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Purpose: Temporal summation (TS) of pain occurs when pain increases over repeated presentations of identical noxious stimuli. TS paradigms can model central sensitization, a state of hyperexcitability in nociceptive pathways that promotes chronic pain onset and maintenance. Many experimenters use painful heat stimuli to measure TS (TS-heat); yet, TS-heat research faces unresolved challenges, including difficulty evoking summation in up to 30-50% of participants. Moreover, substantial variability exists between laboratories regarding the methods for evoking and calculating TS-heat. Patients and Methods: To address these limitations, this study sought to identify optimal parameters for evoking TS-heat in healthy participants with a commercially available constant contact heat stimulator, the Medoc TSA-II. Working within constraints of the TSA-II, stimulus trains with varying parameters (eg, stimulus frequency, baseline temp, peak temp, peak duration, testing site) were tested in a sample of 32 healthy, chronic pain-free participants to determine which combination best evoked TS-heat. To determine whether TS scoring method altered results, TS-heat was scored using three common methods. Results: Across all methods, only two trains successfully evoked group-level TS-heat. These trains shared the following parameters: site (palmar hand), baseline and peak temperatures (44°C and 50°C, respectively), and peak duration (0.5 s). Both produced summation that peaked at moderate pain (~50 out of 100 rating). Conclusion: Future TS-heat investigations using constant contact thermodes and fixed protocols may benefit from adopting stimulus parameters that include testing on the palmar hand, using 44°C baseline and 50°C peak temperatures, at ≥0.33 Hz stimulus frequency, and peak pulse durations of at least 0.5 seconds.
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The CRESST experiment employs cryogenic calorimeters for the sensitive measurement of nuclear recoils induced by dark matter particles. The recorded signals need to undergo a careful cleaning process to avoid wrongly reconstructed recoil energies caused by pile-up and read-out artefacts. We frame this process as a time series classification task and propose to automate it with neural networks. With a data set of over one million labeled records from 68 detectors, recorded between 2013 and 2019 by CRESST, we test the capability of four commonly used neural network architectures to learn the data cleaning task. Our best performing model achieves a balanced accuracy of 0.932 on our test set. We show on an exemplary detector that about half of the wrongly predicted events are in fact wrongly labeled events, and a large share of the remaining ones have a context-dependent ground truth. We furthermore evaluate the recall and selectivity of our classifiers with simulated data. The results confirm that the trained classifiers are well suited for the data cleaning task.
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CRESST is a leading direct detection sub-GeVc-2 dark matter experiment. During its second phase, cryogenic bolometers were used to detect nuclear recoils off the CaWO4 target crystal nuclei. The previously established electromagnetic background model relies on Secular Equilibrium (SE) assumptions. In this work, a validation of SE is attempted by comparing two likelihood-based normalisation results using a recently developed spectral template normalisation method based on Bayesian likelihood. Albeit we find deviations from SE in some cases we conclude that these deviations are artefacts of the fit and that the assumptions of SE is physically meaningful.
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Research has documented neurophysiological indicators of anticipation (Stimulus Preceding Negativity [SPN]) and perception (Late Positive Potential [LPP]) of threat, yet little is known as to how self-focused attention manipulations influence emotion processing within the context of cued picture viewing. With self-referent attention moderating attention to external stimuli, it is necessary to document how self-focused attention impacts attention and the ability to emotionally process external threat. The goal of the present study was to evaluate the impact of self-focused attention on the anticipation and perceptual processing of unpleasant pictures within a cued-picture viewing paradigm among 33 participants. Overall, the results suggest that the self-focused attention manipulations disrupted anticipation but not processing of pictures, as indexed by the SPN and LPP respectively. Self-focused attention appears to disrupt the preparatory attention for upcoming unpleasant stimuli, potentially through loading cognitive resources or activation of associative defensive responding. Collectively, these findings demonstrate the impact of self-focused attention within the context of emotional picture processing and suggest further areas of investigation.
