Flow cytometry identifies changes in peripheral and intrathecal lymphocyte patterns in CNS autoimmune disorders and primary CNS malignancies.

BACKGROUND: Immune dysregulation is a hallmark of autoimmune diseases of the central nervous system (CNS), characterized by an excessive immune response, and primary CNS tumors (pCNS-tumors) showing a highly immunosuppressive parenchymal microenvironment. METHODS: Aiming to provide novel insights into the pathogenesis of CNS autoimmunity and cerebral tumor immunity, we analyzed the peripheral blood (PB) and cerebrospinal fluid (CSF) of 81 autoimmune limbic encephalitis (ALE), 148 relapsing-remitting multiple sclerosis (RRMS), 33 IDH-wildtype glioma, 9 primary diffuse large B cell lymphoma of the CNS (CNS-DLBCL), and 110 controls by flow cytometry (FC). Additionally, an in-depth immunophenotyping of the PB from an independent cohort of 20 RRMS and 18 IDH-wildtype glioblastoma patients compared to 19 controls was performed by FC combined with unsupervised computational approaches. RESULTS: We identified alterations in peripheral and intrathecal adaptive immunity, mainly affecting the T cell (Tc) but also the B cell (Bc) compartment in ALE, RRMS, and pCNS-tumors compared to controls. ALE, RRMS, and pCNS-tumors featured higher expression of the T cell activation marker HLA-DR, which was even more pronounced in pCNS-tumors than in ALE or RRMS. Glioblastoma patients showed signs of T cell exhaustion that were not visible in RRMS patients. In-depth characterization of the PB revealed differences mainly in the T effector and memory compartment between RRMS and glioblastoma patients and similar alterations in the Bc compartment, including atypical Bc, CD19+CD20- double negative Bc, and plasma cells. PB and CSF mFC together with CSF routine parameters could reliably differentiate ALE and RRMS from pCNS-tumors facilitating early diagnosis and treatment. CONCLUSIONS: ALE, RRMS, and pCNS-tumors show distinct but partially overlapping changes mainly in HLA-DR+ Tc, memory Tc, exhausted Tc, and Bc subsets providing insights into disease pathogenesis. Moreover, mFC shows diagnostic potential facilitating early diagnosis and treatment.

K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions.

BACKGROUND: The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months. METHODS AND RESULTS: PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-alpha-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-alpha-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types. CONCLUSIONS: In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.

In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions.

The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.

Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial.

BACKGROUND: Emerging evidence suggests that stem cells and progenitor cells derived from bone marrow can be used to improve cardiac function in patients after acute myocardial infarction. In this randomised trial, we aimed to assess whether intracoronary transfer of autologous bone-marrow cells could improve global left-ventricular ejection fraction (LVEF) at 6 months' follow-up. METHODS: After successful percutaneous coronary intervention (PCI) for acute ST-segment elevation myocardial infarction, 60 patients were randomly assigned to either a control group (n=30) that received optimum postinfarction medical treatment, or a bone-marrow-cell group (n=30) that received optimum medical treatment and intracoronary transfer of autologous bone-marrow cells 4.8 days (SD 1.3) after PCI. Primary endpoint was global left-ventricular ejection fraction (LVEF) change from baseline to 6 months' follow-up, as determined by cardiac MRI. Image analyses were done by two investigators blinded for treatment assignment. Analysis was per protocol. FINDINGS: Global LVEF at baseline (determined 3.5 days [SD 1.5] after PCI) was 51.3 (9.3%) in controls and 50.0 (10.0%) in the bone-marrow cell group (p=0.59). After 6 months, mean global LVEF had increased by 0.7 percentage points in the control group and 6.7 percentage points in the bone-marrow-cell group (p=0.0026). Transfer of bone-marrow cells enhanced left-ventricular systolic function primarily in myocardial segments adjacent to the infarcted area. Cell transfer did not increase the risk of adverse clinical events, in-stent restenosis, or proarrhythmic effects. INTERPRETATION: Intracoronary transfer of autologous bone-marrow-cells promotes improvement of left-ventricular systolic function in patients after acute myocardial infarction.

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.

Haematopoietic stem cells improve cardiac function after infarction without permanent cardiac engraftment.

