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Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the Drosophila sleep homeostat (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation). We report that dFB neurons can distinguish between different types of waking and can change their physiological response-properties accordingly. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons themselves can determine their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

Falling asleep at the wrong time can place an individual at risk of immediate physical harm. However, not sleeping degrades cognition and adaptive behavior. To understand how animals match sleep need with environmental demands, we used live-brain imaging to examine the physiological response properties of the dorsal fan-shaped body (dFB) following interventions that modify sleep (sleep deprivation, starvation, time-restricted feeding, memory consolidation) in Drosophila. We report that dFB neurons change their physiological response-properties to dopamine (DA) and allatostatin-A (AstA) in response to different types of waking. That is, dFB neurons are not simply passive components of a hard-wired circuit. Rather, the dFB neurons intrinsically regulate their response to the activity from upstream circuits. Finally, we show that the dFB appears to contain a memory trace of prior exposure to metabolic challenges induced by starvation or time-restricted feeding. Together, these data highlight that the sleep homeostat is plastic and suggests an underlying mechanism.

Neuropeptides control rhythmic behaviors, but the timing and location of their release within circuits is unknown. Here imaging in the brain shows that synaptic neuropeptide release by Drosophila clock neurons is diurnal, peaking at times of day that were not anticipated by prior electrical and Ca2+ data. Furthermore, hours before peak synaptic neuropeptide release, neuropeptide release occurs at the soma, a neuronal compartment that has not been implicated in peptidergic transmission. The timing disparity between release at the soma and terminals results from independent and compartmentalized mechanisms for daily rhythmic release: consistent with conventional electrical activity-triggered synaptic transmission, terminals require Ca2+ influx, while somatic neuropeptide release is triggered by the biochemical signal IP3. Upon disrupting the somatic mechanism, the rhythm of terminal release and locomotor activity period are unaffected, but the number of flies with rhythmic behavior and sleep-wake balance are reduced. These results support the conclusion that somatic neuropeptide release controls specific features of clock neuron dependent behaviors. Thus, compartment specific mechanisms within individual clock neurons produce temporally and spatially partitioned neuropeptide release to expand the peptidergic connectome underlying daily rhythmic behaviors.Significance StatementIt is believed that electrical activity simultaneously stimulates widespread release sites in single neurons to elicit neuropeptide dependent behaviors. However, optically detecting neuropeptide release in the intact brain shows that clock neurons release neuropeptides from different sites at different times of the day. This is possible because one neuronal compartment, the soma, uses biochemical signaling instead of electrical activity to evoke release. Disrupting somatic release affects specific features of circadian locomotor activity and sleep. Thus, neuropeptide release is elicited by independent triggers from distinct parts of clock neurons to engage different regions of behavior regulating circuitry. This strategy for expanding the connectome may be used for other neuropeptide dependent behaviors, such as feeding and pain perception.

To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.-Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

Despite regulatory issues surrounding the use of animal-derived cell culture supplements, most clinical cardiac cell therapy trials using mesenchymal stromal cells (MSCs) still rely on fetal bovine serum (FBS) for cell expansion before transplantation. We sought to investigate the effect of human serum from heart failure patients (HFS) on cord blood MSCs (CB-MSCs) during short-term culture under regular conditions and during simulated acute and chronic stress. Cell survival, proliferation, metabolic activity, and apoptosis were quantified, and gene expression profiles of selected apoptosis and cell cycle regulators were determined. Compared to FBS, HFS and serum from healthy donors (CS) showed similar effects by substantially increasing cell survival during chronic and acute stress and by increasing cell yields 5 days after acute stress. Shortly after the termination of acute stress, both HFS and CS resulted in a marked decrease in apoptotic cells. Transcriptome analysis suggested a decrease in TNF-mediated induction of caspases and decreased activation of mitochondrial apoptosis. Our data confirm that human serum from both healthy donors and heart failure patients results in increased cell yields and increased resistance to cellular stress signals. Therefore, we consider autologous serum a valid alternative to FBS in cell-based therapies addressing severe heart disease.

For more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation (“cellular cardiomyoplasty”), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.