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Eletroencefalografia , Potenciais Evocados , Humanos , Potenciais Evocados/fisiologia , Eletroencefalografia/métodos , Estimulação Luminosa , Emoções/fisiologia , MotivaçãoRESUMO
The most widely accepted definition of pain considers it a sensory and emotional experience associated with potential or actual physical harm. However, research tends to generalize findings from predominantly European American samples thereby assuming universality across cultures. Because of the high prevalence of pain within the AI group, it is important to consider whether their conceptualization of pain is similar to the universal definition. To accomplish this aim, a semi-structured interview was conducted with 152 AIs (primarily Southern Plains and eastern Oklahoma tribes) and 150 NHWs. Both groups were asked questions including what words describe hurtful experiences, the purpose of painful experiences, individual and culture-specific meanings of pain, and what constituted the opposite of pain. Many similarities were found between groups as well as differences. For example, NHWs used the word pain more often to describe physically hurtful experiences and were more likely to consider pain to be a signal or warning of an abnormality or pathology. By contrast, only AIs reported culture-specific meanings of pain, such as references to AI rituals or ceremonies. These observed differences are attenuated by small effect sizes. These findings are important to consider when hypothesizing the differences in pain among cultural groups.
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Indígenas Norte-Americanos , Dor , Humanos , Indígenas Norte-Americanos/psicologia , Oklahoma/epidemiologia , População Branca , Indígena Americano ou Nativo do AlascaRESUMO
Native Americans (NAs) experience higher rates of chronic pain. To examine the mechanisms for this pain inequity, we have previously shown that NAs report higher levels of pain-related anxiety and pain catastrophizing, which are in turn related to pronociceptive (pain-promoting) processes. But, it is currently unclear why NAs would report greater pain-related anxiety and catastrophizing. Given that NAs are also more likely to experience adverse life events (ALEs) and associated psychological distress, it was hypothesized that higher anxiety/catastrophizing in NAs would be partially explained by higher rates of ALEs and psychological distress. Structural equation modeling was used to analyze these pathways (NA ethnicity â ALEs â psychological distress â pain anxiety/catastrophizing) in 305 healthy, pain-free adults (N = 155 NAs, N = 150 non-Hispanic Whites [NHWs]). Pain-related anxiety and situational pain catastrophizing were assessed in response to a variety of painful tasks. The Life Events Checklist was used to assess cumulative exposure to ALEs that directly happened to each participant. A latent psychological distress variable was modeled from self-reported perceived stress and psychological symptoms. Results found that NAs experienced more ALEs and greater psychological distress which was associated with higher rates of pain-related anxiety and pain catastrophizing. Notably, NAs did not report greater psychological distress when controlling for ALE exposure. This suggests that a higher risk of chronic pain in NAs may be due, in part, to psychological distress, pain-related anxiety, and pain catastrophizing that are promoted by exposure to ALEs. These results highlight several targets for intervention to decrease NA pain risk.
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Dor Crônica , Estresse Psicológico , Adulto , Dor Crônica/psicologia , Cognição , Humanos , Oklahoma/epidemiologia , Estresse Psicológico/psicologia , Indígena Americano ou Nativo do AlascaRESUMO
Adverse life events (ALEs) are a risk factor for chronic pain; however, mechanisms underlying this association are not understood. This study examined whether cumulative ALE exposure impairs endogenous inhibition of pain (assessed from pain report) and spinal nociception (assessed from nociceptive flexion reflex; NFR) in healthy, pain-free Native Americans (nâ¯=â¯124) and non-Hispanic Whites (nâ¯=â¯129) during a conditioned pain modulation (CPM) task. Cumulative ALE exposure was assessed prior to testing by summing the number of potentially traumatic events experienced by each participant across their lifespan. Multilevel modeling found that ALEs were associated with NFR modulation during the CPM task even after controlling for general health, body mass index, sex, age, blood pressure, sleep quality, stimulation intensity, stimulus number, perceived stress, and psychological distress. Low exposure to ALEs was associated with NFR inhibition, whereas high exposure to ALEs was associated with NFR facilitation. By contrast, pain perception was inhibited during the CPM task regardless of the level of ALE exposure. Race/ethnicity did not moderate these results. Thus, ALEs may be pronociceptive for both Native Americans and non-Hispanic Whites by impairing descending inhibition of spinal nociception. This could contribute to a chronic pain risk phenotype involving latent spinal sensitization. PERSPECTIVE: This study found that adverse life events were associated with impaired descending inhibition of spinal nociception in a sample of Native Americans and non-Hispanic Whites. These findings expand on previous research linking adversity to chronic pain risk by identifying a proximate physiological mechanism for this association.