BACKGROUND: Transplantation of bone marrow derived adult stem cells (BMC) improves cardiac function after acute myocardial infarction (MI). However, the cell population mediating myocardial recovery and the fate of the transplanted cells are still controversial. AIMS: We determined the effects of Sca-1+ c-kit+ lin- haematopoietic BMC on cardiac function after MI and the cell fate after transplantation. METHODS: Sca-1+ c-kit+ lin- BMC of male donor C57BL/6 mice were transplanted by intravenous injection into syngenic females after permanent MI. LV dimensions and function were determined by echocardiography and cardiac magnetic resonance imaging, transplanted BMC were identified by Y chromosome DNA in situ hybridization. RESULTS: BMC treatment completely prevented LV dilation (LV end-diastolic volume BMC 70 +/- 16 microl vs. control 122 +/- 41 microl; p < 0.05) and improved fractional shortening (BMC 22.9 +/- 8% vs. control 15.4 +/- 8.4%; p < 0.05) and ejection fraction BMC 68.2 +/- 6.6% vs. control 52 +/- 14.3%; p < 0.05) as early as 3 days after transplantation, but did not decrease infarct size (BMC 27 +/- 6% vs. control 28 +/- 7%, p = n.s.). After 4 weeks, only sporadic cells of male origin were identified in infarcted hearts (< 0.01% of periinfarct cells). CONCLUSION: Intravenous injection of Sca-1+ c-kit+ lin- in BMC after MI improves LV dimensions and function without evidence for long term engraftment.

Objectifying acupuncture effects by lung function and numeric rating scale in patients undergoing heart surgery.

Rationale. Poststernotomy pain and impaired breathing are common clinical problems in early postoperative care following heart surgery. Insufficiently treated pain increases the risk of pulmonary complications. High-dose opioids are used for pain management, but they may cause side effects such as respiratory depression. Study Design. We performed a prospective, randomized, controlled, observer-blinded, three-armed clinical trial with 100 patients. Group 1 (n = 33) and Group 2 (n = 34) received one 20 min session of standardized acupuncture treatment with two different sets of acupoints. Group 3 (n = 33) served as standard analgesia control without additional intervention. Results. Primary endpoint analysis revealed a statistically significant analgesic effect for both acupuncture treatments. Group 1 showed a mean percentile pain reduction (PPR) of 18% (SD 19, P < 0.001). Group 2 yielded a mean PPR of 71% (SD 13, P < 0.001). In Group 1, acupuncture resulted in a mean forced vital capacity (FVC) increase of 30 cm(3) (SD 73) without statistical significance (P = 0.303). In Group 2, posttreatment FVC showed a significant increase of 306 cm(3) (SD 215, P < 0.001). Conclusion. Acupuncture revealed specific analgesic effects after sternotomy. Objective measurement of poststernotomy pain via lung function test was possible.

Severe right heart failure after heart transplantation. A single-center experience.

We reviewed our heart transplantation recipient population, using hard criteria defining severe right heart failure (RHF), and analyzed possible risk factors for outcome after RHF. Between 1983 and 1998 621 cardiac transplantations were performed at our institution. RHF was defined by the necessity to implant an assist device or echocardiographically confirmed right ventricular ballooning with concomitant end organ failure. RHF patients were compared with a matched control group. Thirty-five patients (5.9%) with severe RHF after transplantation fulfilled inclusion criteria. Of these, 32 patients died, while none of the control patients died (P < 0.001). Increased preoperative pulmonary capillary wedge (P = 0.005) and mean pulmonary artery pressure (P = 0.006) were identified as significant risk factors for severe RHF. Severe RHF as defined in our study is irreversible in almost every case without differences among therapeutical concepts. Hence, improvement of postoperative outcome necessitates avoidance or aggressive therapy of possible risk factors.

Impact of multivessel coronary artery disease on outcome after isolated minimally invasive bypass grafting of the left anterior descending artery.