Background If compression bandaging is not performed in a professional manner, the objectives of the therapy may not be achieved and side effects or complications may result. Objectives This cross-sectional observational survey examines the handling of the treatment options: short-stretch bandages with padding, multicomponent compression systems, and adaptive compression bandages. Participants and methods During several training sessions on the topic of compression therapy, 137 participants performed compression bandagings on each other. In this regard, they were asked to achieve a predetermined pressure range (short-stretch bandages: 50-60 mm Hg, multicomponent compression systems: 40-50 mm Hg, adaptive compression bandage: 35-45 mm Hg). To evaluate the efficiency, the time used for application, the achieved pressure value, and the comfort were determined. Results Of the 302 bandagings (n = 137 participants), 28.4% lay within the given target pressure value range. This included 11.2% of performed short-stretch bandages, 35.2% of multicomponent compression systems, and 85.0% of adaptive compression bandages. Significant differences in the mean deviations are found between the treatment options. The bandage was described as being comfortable by 37.7% of users of short-stretch bandages with padding, by 65.0% of those wearing a multicomponent compression system, and by 94.6% of participants with an adaptive compression bandage. Conclusions In practice, short-stretch bandages are still the most frequently used care option for the creation of a phlebological compression bandage. In this survey, they proved to be unsafe, time-consuming, and uncomfortable in relation to other treatment options. Multicomponent compression systems and adaptive compression bandages are treatment options that may be a contemporary alternative which also bares more comfort for the patient.

Background Chronic lymphedema is characterized by a continuous need for medical treatment, many comorbidities, and impaired quality of life. In Germany, about 4.5 million patients are affected by lymphedema. Thus, lymphedema causes high direct and indirect costs, even more in case of complications such as erysipelas and ulcers. Objective The aim of this study was to determinate the costs of illness of community lymphedema patients living in the metropolitan area of Hamburg, Germany. Methods An observational cross-sectional study in patients with lymphedema and combined lipolymphedema of any origin was performed analysing direct and indirect costs for the patients, the statutory health insurance, and society. Results In total, 348 patients (90.8% female) were examined and interviewed. The mean age of the patients was 57.3 ± 14.5 years. On average, the total costs per patient and year were € 5784, of which € 4445 (76.0%) were direct costs and € 1338 indirect costs. Within the direct medical costs, € 3796 were accounted for the statutory health insurances and € 649 for the patient. The main cost drivers were costs for manual decongestive therapy and disability costs. Conclusion Chronic lymphedema is associated with high direct and indirect costs. This community based study is the first cost analysis of chronic lymph- and combined lipolymphedema giving insights to economic impact of lymphedema treatment. There is a high need for structured disease management programs in order to diagnose and treat lymphedema early and to avoid complications, thus limiting socioeconomic burden. This article is protected by copyright. All rights reserved.

Objective Enriched populations of proliferative induced cardiomyocyte precursors (iCMPs) generated from cardiac fibroblasts (CF) could represent a potential population for use in therapeutic application. We developed a protocol for generation of iCMPs from cardiac fibroblasts by genetic reprogramming followed by transcriptional selection and induced maturation. Methods Cardiac fibroblasts were reprogrammed into induced cardiomyocyte precursor cells (iCMPs) via transduction with cardiomyogenesis-related transcription factors Gata4, Mef2c, TBX5 and Myocd. Pure population of iCMPs were obtained by molecular beacons (MB) based selection of MYH6/7 expression. The transcriptome was profiled by RNA sequencing. For maturation iCMPs were treated with 5'-azacytidine and cultured in medium containing ascorbic acid and TGFβ1. The cells were monitored regularly for formation of sarcomere structures, cardiac marker expression and contractions. iCMPs were transplanted into MI mice and monitored for 6 weeks. At the end of 6 weeks, the mice were sacrificed and hearts were explanted and cryopreserved to examine the heart structure and scare size. Cardiac function was monitored by echocardiography. Results iCMPs expressed troponin T, α-actinin and myosin heavy chain (MHC) protein observed by immunocytology. These iCMPs could robustly proliferative and maintain a stable phenotype. Global transcriptome analysis revealed that the iCMP gene expression profile is unique from those of the parental CFs and adult CMs. With Gene Ontology (GO) analysis, we found that iCMPs show upregulation of genes associated with cardiac development, differentiation and morphogenesis while they showed downregulation of genes associated to cell-proliferation in comparison to their parental CFs. Evaluation of selected gene sets showed downregulation of non-myocyte genes, upregulation of transcription factors and upregulation of certain functional and structural genes like ion channel genes and contractile genes. Maturation studies showed that iCMPs gradually changed morphology after stimulation with 5-Aza, as seen in immunofluorescence staining. Cells expressing cTnT showed formation of sarcomeric striations. In vivo translational studies showed reduction of heart function in control groups treated with PBS or CF whereas the iCMPs group showed an improvement in function, reflected by left ventricular ejection fraction and fractional shortening as evaluated by echocardiography. Additionally we noticed an improvement in left ventricular wall thickness at diastole in mice transplanted with iCMPs. Conclusions Gene expression pattern suggests that these iCMPs represent an intermediate state of cardiogenic population that can be expanded to yield therapeutic cell doses. When transplanted to mouse hearts following myocardial infarction, they improved cardiac function.