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Indígena Americano ou Nativo do Alasca/etnologia , Acontecimentos que Mudam a Vida , Inibição Neural/fisiologia , Nociceptividade/fisiologia , Dor/fisiopatologia , Trauma Psicológico/fisiopatologia , Reflexo/fisiologia , Medula Espinal/fisiopatologia , Adulto , Feminino , Humanos , Masculino , Dor Nociceptiva/etnologia , Dor Nociceptiva/fisiopatologia , Oklahoma/etnologia , Dor/etnologia , Trauma Psicológico/etnologia , Fatores de Risco , População Branca/etnologiaRESUMO
INTRODUCTION: Evidence suggests Native Americans (NAs) experience higher rates of chronic pain than the general US population, but the mechanisms contributing to this disparity are poorly understood. Recently, we conducted a study of healthy, pain-free NAs (n = 155), and non-Hispanic whites (NHWs, n = 150) to address this issue and found little evidence that NAs and NHWs differ in pain processing (assessed from multiple quantitative sensory tests). However, NAs reported higher levels of pain-related anxiety during many of the tasks. OBJECTIVE: The current study is a secondary analysis of those data to examine whether pain-related anxiety could promote pronociceptive processes in NAs to put them at chronic pain risk. METHODS: Bootstrapped indirect effect tests were conducted to examine whether pain-related anxiety mediated the relationships between race (NHW vs NA) and measures of pain tolerance (electric, heat, ischemia, and cold pressor), temporal summation of pain and the nociceptive flexion reflex (NFR), and conditioned pain modulation of pain/NFR. RESULTS: Pain-related anxiety mediated the relationships between NA race and pain tolerance and conditioned pain modulation of NFR. Exploratory analyses failed to show that race moderated relationships between pain-related anxiety and pain outcomes. CONCLUSION: These findings imply that pain-related anxiety is not a unique mechanism of pain risk for NAs, but that the greater tendency to experience pain-related anxiety by NAs impairs their ability to engage descending inhibition of spinal nociception and decreases their pain tolerance (more so than NHWs). Thus, pain-related anxiety may promote pronociceptive processes in NAs to place them at risk for future chronic pain.
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Native Americans (NAs) have a higher prevalence of chronic pain than other U.S. racial/ethnic groups, but there have been few attempts to understand the mechanisms of this pain disparity. This study used a comprehensive battery of laboratory tasks to assess peripheral fiber function (cool/warm detection thresholds), pain sensitivity (eg, thresholds/tolerances), central sensitization (eg, temporal summation), and pain inhibition (conditioned pain modulation) in healthy, pain-free adults (N = 155 NAs, N = 150 non-Hispanic Whites [NHWs]). Multiple pain stimulus modalities were used (eg, cold, heat, pressure, ischemic, and electric), and subjective (eg, pain ratings and pain tolerance) and physiological (eg, nociceptive flexion reflex) outcomes were measured. There were no group differences on any measure, except that NAs had lower cold-pressor pain thresholds and tolerances, indicating greater pain sensitivity than NHWs. These findings suggest that there are no group differences between healthy NAs and NHWs on peripheral fiber function, central sensitization, or central pain inhibition, but NAs may have greater sensitivity to cold pain. Future studies are needed to examine potential within-group factors that might contribute to NA pain risk.
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Indígena Americano ou Nativo do Alasca , Sensibilização do Sistema Nervoso Central/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Nociceptividade/fisiologia , Limiar da Dor/fisiologia , Dor/etnologia , População Branca , Adolescente , Adulto , Feminino , Humanos , Inibição Psicológica , Masculino , Oklahoma , Dor/fisiopatologia , Limiar da Dor/etnologia , Somação de Potenciais Pós-Sinápticos/fisiologia , Sensação Térmica/fisiologia , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA). EXPERIMENTAL APPROACH: The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured. KEY RESULTS: The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC(50) in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure-activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%. CONCLUSIONS AND IMPLICATIONS: Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.
