Karin Macfelda

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation

AIMS Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

Combining light microscopy, dielectric spectroscopy, MALDI intact cell mass spectrometry, FTIR spectromicroscopy and multivariate data mining for morphological and physiological bioprocess characterization of filamentous organisms

Along with productivity and physiology, morphological growth behavior is the key parameter in bioprocess design for filamentous fungi. Lacking tools for fast, reliable and efficient analysis however, fungal morphology is still commonly tackled by empirical trial-and-error techniques during strain selection and process development procedures. Bridging the gap, this work presents a comprehensive analytical approach for morphological analysis combining automated high-throughput microscopy, multi-frequency dielectric spectroscopy, MALDI intact cell mass spectrometry and FTIR spectromicroscopy. Industrial fed-batch production processes were investigated in fully instrumented, automated bioreactors using the model system Penicillium chrysogenum. Physiological process characterization was based on the determination of specific conversion rates as scale-independent parameters. Conventional light microscopic morphological analysis was based on holistic determination of time series for more than 30 morphological parameters and their frequency distributions over the respective parameter range by automated high-throughput light microscopy. Characteristic protein patterns enriched in specific morphological and physiological states were further obtained by MALDI intact cell mass spectrometry. Spatial resolution of molecular biomass composition was facilitated by FTIR spectromicroscopy. Real-time in situ monitoring of morphological process behavior was achieved by linking multi-frequency dielectric spectroscopy with above outlined off-line methods. Data integration of complementing orthogonal techniques for morphological and physiological analysis together with multivariate modeling of interdependencies between morphology, physiology and process parameters facilitated complete bioprocess characterization. The suggested approach will thus help understanding morphological and physiological behavior and, in turn, allow to control and optimize those complex processes.

In Vivo Activity of a Novel Polymeric Guanidine in Experimental Skin Infection with Methicillin-Resistant Staphylococcus aureus

ABSTRACT The in vivo efficacy of the novel polymeric guanidine AKACID Plus was evaluated in a guinea pig model of experimental skin infection with methicillin-resistant Staphylococcus aureus (MRSA). Topical application of AKACID Plus at concentrations of ≥0.5% was as effective as mupirocin 2% cream in the treatment of superficial skin infection with MRSA.

Hypoxia-inducible factor-1β (HIF-1β) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions

Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1β. The alpha subunit is continuously degraded under normoxia and becomes stabilized under reduced oxygen supply. In contrast, HIF-1β is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1β in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1β was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1β induction, thus arguing for HIF-1β de novo synthesis rather than protein stabilization by heterodimerization. A Hens egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1β and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1β under hypoxic conditions for the first time. The results indicate a novel cell specific mechanism which might prevent HIF-1β to become a limiting factor.

3D Printable Biophotopolymers for in Vivo Bone Regeneration

The present study investigated two novel biophotopolymer classes that are chemically based on non-toxic poly (vinyl alcohol). These vinylesters and vinylcarbonates were compared to standard acrylates in vitro on MC3T3-E1 cells and in vivo in a small animal model. In vitro, both vinylester and vinylcarbonate monomers showed about tenfold less cytotoxicity when compared to acrylates (IC50: 2.922 mM and 2.392 mM vs. 0.201 mM) and at least threefold higher alkaline phosphatase activity (17.038 and 18.836 vs. 5.795, measured at [10 mM]). In vivo, polymerized 3D cellular structures were implanted into the distal femoral condyle of 16 New Zealand White Rabbits and were observed for periods from 4 to 12 weeks. New bone formation and bone to implant contact was evaluated by histomorphometry at end of observation. Vinylesters showed similar rates of new bone formation but significantly less (p = 0.002) bone to implant contact, when compared to acrylates. In contrast, the implantation of vinylcarbonate based biophotopolymers led to significantly higher rates of newly formed bone (p < 0.001) and bone to implant contact (p < 0.001). Additionally, distinct signs of polymer degradation could be observed in vinylesters and vinylcarbonates by histology. We conclude, that vinylesters and vinylcarbonates are promising new biophotopolymers, that outmatch available poly(lactic acid) and (meth)acrylate based materials.

Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae.