OBJECTIVES: Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways. METHODS: Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex ® immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells ( poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK) 1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic). RESULTS: CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs. CONCLUSIONS: The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 μm (15–157 μm) by laser diffraction, and 57 μm (20–182 μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, −72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) inDrosophila. Null mutants ofent2are lethal during late larval/early pupal stages, indicating thatent2is essential for normal development. Hypomorphic mutant alleles ofent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock downent2expression in specific regions of the CNS and show thatent2is required in the α/β lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated inent2mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR1) into theent2mutant background. The results of the present study provide the first evidence of a role forent2function inDrosophilaand suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

Verhaltenstherapeutische Kurse zur Raucherentwohnung konnen als praventive Satzungsleistungen nach § 20 SGB V und § 20a SGB V von gesetzlichen Krankenkassen angeboten werden. In der vorliegenden Untersuchung sollen die Angebots- und Erstattungssituation dieser Masnahmen analysiert werden. Die Querschnittsbefragung wurde als Vollerhebung der gesetzlichen Krankenkassen (n=219) durchgefuhrt. Die Antwortrate betragt 45% (n=98). Uber 90% dieser Krankenkassen bieten interne oder externe verhaltensbezogene (uberwiegend gruppenbasierte) Entwohnungskurse im Rahmen des § 20 SGB V an. Der Anteil erstatteter Kosten bewegt sich zwischen 80% und 100% (maximal 75–150 EUR). Die obere Grenze gilt fur die intern angebotenen Kurse. Etwa ein Drittel der befragten Kassen bietet eine Telefonberatung oder internetbasierte Angebote zur Begleitung der Tabakentwohnung an. Die grose Mehrheit der Kassen ist zudem im Bereich der Raucherentwohnung nach § 20a SGB V (betriebliche Gesundheitsforderung) aktiv. Die meisten Krankenkassen bieten verhaltensbezogene Entwohnungskurse an und/oder erstatten diese entweder vollstandig oder zu einem wesentlichen Teil. Nach internationalen Erfahrungen kann eine Kostenubernahme dabei zu einer hoheren Inanspruchnahme und besseren Abstinenzraten fuhren. Vor dem Hintergrund der verfugbaren Evidenz zur Effektivitat und Wirtschaftlichkeit ist es notwendig, die bestehenden Entwohnungsangebote (auch in Kombination mit unterstutzenden Masnahmen) in methodisch hochwertigen gesundheitsokonomischen Evaluationen zu untersuchen.

