A multifunctional bioreactor for three-dimensional cell (co)-culture

Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown. Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4',6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14. The optimized seeding density in the matrix was 2.0 x 10(7) cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2-183.7 +/- 18.4 mmHg) and physiological pH values (7.4 +/- 0.02) and a constant level of pCO2 (43.1 +/- 2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82 +/- 6.7% living cells. No significant differences were found between spatial separated bioreactor compartments. Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.     

Energetics of karate kumite

It is speculated that anaerobic metabolism is the predominant source of energy in karate kumite. However, no experimental proof is currently available. The metabolic cost and fractions of aerobic and anaerobic energy of karate kumite fighting were investigated. Ten male nationally or internationally ranked karateka [means (SD) age 26.9 (3.8) years, height 1.80 (0.08) m, mass 77.2 (12.8) kg] performed two to four fights scheduled and judged like a championship. Oxygen uptake was measured continuously with a portable spirometric device. Blood lactate was determined immediately before, and minute by minute after, each fight. Aerobic, anaerobic alactic and anaerobic lactic energy were calculated from oxygen uptake during the fight (VO₂), the fast component of the post-fight oxygen uptake (VO_2PCr) above resting values and changes in blood lactate concentration (Net-BLC), respectively. Altogether, 36 fights lasting 267 (61) s were analysed. The referees decisions caused an activity-to-break ratio of approximately 2:1. VO₂, VO_2PCr, and Net-BLC per fight were 165.3 (52.4) ml^.kg^–1, 32.2 (7.2) ml^.kg^–1and 4.2 (1.9) mmol^.l^–1; the overall energy cost above rest was 334.3 (86.3) kJ per fight. Fractions of aerobic, anaerobic alactic, and lactic energy sources were 77.8 (5.8)%, 16.0 (4.6)%, and 6.2 (2.4)%, respectively. The results indicate a high metabolic rate in karate kumite. However, the acyclic activity profile implies that aerobic metabolism is the predominant source of energy and there is anaerobic supplementation, mainly by high-energy phosphates.     

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid

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Role of ventricular assist devices in the German heart allocation system

The Eurotransplant (ET) allocation algorithm, newly implemented in 2000, gives priority for heart transplantation (HTx) to patients with high urgency (HU) status, but now this status is rescinded upon ventricular assist device (VAD) implantation and only regained if severe complications occur during mechanical circulatory support (MCS). We studied the effects of this change on the patients in our institute who were waiting for HTx with MCS. The median duration of MCS until HTx in adult patients gradually increased from 3.1 months in 1994, reaching a peak of 16.7 months in 2000, and then gradually decreased to 6.0 months in 2003. Among the patients with VAD implantation as a bridge to HTx, two patients were on MCS for more than 1 year (the longest duration of MCS being 1.6 years) at the end of 1999, and this figure increased to nine patients and a maximum MCS duration of 3.7 years at the end of 2003. These data imply that the patients in whom a complication occurred in the early phase of MCS and who had overcome this complication underwent HTx early with HU status, and those who were stable during MCS waited a long time for HTx. Furthermore, the number of patients in the latter group is increasing. The new allocation algorithm imposes on patients with MCS waiting for HTx who are relatively young and free from complications and serious coexisting disease, very long-term MCS without an end to VAD bridging, which is almost equivalent to destination therapy. Part of this paper was presented at the 42nd JSAO Conference (Tokyo, October 5–7, 2004)     

The Importance of Ventilation in Exercise-Induced Asthma

The degree of post treadmill-running decrease in pulmonary function (Exercise-Induced Asthma) in 11 adult asthmatics was compared with the decrease in pulmonary function followed by resting isocapnic hyperventilation. It was checked that ventilation during the hyperventilation was kept identical to the ventilation during treadmill-running by continuous recording of respiratory frequency, minute ventilation, tidal volume and accumulated ventilation. The temperature of the inspired air was identical in the two situations and the relative humidity was 40% during treadmill-running and 15% during hyperventilation. The average accumulated ventilation during treadmill-running and hyperventilation was 411 1/6 min in both events. The decrease in peak expiratory flow after treadmill-running was 25% and after isocapnic hyperventilation 24%. It is concluded that the ventilation is of more importance for the decrease in pulmonary function after exercise, than the work load.     

Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Characterization of monocyte-derived dendritic cells in recent-onset diabetes mellitus type 1

Dendritic cells (DC) may play an important role in the immunopathogenesis of type 1 diabetes mellitus (DM-1). In this study, we have analyzed phenotypical changes during cytokine-driven maturation from CD14+ monocytes to mature DC and DC-dependent T-cell stimulation in recent-onset pediatric DM-1 patients and healthy controls. DC maturation was monitored by flow cytometric analyses for the expression of surface markers (HLA-DR, CD1a, CD40, CD80, CD86, CD83, CD14, CD32, mannose-receptor, and CD11c). Flow cytometric analysis of isolated peripheral blood monocytes did not reveal apparent differences between patients and controls. During DC maturation no obvious differences in the expression patterns of surface markers over time or evidence for maturation impairments in DM-1 patients could be appreciated. Solely, a marginal, but significant, transient down-regulation of CD1a on Day 3 (mean MDFI 3.82 vs 7.25; P = 0.021), which was accompanied by an increase of IL-6, could be observed. The comparison of mature DCs (Day 10) between patients and controls indicated no significant differences, except for CD83 (mean MDFI 1.7 vs 1.5; P = 0.042) and CD80 (mean MDFI 15.92 vs 12.73; P = 0.042). Moreover, no difference in T-cell stimulatory capacity was seen. In conclusion, our analysis of a cohort of recent-onset DM-1 patients and controls does not support a role for disease-related alterations in cytokine-driven maturation of monocyte-derived DC.     

Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.     

New polymers with coronand structure

New polymers with an alternating sequence of non-polar aliphatic chains and coronands were synthesized via Diels-Alder addition polymerization of bis(2-pyrones) with bismaleimides; number-average molecular weight M?_n: 18000 (vapour pressure osmometry), glass transition temperature T?_g: 250°C. The chemical structures of these polymers are supported by NMR spectra and model reactions. The reaction mechanism is discussed.