Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system.

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300µM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4µl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300µM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Study of fluid dynamics at the boundary wall of a microchannel by Bloch surface waves.

Understanding how a fluid flows at the boundaries when it is confined at the microscale/nanoscale is crucial for a broad range of engineering and biology applications. We propose an experimental technique based on Bloch surface waves sustained by a one-dimensional photonic crystal to evaluate the speed of the contact line, i.e., the triple junction separating three phases, in the low Reynold's number regime, and with a nanometric resolution. Here, we report on the experimental characterization of the translatory motion of the contact line that separates two water solutions with a relatively high refractive index mismatch (7.35×10-3) and its slipping over a solid surface. The advantages are the relative simplicity and economy of the experimental configuration.

[Lipid therapy in daily routine]. / Lipidtherapie im Alltag.

Patients with increased cardiovascular risk profile are frequently seen in general practice. Comprehensive management of modifiable risk factors, in particular dyslipidemia, is mandatory. Many studies in clinical practice have shown a gap between the recommendations in clinical guidelines and the actual situation. Current data on the management situation of patients with high cardiovascular risk is provided by the prospective registry LIMA. Primary care physicians in 2,387 offices throughout Germany documented 13,924 patients with coronary artery disease (CAD), diabetes mellitus or peripheral arterial disease (PAD). Treatment with simvastatin 40 mg was an inclusion criterion. Physicians documented drug utilization, laboratory values (lipids, blood glucose), blood pressure and clinical events over one year and received feedback about the target value attainment of their patients after data entry. Mean age of the patients was 65.7 years, and 61.6 % were men. CAD was reported in 70.6 %, diabetes mellitus in 58.2 % and PAD in 14.9 %. Most patients (68 %) received simvastatin as monotherapy also after the inclusion visit; 20.6 % of patients received in addition the cholesterol absorption inhibitor (ezetimibe) in the first 6 months, and 23.3 % in the second 6 months. Patients achieved the LDL-cholesterol target value in 31.8 % at entry and 50.0 % after one year. The blood pressure target < 140 /90 mmHg was reached by 65.8 % after one year. Of patients with diabetes mellitus 40.0 % reached an HbA1c value below 6.5 %. Clinical events (death, hospitalization, (cardio-) vascular events, and dialysis) were reported by 11.7 % of patients between entry and Month 6, and by 12.0 % between Month 7 and 12. In daily practice comprehensive management of risk factors in patients at high cardiovascular risk remains a challenge. For normalization of increased LDL cholesterol values addition of ezetimibe to existing statin therapy improves the chances of patients for target level attainment.

The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes.

Adeno-associated virus type 2 (AAV2) capsid assembly requires the expression of a virally encoded assembly-activating protein (AAP). By providing AAP together with the capsid protein VP3, capsids are formed that are composed of VP3 only. Electron cryomicroscopy analysis of assembled VP3-only capsids revealed all characteristics of the wild-type AAV2 capsids. However, in contrast to capsids assembled from VP1, VP2, and VP3, the pores of VP3-only capsids were more restricted at the inside of the 5-fold symmetry axes, and globules could not be detected below the 2-fold symmetry axes. By comparing the capsid assembly of several AAV serotypes with AAP protein from AAV2 (AAP-2), we show that AAP-2 is able to efficiently stimulate capsid formation of VP3 derived from several serotypes, as demonstrated for AAV1, AAV2, AAV8, and AAV9. Capsid formation, by coexpressing AAV1-, AAV2-, or AAV5-VP3 with AAP-1, AAP-2, or AAP-5 revealed the ability of AAP-1 and AAP-2 to complement each other in AAV1 and AAV2 assembly, whereas for AAV5 assembly more specific conditions are required. Sequence alignment of predicted AAP proteins from the known AAV serotypes indicates a high degree of homology of all serotypes to AAP-2 with some divergence for AAP-4, AAP-5, AAP-11, and AAP-12. Immunolocalization of assembled capsids from different serotypes confirmed the preferred nucleolar localization of capsids, as observed for AAV2; however, AAV8 and AAV9 capsids could also be detected throughout the nucleus. Taken together, the data show that AAV capsid assembly of different AAV serotypes also requires the assistance of AAP proteins.