Ziel: Hochvolumige Datensatze der bildgebenden Diagnostik (Direktradiografie, Multi-Slice-CT etc.) sichern die diagnostische Betreuung. Die bildgebende Diagnostik hat als Querschnittsfach Schrittmacherfunktion fur effektive Workflow-Szenarien ubernommen. Fur ein effektives Datenmanagement sind hierfur seit Jahren Konzepte zur Datenkompression diskutiert worden. Im Februar 2008 hat eine Konsensuskonferenz der Deutschen Rontgengesellschaft stattgefunden. Es wurden einzelne Datenkompressionstechniken, Kompressionsfaktoren und deren Organbezug tabellarisch als Empfehlung zusammengestellt. Material und Methoden: Unsere Arbeit gibt eine Gesamtubersicht uber den Literaturstand zur Datenkompression, Technologie (JPEG und JPEG 2000) und Organbezug und analysiert unterschiedliche Workflow-Szenarien. Dies war Grundlage der Konsensuskonferenz. Die Studien wurden in 4 Level (0 – 3) in Abhangigkeit zu ihrer Evidenz eingeteilt. Fur den hochsten Level 3 konnten 51 Studien ausgewertet werden. Ergebnisse: Mit Ausnahme der Schadel-CT wird ein einheitlicher Kompressionsfaktor von 1 : 8 empfohlen. Schadel-CT konnen ohne diagnostischen Qualitatsverlust mit einem Kompressionswert von 1:5 komprimiert werden. Aus Workflow-Sicht empfehlen wir, Kompressionen an den Modalitaten (CT etc.) vorzunehmen. PACS-basierte Kompressionen sind jedoch derzeit ublich. In diesen Fallen werden allerdings nicht alle Workflow-Vorteile genutzt. Schlussfolgerung: Aus der Literaturubersicht hinsichtlich Technik, Organbezug und unserer Empfehlung zum Workflow ergibt sich die Forderung an die Industrie, die bildgebenden Modalitaten mit einem Kompressionsfilter auszustatten. Es gilt, dass grundsatzlich pro Bilddatensatz nur einmal komprimiert wird. Purpose: Today healthcare policy is based on effectiveness. Diagnostic imaging became a ”pacesetter” due to amazing technical developments (e. g. multislice CT), extensive data volumes, and especially the well defined workflow-orientated scenarios on a local and (inter)national level. To make centralized networks sufficient, image data compression has been regarded as the key to a simple and secure solution. In February 2008 specialized working groups of the DRG held a consensus conference. They designed recommended data compression techniques and ratios. Material und Methoden: The purpose of our paper is an international review of the literature of compression technologies, different imaging procedures (e. g. DR, CT etc.), and targets (abdomen, etc.) and to combine recommendations for compression ratios and techniques with different workflows. The studies were assigned to 4 different levels (0 – 3) according to the evidence. 51 studies were assigned to the highest level 3. Results: We recommend a compression factor of 1 : 8 (excluding cranial scans 1:5). For workflow reasons data compression should be based on the modalities (CT, etc.). PACS-based compression is currently possible but fails to maximize workflow benefits. Only the modality-based scenarios achieve all benefits. Conclusion: Imaging equipment manufacturers are encouraged to improve the compression technology of their imaging devices (e. g. freely selectable compression ratios in the output filter). Double compression should be strictly avoided. Lossless compression formats should be switched off.

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.

A prior hyperthermic stress (heat shock) can induce thermoprotection of neuromuscular transmission in Locusta migratoria extensor tibiae muscle measured 4 h after the onset of the heat shock. It is not clear what effect an acute hyperthermic stress may have on the nervous system's ability to tolerate thermal stress, that is, before increased expression of heat-shock proteins. We found that over consecutive thermal stress tests, failure temperature was not altered in either heat-shock or control animals. This suggests that protective mechanisms are not established in the short term (within one hour). Various members of the heat-shock protein family interact with elements of the cytoskeleton. We found that preexposure of the preparation to cytoskeletal stabilizing drugs induced thermoprotection, while preexposure to cytoskeletal disrupting drugs disrupted the ability to confer and maintain thermoprotection. We conclude that thermoprotection relies on a stable cytoskeleton and suggest that members of the heat shock protein family are involved.

Background/Aims: Cell-based therapies may be useful for treating ischemic diseases, but the underlying mechanisms are incompletely understood. We investigated the impact of cord blood mesenchymal stromal cell (CBMSC)- or fibroblast (FB)-secreted factors on starved endothelial cells and determined the relevant intracellular signaling pathways. Methods: HUVECs were subjected to glucose/serum deprivation (GSD) in hypoxia or normoxia, in presence of CBMSC- or FB-conditioned medium (CM). Viability and proliferation were determined via WST-8 conversion and BrdU incorporation. Apoptosis was quantified by annexin V/ethidium homodimer-III staining, nuclear fragmentation and cell morphology. mRNA expression and protein phosphorylation were determined by real-time qPCR and western blot. Experiments were repeated in presence of small-molecule inhibitors. Results: The negative impact of GSD was most pronounced at 21% O2. Here, medium of CBMSCs and FBs increased viability and proliferation and reduced apoptosis of HUVECs. This was associated with increased STAT3 and ERK1/2 phosphorylation and BCL-2 expression. Under STAT3 inhibition, the beneficial effect of CBMSC-CM on viability and BCL-2 expression was abolished. Conclusion: Factors released by CBMSCs protect endothelial cells from the deleterious impact of GSD by activation of the STAT3 survival pathway. However, this phenomenon is not CBMSC-specific and can be reproduced using juvenile fibroblasts.