BACKGROUND: The outcome in patients treated surgically for coronary artery disease is known to be influenced by the extent of the disease. Whether this factor also has an effect in patients undergoing isolated minimally invasive revascularization of the left anterior descending (LAD) artery using the internal thoracic artery (ITA) (MIDCAB) has not been looked at. Thus, this study sought to evaluate the impact of multivessel disease (MVD) on midterm outcome after MIDCAB. METHODS: From 1996 to 1999, 411 patients received a MIDCAB at our institution and were now followed up. Isolated disease of the LAD (SVD -single vessel disease) was presented in 262 patients (63.7%) and 149 patients (36.3%) had MVD at the time of operation. The reasons for apparent incomplete revascularization in patients with MVD were very small target vessels (< 1.0-mm diameter), stenoses of less than 50%, distal localization of the stenoses, long-term patency after angioplasty, or an extensive risk for sternotomy and(or) cardiopulmonary bypass. The midterm outcome was evaluated by questionnaires sent to the patients and their physicians. RESULTS: The mean follow-up was 29.4 +/- 11.1 months. The incidence of myocardial infarction was significantly higher in MVD as compared to SVD patients (8.1% vs 1.9%, p = 0.04). Patients with MVD had significantly more subsequent percutaneous transluminal coronary angioplasty (10.7% vs 5.3%, p = 0.049) and a similar number of repeat surgical revascularizations as compared to SVD patients. Patients with MVD had a significantly higher total 3-year mortality as compared to SVD patients by Kaplan-Meier estimate (8.7% vs 3.1%, relative risk [RR] = 2.56, p = 0.011). The 3-year cardiac mortality was significantly higher in patients with MVD as compared to SVD (4.0% vs 0.4%, RR = 9.48, p = 0.0054). After adjustment of baseline characteristics by Cox regression analysis, the 3-year risk of cardiac death was significantly higher in the MVD groups (RR = 2.2, confidence interval [CI] 95%: 1.8 to 4.65, p = 0.029). CONCLUSIONS: Patients with isolated disease of the LAD appear to benefit from ITA grafting in the form of a MIDCAB procedure. Here, it should be an approach of choice. The results show that MVD is an independent risk factor for outcome in patients undergoing a MIDCAB procedure. Nevertheless, the midterm morbidity and mortality in MVD patients after a MIDCAB procedure where the LAD is the only target vessel for interventional or surgical treatment is acceptable despite a higher morbidity than in SVD patients.

Impact of diabetes on outcome following isolated minimally invasive bypass grafting of the left anterior descending artery.

BACKGROUND: The outcome in patients treated by conventional coronary artery bypass grafting (CABG) for coronary artery disease is negatively influenced by the presence of diabetes. The relative effect of diabetes in patients undergoing isolated minimally invasive revascularization of the left anterior descending artery (LAD) using the internal thoracic artery (ITA) has as yet not specifically been looked at. Thus, this study sought to evaluate the impact of diabetes on mid-term outcome following minimally invasive coronary artery bypass grafting (MIDCAB). METHODS: From 1996 to 1999, 411 patients received a MIDCAB procedure at our institution and were now followed up. In this study population there were 63 diabetic patients (15.3%) and 348 nondiabetic patients (84.7%). Isolated proximal stenoses or an occlusion of the LAD were present in 262 patients (63.7%), whereas 149 (36.3%) had multi-vessel disease (MVD) at the time of the MIDCAB procedure. The clinical outcome was evaluated by questionnaires sent to the patients and their physicians. RESULTS: The mean follow-up was 29.4 +/- 11.1 months. The incidence of myocardial infarction was significantly higher in diabetics as compared to nondiabetics (9.5% vs 3.2%, p = 0.034). Diabetics and nondiabetics had similar rates of subsequent revascularization procedures during follow-up. Cumulative total survival of diabetic and nondiabetic patients was not statistically different. The 3-year cardiac mortality was however significantly higher in diabetic than in nondiabetic patients if MVD was initially present (Kaplan-Meier estimate: 10.7% vs 2.5%, relative risk [RR] = 5.5, p = 0.017 by log-rank test). The 3-year cardiac mortality in diabetic and nondiabetic patients with isolated disease of the LAD (single vessel disease [SVD]) was not significantly different. After adjustment of baseline characteristics by Cox regression analysis the 3-year risk of cardiac death was significantly higher in the diabetic group (RR = 1.82, CI 95%:1.2 to 3.3, p = 0.045). CONCLUSIONS: The results support diabetes to be an independent risk factor for outcome in patients with MVD undergoing a MIDCAB procedure in analogy to those undergoing CABG procedures. Diabetics with isolated disease of the LAD, however, benefit out of proportion from this treatment modality.