Joerg Herold

Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties

Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony–stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 ± 6 × 106 differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% ± 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% ± 6% by depleting adherent macrophages. Our findings indicate that bone marrow–derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models.

Efficacy and safety profile of dronedarone in clinical practice. Results of the Magdeburg Dronedarone Registry (MADRE study)

BACKGROUND Dronedarone is a new antiarrhythmic agent that has only recently been approved for the therapy of atrial fibrillation (AF). Results regarding a broader spectrum of patients and experience accumulated in clinical practice are still very scarce. Therefore, we prospectively investigated the efficacy and tolerance of dronedarone in a real life setting. METHODS AND RESULTS The study included 191 patients (85 women) aged 63 ± 9.9 years with a history of paroxysmal or persistent AF. Follow-up time was 14.3 ± 4.9 months. In patients with persistent AF, sinus rhythm was restored using electrical cardioversion prior to dronedarone administration. Each patient underwent standard ECG on a daily basis during the first 4 days of treatment, and on days 7, 30 and 90, resp. After that, the patients had a follow-up visit every three months. Creatinine, creatine kinase, and hepatic enzymes were closely monitored. Clinical history was meticulously taken at multiple follow-up visits. Dronedarone maintained sinus rhythm in 33.5% (95% CI: 27%-40%), and AF recurrence rate was high: 66.5% (95% CI: 60%-73%). Adverse effects occurred in 31.9% (95% CI: 27%-38%) of the patients and necessitated permanent discontinuation of dronedarone in 22% (95% CI: 17%-27%). CONCLUSIONS The results suggest that dronedarone may not be superior to available antiarrhythmic agents and caution against its use as a first line therapy in AF.

Indocyanine green angiography: a new method to quantify collateral flow in mice.

OBJECTIVES Therapeutic augmentation of collateral artery growth (ie, arteriogenesis) is of particular clinical interest for improving blood flow in vascular occlusive disease. Quantification of collateralization in small animal models is difficult, however, and the commonly used technique of laser Doppler perfusion imaging (LDPI) has always been criticized. Therefore, a new method, termed indocyanine green angiography (ICGA), was established for in vivo imaging of arteriogenesis in mice and compared with LDPI. METHOD Using the accepted model of ligation of the left femoral artery of 45 C57BL6 mice, we determined arteriogenesis both by LDPI and ICGA, which were applied before and periodically after ligation of the left femoral artery (each group n = 7). Collateral artery growth within the hind limb was additionally verified by histologic workup. RESULTS Determination of flow by ICGA, as represented by maximal pixel intensity (ratio of left/right hind limb) demonstrated a drop from 0.97 +/- 0.06 before ligation to 0.11 +/- 0.12 directly after ligation, which recovered to 0.48 +/- 0.22 after 1 week, to 0.65 +/- 0.11 after 2 weeks, and to 0.59 +/- 0.22 after 3 weeks (n = 7, P < .05). Similarly, flow determined as the perfusion index (slope of pixel intensity, ratio left/right) dropped from 1.18 +/- 0.4 before ligation to 0.02 +/- 0.03 immediately after ligation but recovered to 0.08 +/- 0.01 after 1 week, to 0.17 +/- 0.01 after 2 weeks, and to 0.17 +/- 0.06 after 3 weeks (n = 7, P < .05). Quantification by LDPI demonstrated a drop from 1.06 +/- 0.06 (left/right ratio) before ligation to 0.37 +/- 0.03 immediately after ligation. In contrast to ICGA, perfusion recuperated completely within 1 week to 1.01 +/- 0.14 and tended to be even higher in the ligated than in the unligated hind limb after 2 (1.09 +/- 0.25) and 3 weeks (1.20 +/- 0.29), pointing towards limitations of this technique. Histologic analysis confirmed the significant increase in the number of collaterals. The intraindividual ratio increased from 1.0 +/- 0.05 before ligation to 1.35 +/- 0.10 at 2 weeks and 1.41 +/- 0.08 at 3 weeks after ligation (P < .05). CONCLUSION Our data demonstrate that ICGA represents a potent tool for the quantification of collateral flow in small animal models. The current standard of LDPI seems to rather represent blood movements within the superficial skin but not of the entire hind limb.

Ultrasound guided thrombin injection of pseudoaneurysm of the radial artery after percutaneous coronary intervention.

Thrombin injection is frequently used to occlude iatrogenic pseudoaneurysms in larger vessels, but has never successfully been used in the radial artery location. Here we report the use of this treatment in a patient with radial artery pseudoaneurysm following coronary intervention. After Doppler sonographic visualization of the pseudoaneurysm cavity and its neck, an ultrasound-guided transcutaneous injection of thrombin was carried out. Immediately after the injection, the pseudoaneurysm was completely clotted and Doppler measurement confirmed the stop of blood flow. The result suggests that ultrasound-guided injection of thrombin into a radial artery pseudoaneurysm following coronary intervention is a feasible alternative to surgical intervention.