Background Clinical cardiac cell therapy using autologous somatic stem cells is restricted by age and disease-associated impairment of stem cell function. Juvenile cells possibly represent a more potent alternative, but the impact of patient-related variables on such cell products is unknown. We therefore evaluated the behavior of neonatal cord blood mesenchymal stem cells (CB-MSC) in the presence of serum from patients with advanced heart failure (HF). Methods Human serum was obtained from patients with severe HF (n = 21) and from healthy volunteers (n = 12). To confirm the systemic quality of HF in the sera, TNF-α and IL-6 were quantified. CB-MSC from healthy neonates were cultivated for up to 14 days in medium supplemented with 10% protein-normalized human HF or control serum or fetal calf serum (FCS). Results All HF sera contained increased cytokine concentrations (IL-6, TNF-α). When exposed to HF serum, CB-MSC maintained basic MSC properties as confirmed by immunophenotyping and differentiation assays, but clonogenic cells were reduced in number and gave rise to substantially smaller colonies in the CFU-F assay. Cell cycle analysis pointed towards G1 arrest. CB-MSC metabolic activity and proliferation were significantly impaired for up to 3 days as measured by MTS turnover, BrdU incorporation and DAPI + nuclei counting. On day 5, however, CB-MSC growth kinetics approached control serum levels, though protein expression of cell cycle inhibitors (p21, p27), and apoptosis marker Caspase 3 remained elevated. Signal transduction included the stress and cytokine-induced JNK and ERK1/2 MAP kinase pathways. Conclusions Heart failure temporarily inhibits clonality and proliferation of “healthy” juvenile MSC in vitro. Further studies should address the in vivo and clinical relevance of this finding.

SYNOPSIS. Environmental stresses such as high temperature or low levels of oxygen can lead to structural destabilization of cells, disruption of cellular processes, and, in extreme cases, death. Previous experience of sub-lethal stress can lead to protection during a subsequent stress that may otherwise have been lethal. Synapses are particularly vulnerable to extreme environmental conditions and failure of function at this level may be the primary cause of organismal death. Prior heat shock induces enhanced thermotolerance at neuromuscular junctions in the locust extensor tibiae muscle and in abdominal muscles of larval Drosophila. Synaptic thermoprotection is associated with an increase in short-term plasticity at these synapses. Prior anoxic coma in locusts induces synaptic thermotolerance suggesting that the same protective pathways are activated. It is well established that diverse forms of stress induce the upregulation of cellular chaperones (heat shock proteins; HSPs) that mediate acquired protection. The mechanisms underlying HSP-mediated synaptic protection are currently unknown but evidence is accumulating that stabilization of the cytoskeleton may play an important role.

The rate of contrast injection during i.v. urography may vary considerably (bolus injection or drip infusion). The effect of 5 rates of injection (6, 12, 18, 36 and 72 ml/min) on the contrast density in the renal collecting system was examined over a period of 30 minutes. Measurements showed an inter-individual difference of more than 200% in each group. The intra-individual variations for different rates of injection were very slight; individual concentration in the kidney for a given dose depends only slightly on the rate of injection. There is no statistically significant improvement in contrast values as a result of a bolus injection. The reduced incidence of side effects justifies the use of bolus injections but pressure injections are unnecessary from a diagnostic point view.