ZusammenfassungEINLEITUNG: Nicht-typisierbarer Haemophilus influenzae (NTHi) ist ein wichtiger bakterieller Erreger ambulant erworbener Infektionen der Atemwege und ist in der Regel extrazellulär zu finden. Studien haben jedoch gezeigt, dass NTHi die Fähigkeit besitzt in menschliche Epithelzellen einzudringen, wo er sowohl vor den Angriffen des lokalen Immunsystems als auch zum Teil vor der Abtötung durch Antibiotika geschützt ist. Ziel der vorliegenden Studie war es, die Fähigkeit der 5 klinisch häufig eingesetzten Antibiotika Ampicillin, Azithromycin, Telithromycin, Ciprofloxacin und Moxifloxacin zu bewerten, NTHi innerhalb von bronchialen Epithelzellen abzutöten. METHODEN: Konfluierende humane bronchiale Epithelzellen wurden mit NTHi 77, einem besonders invasiven klinischen Stamm infiziert. Die extrazellulären Bakterien wurden mittels Gentamicin abgetötet. Die intrazellulären Bakterien wurden mit Antibiotika in einer Konzentration von 1 mg/l oder 10 mg/l für 4 h oder 8 h behandelt. Lebensfähige intrazelluläre Bakterien wurden nach Lyse der Epithelzellen gezählt. ERGEBNISSE: Mit Ausnahme von Ampicillin erreichten alle Antibiotika eine signifikante Reduktion der intrazellulären Bakterien in einer Konzentration von 10 mg/l und einer Expositionszeit für 4 h sowie bei 1 mg/l für 8 h. Bei einer Konzentration von 1 mg/l eliminierte Moxifloxacin 94% der intrazellulären NTHi nach 4 h und 98% nach 8 h. Ciprofloxacin, Azithromycin und Telithromycin erreichten nur Killing-Indices < 75 nach 4 h, jedoch eine 86–90%ige Abtötung nach 8 h. Bei 10 mg/l erreichten Moxifloxacin, Ciprofloxacin, Telithromycin and Azithromycin nach einer Expositionszeit von 4 h eine 99,7%, 96,3%, 86,7% und 74,7%ige Eradikation der intrazellulären Bakterien. SCHLUSSFOLGERUNG: Die Ergebnisse demonstrieren die rasche antibakterielle Effektivität von Moxifloxacin gegenüber intrazellulärem NTHi in vitro. Moxifloxacin, welches hohe extrazelluläre und intrazelluläre Wirksamkeit kombiniert, könnte eine vorteilhafte Substanz in der Behandlung von rezidivierenden Atemwegsinfektionen darstellen.SummaryINTRODUCTION: Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of community-acquired respiratory tract infection and is usually found extracellularly, although studies have revealed that NTHi may possess the ability to invade human epithelial cells where it is then protected against attack by the local immune system and partly against the effect of antibiotics. The aim of the present study was to assess the ability of ampicillin, azithromycin, telithromycin, ciprofloxacin and moxifloxacin, five antibiotics in common clinical use, to kill NTHi within bronchial epithelial cells. METHODS: Confluent human bronchial epithelial cells were infected with NTHi 77, a particularly invasive clinical strain. Extracellular bacterial cells were killed with gentamicin and the intracellular bacteria were incubated with antibiotics at concentrations of 1 mg/l or 10 mg/l for 4 h or 8 h. Viable intracellular bacteria were counted after lysis of the epithelial cells. RESULTS: With the exception of ampicillin, all the antibiotics caused significant reduction of intracellular bacteria at concentrations of 10 mg/l and exposure for 4 h or at 1 mg/l for 8 h. At 1 mg/l, moxifloxacin eliminated 94% of intracellular NTHi after 4 h and 98% after 8 h; ciprofloxacin, azithromycin and telithromycin only achieved killing indices below 75 after 4 h but 86–90% killing after 8 h. At 10 mg/l, moxifloxacin, ciprofloxacin, telithromycin and azithromycin were able to achieve 99.7%, 96.3%, 86.7% and 74.7% eradication of intracellular bacteria, respectively, after exposure for 4 h. CONCLUSION: These results demonstrate the rapid antibacterial efficacy of moxifloxacin against intracellular NTHi in vitro. Moxifloxacin, which combines high extracellular and intracellular activities, could be an important tool in the treatment of recurrent respiratory tract infections.

Microcurrent stimulation promotes reverse remodelling in cardiomyocytes

It has been shown that electrical stimulation can improve tissue repair in patients. Imbalances in the extracellular matrix composition induce manifestation of heart failure. Here we investigated the application of microcurrent (MC) to modulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in cardiomyocytes in vitro and in vivo to reverse remodelling in the heart in spontaneous hypertensive rats (SHR).

Bioelectrical signals improve cardiac function and modify gene expression of extracellular matrix components

Beyond the influence of stimulating devices on cardiac excitation, their use in treating patients with heart failure has positive effects on the myocardium at the molecular level. Electrical signals can induce a wide spectrum of effects in living tissue. Therefore, we sought to determine whether applying electrical microcurrent directly to failing hearts leads to functional improvement.

CHANGES OF THE COLLAGEN COMPOSITION IN THE HEART CAUSED BY MICROCURRENT APPLICATION. AN EXPLANATION FOR THE IMPROVEMENT OF CARDIAC FUNCTION BY BI-VENTRICULAR PACING?

Objective Improvement of cardiac function by the unloading effect of a cardiac assist device mainly depends on the duration of heart failure (HF). Patients with a short history of HF ( 5 years) do not show significant cardiac function improvement is that the collagen composition of the extracellular matrix is irreversible. It is successful clinical practice to apply microcurrent in patients with bone fractures and wound healing disturbances in order to improve the healing process by modulation of the collagen synthesis. In order to examine whether microcurrent can also influence the collagen synthesis in the myocardium, the effect of microcurrent application on collagen synthesis of adult cardiomyocytes was investigated. Methods Adult cardiomyocytes were isolated and cultivated in 24 well cell culture plates. Current of different magnitudes (0, 20, 40, 60, 80, 100 :A) was applied via platinum electrodes by a special custom-made device. The whole equipment was incubated under cell culture conditions (+37°C, 5% CO2) over a period of 7 days. Changes of the collagen type I and type III synthesis were analyzed using immunohistochemical staining methods. Collagen type I and type III content was quantified using a special fluorescence confocal laser scanning microscopy system including special analysis software. Results Compared to cardiomyocytes exposed to 0 :A (control cells), collagen type I synthesis of cardiomyocytes showed no significant change after exposure to a moderate current magnitude(40, 60 :A) but a highly significant mean decrease (20.6 %) if exposed to high current (80, 100 :A). Collagen type III revealed a mean increase at moderate current of 29.7 % and a decrease of 25.2 % at high current exposure. As a side effect, we detected an increase in the cell proliferation rate at moderate and high current. Conclusion The results obtained in cell culture suggest that the application of micro-current is able to modulate the synthesis of collagen. In particular, in dependency of the current magnitude collagen type I can be up- or down-regulated. Collagen type I is responsible for the stiffness and the degree of dilatation of the heart. Therefore it can be envisaged that this method -if applied clinically - may help to improve cardiac function, as it helps to heal bone fractures.