Preserving oxygenation during walking in severe chronic obstructive pulmonary disease: noninvasive ventilation versus oxygen therapy.

BACKGROUND: Physical activity is known to cause significant deoxygenation in patients with severe chronic obstructive pulmonary disease (COPD). Although noninvasive positive pressure ventilation (NPPV) has been shown to improve oxygenation and physical activity in these patients, no practical approach for the application of NPPV during walking has yet been established. OBJECTIVE: To elucidate the most effective approach to preserving oxygenation during walking in patients with severe COPD receiving long-term NPPV. METHODS: Three 12-min walking tests were performed in a randomized cross-over design on 3 consecutive days, comparing the usual and double dosages of oxygen versus NPPV plus the usual dosage of oxygen. The ventilator and oxygen tank were placed in a backpack. RESULTS: Eleven patients (FEV(1) 26 +/- 9% predicted) completed the study, while 8 patients refused to walk with NPPV, due to the weight of the ventilatory device (7.3 kg with NPPV vs. 3.1 kg without). PaO(2) changes during walking differed [p = 0.01, repeated-measures (RM)-ANOVA], whereas dyspnea was unchanged. The difference in PaO(2) change was 14.0 +/- 16.6 mm Hg (unadjusted p = 0.0036, critical level = 0.017, RM-ANOVA) in favor of NPPV compared to the usual dosage of oxygen. Changes in FEV(1), tidal volume and inspiratory impedance were in favor of NPPV-aided exercise (all p < 0.05, RM-ANOVA). Walking distance was reduced under NPPV (555 +/- 227 m) compared to the usual (619 +/- 210 m) and double (622 +/- 215 m) dosages of oxygen (p = 0.024, RM-ANOVA). CONCLUSIONS: NPPV plus supplemental oxygen, but not oxygen alone, preserves oxygenation during walking in patients with severe COPD. However, dyspnea and walking distance were not improved due to the burden of carrying the heavy ventilatory equipment in a backpack.

Exercise in severe COPD: is walking different from stair-climbing?

BACKGROUND: It remains unclear whether the 6-min walking test can predict performance during stair-climbing in severe COPD patients. The present study aimed to assess different pathophysiological changes between walking and stair-climbing in these patients. METHODS: Sixteen COPD patients (mean FEV1 33+/-13% predicted) underwent a 6-min walking test and performed stair-climbing (44 steps) in a randomized, cross-over design. Blood gases, blood lactate, lung function parameters, maximal inspiratory mouth, sniff nasal and twitch mouth pressures, blood pressure, heart rate, and Borg Dyspnea Scale (BDS) were measured before and after exercise. RESULTS: The median drop of PaO2 during walking (2.6 mmHg) and stair-climbing (2.4 mmHg) was comparable (p=0.93). However, stair-climbing caused more dyspnea (median BDS 6.5 vs. 5.5, p=0.01), a higher median blood lactate (1.1 vs. 0.3 mmol/l p<0.001), a more pronounced drop in mean pH (-0.05+/-0.02 vs. -0.03+/-0.03, p=0.02) and a higher increase in mean systolic blood pressure (27+/-11 vs. 13+/-16 mmHg; p=0.009). Stair-climbing, but not walking, caused prolonged lung hyperinflation (mean TLC difference 4.4+/-4.7% predicted, p=0.003). There was no relationship between the 6-min walking distance (314+/-104 m) and the time needed for stair-climbing (55+/-33 s), nor were there any differences in inspiratory muscle strength and heart rate. CONCLUSION: Although the drop of PaO2 was comparable, stair-climbing resulted in more prolonged hyperinflation of the lungs, higher blood lactate production and more dyspnea than walking. The walking distance was not related to the time needed to manage stair-climbing. Therefore, pathophysiological changes during the 6-min walking test do not anticipate those during stair-climbing in patients with severe COPD.