The lysosomal transfer of LDL/cholesterol from macrophages into vascular smooth muscle cells induces their phenotypic alteration

AIMS Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

Isolation and transduction of monocytes: promising vehicles for therapeutic arteriogenesis.

Background and aimsAugmentation of collateral vessel growth (arteriogenesis) is of particular clinical interest for the treatment of vascular occlusive disease. Monocytes play a key role for arteriogenesis. They localize to areas of collateral development and create a highly arteriogenic environment. “Homing” of ex vivo genetically engineered monocytes could therapeutically be exploited for augmentation of arteriogenesis. However, isolation and ex vivo transduction of monocytes is problematic.MethodsIn this study, we established a valid method of monocyte isolation from peripheral blood and evaluated different in vitro transduction methods.ResultsOur results revealed that liposomes and electroporation were unsuccessful for monocyte transduction. However, high-efficiency gene transfer (almost 95%) was achieved by adenoviral infection. Subsequent homing of virally transduced monocytes to sites of arteriogenesis could be demonstrated.ConclusionOur study may offer a new method for the augmentation of arteriogenesis, all of which makes the ultimate goal of applying this strategy to humans for therapy of vascular disease eminently attractive.

Efficacy and safety profile of dronedarone in clinical practice. Preliminary results of the Magdeburg Dronedarone Registry

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Das Takotsubo-Syndrom von der Erstbeschreibung bis heute

ZusammenfassungDie stressinduzierte Kardiomyopathie, auch als Takotsubo-Syndrom bekannt, ähnelt einem akuten ST-Hebungsinfarkt bzw. einem akuten Koronarsyndrom in Abwesenheit einer relevanten koronaren Herzerkrankung. Exakte epidemiologische Daten liegen bisher nicht vor, jedoch befällt das Takotsubo-Syndrom vorzugsweise Frauen in der Menopause. Auch die genaue Pathogenese ist noch nicht gesichert. Als häufigster beschriebener Auslöser wird plötzlich einsetzender emotionaler Stress genannt. Es gibt bis dato keine offiziellen Leitlinien für die Therapie des Takotsubo-Syndroms. Eine symptomatische Behandlung mit Aspirin, β-Blockern und Angiotensin-converting-Enzym-Hemmern wird empfohlen. Die ventrikuläre Kinetikstörung bildet sich normalerweise innerhalb von 4–5 Wochen zurück. Das Takotsubo-Syndrom hat im Vergleich zum akuten Herzinfarkt eine günstigere Prognose, allerdings können alle Komplikationen des akuten Herzinfarkts bis hin zum kardiogenen Schock auftreten.AbstractStress-induced cardiomyopathy, also known as takotsubo syndrome, imitates an acute ST elevation myocardial infarction or an acute coronary syndrome, but without concomitant coronary artery disease. It mainly affects postmenopausal women, but no established epidemiologic data of this syndrome are available to date. Furthermore, the underlying etiologies are still largely unknown. The most frequently described trigger is strong emotional stress. Supportive therapy with aspirin, β-blockers and angiotensin-converting enzyme inhibitors is recommended. The abnormal kinetics usually reverse or improve within 4–5 weeks. Compared with acute myocardial infarction, takotsubo cardiomyopathy carries a favorable prognosis. However, severe complications, including ventricular fibrillation and cardiogenic shock, may still occur.Stress-induced cardiomyopathy, also known as takotsubo syndrome, imitates an acute ST elevation myocardial infarction or an acute coronary syndrome, but without concomitant coronary artery disease. It mainly affects postmenopausal women, but no established epidemiologic data of this syndrome are available to date. Furthermore, the underlying etiologies are still largely unknown. The most frequently described trigger is strong emotional stress. Supportive therapy with aspirin, beta-blockers and angiotensin-converting enzyme inhibitors is recommended. The abnormal kinetics usually reverse or improve within 4-5 weeks. Compared with acute myocardial infarction, takotsubo cardiomyopathy carries a favorable prognosis. However, severe complications, including ventricular fibrillation and cardiogenic shock, may still occur.

Twiddler's syndrome in an adolescent patient with ICD during neurological and physical rehabilitation

Sirs: A 20-year-old woman was admitted to our hospital by the emergency physician after resuscitation in cardiac arrest due to a ventricular fibrillation (VF) caused by a non-classified cardiomyopathy with a impaired left ventricular function of about 35% and a long QT-syndrome. As a consequence of cerebral hypoxia the patient suffered from an impaired neurological status with cognitive deficit and sensomotoric disability. After 6 weeks of hospitalization good neurological potential was predicted and a single chamber ICD (Guidant/CPI Prizm VR) was implanted for secondary prevention of ventricular fibrillation [4]. The transvenous defibrillation lead (Guidant, Endotak Reliance) was inserted into the right ventricle via the right subclavian vein, since the left side had been affected earlier by a catheter associated thrombosis. The screw-in lead tip was positioned at the lower interventricular septum with a sensing of 12 mV and a pacing threshold of 0.4 V/0.4 ms. The

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow. Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.