Neuropeptides are found in both mammals and invertebrates and can modulate neural function through activation of G-protein-coupled receptors (GPCRS). The precise mechanisms by which many of these GPCRs modulate specific signaling cascades to regulate neural function are not well defined. We usedDrosophila melanogasteras a model to examine both the cellular and behavioral effects of DPKQDFMRFamide, the most abundant peptide encoded by the dFMRF gene. We show that DPKQDFMRFamide enhanced synaptic transmission through activation of two G-protein-coupled receptors,Fmrf Receptor(FR) andDromyosupressin Receptor-2 (DmsR-2). The peptide increased both the presynaptic Ca2+response and the quantal content of released transmitter. Peptide-induced modulation of synaptic function could be abrogated by depleting intracellular Ca2+stores or by interfering with Ca2+release from the endoplasmic reticulum through disruption of either the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor. The peptide also altered behavior. Exogenous DPKQDFMRFamide enhanced fictive locomotion; this required both theFRandDmsR-2. Likewise, both receptors were required for an escape response to intense light exposure. Thus, coincident detection of a peptide by two GPCRs modulates synaptic function through effects of Ca2+-induced Ca2+release, and we hypothesize that these mechanisms are involved in behavioral responses to environmental stress.

Regenerative medicine encompasses “tissue engineering” – the in vitro fabrication of tissues and/or organs using scaffold material and viable cells – and “cell therapy” – the transplantation or manipulation of cells in diseased tissue in vivo. In the cardiovascular system, tissue engineering strategies are being pursued for the development of viable replacement blood vessels, heart valves, patch material, cardiac pacemakers and contractile myocardium. Anecdotal clinical applications of such vessels, valves and patches have been described, but information on systematic studies of the performance of such implants is not available, yet. Cell therapy for cardiovascular regeneration, however, has been performed in large series of patients, and numerous clinical studies have produced sometimes conflicting results. The purpose of this chapter is to summarize the clinical experience with cell therapy for diseases of the cardiovascular system, and to analyse possible factors that may influence its outcome.

Maintenance of synaptic transmission requires regulation of intracellular Ca2+in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca2+indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na+/Ca2+exchanger or removal of extracellular Ca2+prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca2+clearance—inactivation of the endoplasmic reticulum Ca2+-ATPase with thapsigargin and inhibition of the plasma membrane Ca2+-ATPase with high pH—significantly accelerated the temperature-induced rise in resting Ca2+concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca2+-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca2+] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca2+regulation. In each of these cases, Ca2+elevations evoked by an electrical stimulation of the nerve (evoked Ca2+responses) failed when resting Ca2+remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.

Modulation of neuromuscular transmission at invertebrate neuromuscular junctions has been studied in detail mainly in arthropods, particularly in decapod crustaceans and in two orders of insects. Alterations in neurotransmission most often reflect changes in the presynaptic motor nerve terminal of the neuromuscular junctions. Invertebrate motor nerve terminals typically possess numerous small synaptic contacts with group-specific characteristics. Changes in synaptic performance may involve altered probability of neurotransmitter release at individual synaptic contacts, increases or decreases in the number of responsive synapses, or both. Modulation of synaptic performance can be initiated in several ways. The most significant inducing agents are alterations in nerve impulse activity in the transmitting motor neuron, synaptic transmission from another neuron that affects the neuromuscular junction (especially presynaptic inhibition), and circulating neurohormones or local neuromodulators, in particular amines and neuropeptides. Many forms of modulation are linked to regulation of intracellular Ca2+ in the presynaptic nerve terminal, but in some cases second-messenger pathways are activated without significant changes in presynaptic Ca2+.

The lymphographic findings in 20 patients with AIDS-most in clinical stage IV-can be divided into four categories. In six there were no specific changes and in a further six the storage pattern was that of sinus histiocytosis. One patient showed transition to malignant lymphoma and another patient had atypical mycobacterial infection of the retroperitoneal lymph nodes. Six patients showed a lymphographic pattern of scarring. The results of this study show that lymphography is a sensitive method that can provide important additional information.

We examined the thermosensitivity of calcium regulation in Drosophila larval neuromuscular junctions, testing effects of prior heat shock and Hsp70 expression. Motor neurons were loaded with either the ratiometric indicator Fura-dextran or the nonratiometric indicator Oregon Green bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid to monitor parameters of calcium regulation as temperature increased. Nerve terminals treated to a prior heat shock, and those of transgenic flies expressing higher than normal levels of Hsp70, were better able to maintain near-normal resting calcium concentrations, calcium influx, and calcium clearance at higher temperatures. Synaptic transmission was also protected by prior heat shock and by higher than normal Hsp70 expression. Thus the thermal limit of synaptic transmission may be directly linked to the stability of calcium regulation.