Diabetic polyneuropathy is associated with respiratory muscle impairment in type 2 diabetes.

AIMS/HYPOTHESIS: Diabetes has a major negative effect on intensive care unit outcome. This has been partly attributed to impaired respiratory neuromuscular function. However, data on respiratory neuromuscular involvement in diabetes are lacking. This study therefore aimed to assess respiratory neuromuscular function related to diabetic polyneuropathy in patients with type 2 diabetes. METHODS: Respiratory neuromuscular function was assessed by the use of volitional tests and twitch mouth (TwPmo) and twitch transdiaphragmatic (TwPdi) pressures during non-volitional bilateral anterior magnetic phrenic nerve stimulation in 21 male type 2 diabetic patients without pulmonary disease and in 23 healthy, well-matched controls (forced expiratory volume in 1 s 103 +/- 11 vs 103 +/- 12% predicted; p = 0.9). RESULTS: Both volitionally assessed maximal inspiratory and expiratory mouth pressures, and sniff nasal and transdiaphragmatic pressures were comparable between diabetic patients and controls (p > 0.1 for all). TwPmo was reduced in diabetic patients compared with controls (1.3 +/- 0.5 vs 1.0 +/- 0.4 kPa; p = 0.04), while TwPdi was comparable (1.7 +/- 0.5 vs 1.6 +/- 0.7 kPa; p = 0.6). Following subgroup analysis, patients with no or mild polyneuropathy (n = 10) as assessed by neurological disability scoring had normal respiratory neuromuscular function, whereas patients with moderate or severe polyneuropathy (n = 11) presented with markedly impaired respiratory neuromuscular function as indicated by TwPmo (1.3 +/- 0.4 vs 0.8 +/- 0.3 kPa; p = 0.01) and TwPdi (1.9 +/- 0.6 vs 1.1 +/- 0.4 kPa; p < 0.01). CONCLUSIONS/INTERPRETATION: With regard to volitional tests, diabetes does not affect respiratory neuromuscular function. In contrast, the application of non-volitional phrenic nerve stimulation provides strong evidence that diabetic polyneuropathy, as simply assessed by neurological disability scoring, is associated with substantially impaired respiratory neuromuscular function in type 2 diabetic patients.

[Medicamentous prevention of symptomatic paroxysmal atrial fibrillation/flutter onset. Goals and design of the SOPAT Study. Executive committee of the investigators representing trial physicians]. / Die medikamentöse Anfallsprophylaxe bei symptomatischem paroxysmalem Vorhofflimmern/-flattern. Ziele und Design der SOPAT-Studie. Investigators in Vertretung der Prüfärzte.

The indication to treat symptomatic paroxysmal atrial fibrillation is discussed controversely. Successful medical treatment may result in the reduction of symptoms by improving hemodynamics in a reduction of thromboembolic events. However, several antiarrhythmic drugs are also known to increase the risk of proarrhythmic events. A randomized, double-blind, and placebo-controlled multicenter trial with 1000 patients to be recruited was designed to compare the effects of two antiarrhythmic drugs frequently used in Germany for the treatment of atrial fibrillation, Sotalol and the fixed combination of chinidin and verapamil (Cordichin). Patients with symptomatic paroxysmal atrial fibrillation/atrial flutter will be observed for a period of one year. The occurrence of paroxysmal atrial fibrillation is documented by transtelephonic ECG monitoring. Patients with document an ECG once daily, and recording is mandatory in case of symptoms. ECGs are transmitted to a central data base for analysis. This clinical trial is designed to answer the following questions: (1) What is the average rate of spontaneous events of symptomatic atrial fibrillation? (2) Is it possible to reduce the frequency of symptomatic events by chronic antiarrhythmic drug administration? (3) What is the long-term frequency for the occurrence of severe side-effects under antiarrhythmic medication? The primary endpoint is defined as the time to first recurrence of symptomatic arrhythmia after reaching steady-state plasma concentrations of the study medication. The trial started in November 1997 and is planned to be finished by the end of 1999.

