry (MEA, Multiplate(®) ) induced by arachidonic acid (AA) (1.0 mm), adenosine diphosphate (ADP) (10 µm) and collagen (1.0 µg mL(-1) ). RESULTS: Immature platelet levels significantly correlated with MEA (r = 0.31-0.36, P-values < 0.0001) and the platelet activation marker sP-selectin (r = 0.19, P = 0.014). Contrary to the VerifyNow(®) test, MEA significantly correlated with variations in platelet count (r = 0.45-0.68, P-values < 0.0001). Among patients with residual platelet reactivity according to AA, there were significantly more diabetics (61% vs. 41%, P = 0.027) and higher levels of sP-selectin (77.7 ± 29 vs. 70.2 ± 25 ng mL(-1) , P = 0.070) and serum thromboxane B(2) (0.81 [0.46; 1.70] vs. 0.56 [0.31; 1.12] ng mL(-1) , P = 0.034). In a multivariate regression analysis, immature platelet levels were determined by thrombopoietin levels (P < 0.001), smoking (P = 0.020) and type 2 diabetes (P = 0.042). CONCLUSIONS: The antiplatelet effect of aspirin was reduced in CAD patients with an increased platelet turnover. Once-daily dosing of aspirin might not suffice to adequately inhibit platelet aggregation in patients with an increased platelet turnover.

Cardiac sarcoidosis and heart transplantation: a report of four consecutive patients.

Heart transplantation (HTx) is a well-established treatment for severe cardiac failure. However, HTx for cardiac sarcoidosis is rare; less than 80 patients have been reported worldwide. In many patients, the diagnosis was not made prior to HTx. The aim of this study was to describe the use of HTx in the treatment of severe cardiac sarcoidosis. The series was comprised of four Caucasian patients (1 male, 3 females), aged 25-57 years. HTx were performed in the period 1990-2006. None of the patients had clinically overt extra-cardiac sarcoidosis. In one patient the diagnosis of sarcoidosis was proven prior to HTx. In three patients, all with dilated cardiomyopathy due to myocardial sarcoidosis, the final diagnosis was obtained by examination of the explanted heart. Arrythmias (supraventricular and ventricular), heart block, mitral valve insufficiency and dilated cardiomyopathy were prominent clinical features. None of the patients had recurrence of sarcoid disease in the allograft. Two patients are long-term survivors and two are deceased, one of primary graft failure, the other from Cytomegalovirus myocarditis. In conclusion, HTx is a viable treatment for cardiac sarcoidosis with end stage cardiac failure. Cardiac sarcoidosis is difficult to diagnose. Myocardial biopsy has a low diagnostic sensitivity. However, when the newest non-invasive diagnostic methods, including magnetic resonance imaging and positron emission tomography, are applied, an endomyocardial biopsy should not be mandatory to make a diagnosis of cardiac sarcoidosis.

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy.

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.

The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation.

PURPOSE: Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. METHODS AND RESULTS: Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. CONCLUSION: It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.

Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention.

OBJECTIVE: To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI). DESIGN: In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45-/CD34-), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA). RESULTS: Circulating CD45-/CD34- cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28. CONCLUSIONS: Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells.

Repeatability of cystometry and pressure-flow parameters in female patients.

The repeatability of cystometric and pressure-flow data was studied in 31 female patients. The measured parameters had poor correlation coefficients, and logarithmic transforming of the data was generally not warranted. Previous studies and statistical methods of reliability measurement are reviewed. It is suggested as a subject for future studies, that urodynamic measurements are repeated in each patient so that confidence limits can be established within disease entities.

Validation of a computer version of the patient-administered Danish prostatic symptom score questionnaire.

OBJECTIVE: To validate a computer version of the Danish Prostatic Symptom Score (DAN-PSS) questionnaire and compare it with the paper version. MATERIAL AND METHODS: Ninety-three male patients (aged 25-87 years), referred to a department of urology for lower urinary tract symptoms (LUTS), filled in a personal computer (PC) version and a paper version of the DAN-PSS questionnaire. Subsequently they answered a questionnaire concerning their preferences and computer experience. RESULTS: A fair correlation between the total scores from the paper and PC versions was found. The differences were independent of both total score and age. In the PC version all LUTS questions were answered while 9.8% were left blank in the paper version. The sexual questions were answered by 71% of patients in the paper version and by 87.5% in the PC version. For the questions in the PC version a learning curve was observed in terms of the time taken to answer the questions. Previous computer experience did not influence answering time, difference in score between paper and PC versions or total score. Almost all patients preferred the PC version to the paper version. CONCLUSION: The PC scores are reasonably reliable judging by comparison with previously validated traditional paper scores. The PC questionnaire had a higher response rate and was preferred by the majority of patients.

Macrophage and lymphocyte chimerism in bronchoalveolar lavage cells from human lung allograft recipients.

BACKGROUND: Chimerism is suggested to predict a more favourable prognosis in solid organ transplantation. MATERIAL AND METHOD: Forty-eight bronchoalveolar lavages from 10 patients (5 females and 5 males) who had received sex-mismatched donor lungs were monitored for varying periods of time, of up to 2 years, at regular intervals (median 3.0 (0.5-24) months). To investigate the chimerism in macrophages and lymphocytes in bronchoalveolar lavage cells a cloned 2.12 kilobase large biotinylated Y-chromosome-specific DNA-probe was used for in situ hybridization. RESULTS: Donor macrophages disappeared in seven patients within the first 6 months after surgery (median 3.0 (1-6) months). But 15% donor macrophages could be detected in one patient 1 year and 10% in 2 patients two years after surgery. Donor lymphocytes disappeared in all patients within 3 months (median 1 (0.5-3) months). There was no correlation between periods or severity of acute rejection and percentage of donor macrophages and donor lymphocytes in bronchoalveolar lavage. None of the patients developed obliterative bronchiolitis. CONCLUSION: Macrophage chimerism in lung may exist for several years. Whilst our results do not elucidate the role of local macrophage chimerism, they do not currently support the view that chimerism prevents rejection.