[The value of physical activity for prevention of coronary heart disease]. / Stellenwert der körperlichen Aktivität zur Prvention der KHK.

The usefulness of exercise as a preventive measure is discussed on the basis of a review of epidemiological studies. While no preventive effect has been reliably shown for physical activity at the workplace, physical exercise during leisure time does have such an effect. The incidence of myocardial infarction can be reduced by some 50 to 60%, although the contributions of protection and selection cannot be differentiated. The present data, however, militate against the possibility that, among those actively practicing sport, a primarily positive health selection effect is operative. The optimum of physical exercise would appear to be defined by a daily calorie consumption of 300 to 400 kcal (1250-1800 kJ) per day. Within the framework of secondary prevention, physical exercise can result in a reduction in mortality rate of 20%. The possibility of exercise related incidence prompts the requirement for a thorough physical check-up of those in the relevant risk groups.

Influence of verapamil therapy on left ventricular performance at rest and during exercise in hypertrophic cardiomyopathy.

To determine the hemodynamic effect of verapamil at rest and during exercise, 18 patients with hypertrophic cardiomyopathy were studied before and after 7 weeks of treatment with oral verapamil (maximal dose, 720 mg/day). At rest and at peak exercise, verapamil produced a significant increase in left ventricular (LV) systolic performance in terms of stroke volume index (rest, from 43 +/- 11 to 53 +/- 11 ml/m2, p less than 0.001; exercise, from 46 +/- 11 to 51 +/- 10 ml/m2, p less than 0.01), whereas heart rate decreased (rest, from 81 +/- 14 to 70 +/- 11 min-1, p less than 0.001; exercise, from 150 +/- 21 to 141 +/- 18 min-1, p less than 0.01). Cardiac index at rest and during exercise remained unchanged. Systolic vascular resistance did not change at rest, but decreased significantly during exercise (974 +/- 243 to 874 +/- 174 dynes s cm-5; p less than 0.05). After verapamil administration, pulmonary artery pressures did not change at rest, but decreased significantly during exercise. This was probably due to a shift in the LV pressure-volume relation. The improvement in LV hemodynamics was associated with a significant increase in exercise capacity. The findings of this study indicate that in patients with hypertrophic cardiomyopathy, hemodynamic improvement at rest and during exercise can be achieved by chronic administration of verapamil.

[Nephrotic syndrome as indication for one-stage bilateral renal embolisation (author's transl)]. / Nephrotisches Syndrom als Indikation zur einzeitigen bilateralen Nierenembolisation.

A 42-year-old patient suffered from a nephrotic syndrome due to primary perireticular amyloidosis. One year after diagnosis the renal protein loss increased to 40 g/d within a short time so that sufficient substitution was no longer possible. The creatinine clearance was 7-10 ml/min so that haemodialysis was performed as a vital measure to remove excessive fluid. Two weeks later, as an alternative to surgical bilateral nephrectomy, simultaneous bilateral renal embolisation with Ethibloc was done without complications. A small artery to the left lower pole was spared from embolisation. Computer tomographic follow-up showed a residual parenchymal perfusion in the subcapsular and left lower pole areas so that metabolic functions of the renal parenchyma persisted. Plasma renin activity decreased to subnormal levels, the erythropoietin plasma level remained in the lower range of normal. "Medical binephrectomy" should be considered more often as a therapeutic alternative to surgery in therapy-resistant nephrotic syndrome on account of its advantage of preserved metabolic renal function.