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Inibidores da Angiogênese/farmacologia , Oligopeptídeos/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos SCID , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Oligopeptídeos/uso terapêutico , Tubulina (Proteína)/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Afterbirth tissues, which include the umbilical cord, placenta, amnion, and cord blood, are usually discarded. Recent progress in regenerative medicine suggests that we re-evaluate these tissues and assess their therapeutic potential. METHODS: Firstly the unique properties of afterbirth tissues and their current use in regenerative medicine are summarised. Then we introduce the cooperation of our institutions and our experiences regarding the collection and utilisation of afterbirth tissues. RESULTS: A literature survey suggests that besides the well-known transplantation of hematopoietic stem cells from cord blood, afterbirth tissues were also used as a source of stem cells, progenitor cells, differentiated cells, and blood vessels for tissue engineering purposes. According to our own experience, the two participating OB/GYN departments and the blood donation service were able to organise a sufficient supply of umbilical cords for research purposes. The yield correlated with incentives for the midwives. A total of more than 4,300 cords was collected for experiments designed to create small caliber vessel grafts. The contamination rate was low. Birth mode significantly affected umbilical vein function, whereas ischaemia for up to 40 h did not have any deleterious effects. Umbilical veins were cryopreserved with a moderate loss of function. Fresh umbilical veins were endothelium-denuded and reseeded with endothelial cells harvested from coronary artery disease patients to generate an autologous surface. CONCLUSIONS: Afterbirth tissues have unique properties which make them ideally suited for regenerative medicine. These tissues can be procured and utilised in research facilities even in the absence of an in-house birthing centre.
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Âmnio , Sangue Fetal , Placenta , Medicina Regenerativa/métodos , Cordão Umbilical , Veias Umbilicais , Comportamento Cooperativo , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Células Endoteliais , Feminino , Alemanha , Transplante de Células-Tronco Hematopoéticas , Humanos , Recém-Nascido , Comunicação Interdisciplinar , Gravidez , Pesquisa , Células-Tronco , Doadores de Tecidos , Engenharia Tecidual/métodos , Preservação de Tecido/métodosRESUMO
Gold standard for in vitro toxicity tests and drug screenings is primary human hepatocytes (hHeps). Because of their limited availability efforts have been made to provide alternatives, e.g., monocyte-derived NeoHepatocytes. In the past years it has been critically discussed if gaining hepatocyte features is associated with trans-differentiation of monocytes or their activation towards a macrophage phenotype. Generating NeoHepatocytes in the presence of six different human AB sera, fetal calf serum (FCS) or autologous serum showed that yield and quality of NeoHepatocytes is inversely correlated to macrophage activation. Using autologous serum constantly the highest amount of cells with the best metabolic capacity was obtained. Focus of this study was to further analyze bio-transformation capacity of the optimized NeoHepatocytes for use as in vitro toxicity test-system. Treatment of the optimized NeoHepatocytes with two different pro-teratogenic substances with corresponding metabolites and eight known hepatotoxins showed comparable toxicity to hHeps. Bio-transformation rates, assessed by testosterone metabolism, were comparable in both cell types. Our data reveal that use of autologous serum reduced macrophage activation which improved yield and function of NeoHepatocytes resulting in bio-transformation and toxicity profiles comparable to hHeps. Thus, their easy accessibility makes them an ideal candidate for in vitro toxicity studies.
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Hepatócitos/efeitos dos fármacos , Monócitos/metabolismo , Testes de Toxicidade/métodos , Animais , Bovinos , Transdiferenciação Celular , Sangue Fetal/metabolismo , Hepatócitos/metabolismo , Humanos , Macrófagos/metabolismo , Teratogênicos/toxicidade , Testosterona/metabolismoRESUMO
Hepatocyte-transplantation is a therapeutic approach for diverse acute and chronic liver diseases. As availability of primary cells is limited, there is an increasing demand for hepatocyte-like cells (e.g., neohepatocytes generated from peripheral blood monocytes). The aim of this study was to evaluate the effects of six different human AB sera, fetal calf serum, or autologous serum on production of neohepatocytes. The yield and quality of neohepatocytes varied considerably depending on the different sera. Using autologous sera for the whole production process we constantly generated the highest amount of cells with the highest metabolic activity for phase I (e.g., CYP1A1/2, CYP3A4) and phase II enzymes (e.g., glutathione-S-transferase). Moreover, similar effects were seen examining glucose and urea metabolism. Especially, glucose-6-phosphatase and PAS staining showed distinct serum-dependent differences. The role of macrophage activation was investigated by measuring the secretion of TNF-α, TGF-ß, and RANKL, MMP activity, as well as mRNA levels of different interleukins in programmable cells of monocytic origin (PCMO). Our data clearly demonstrate that the use of autologous serum reduced initial macrophage activation in PCMOs and subsequently improved both yield and function of differentiated neohepatocytes. The autologous approach presented here might also be useful in other stem cell preparation processes where cell activation during generation shall be kept to a minimum.
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Transplante de Células , Hepatócitos/citologia , Hepatócitos/metabolismo , Monócitos/citologia , Soro/metabolismo , Cloreto de Amônio/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Hepatócitos/enzimologia , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Ativação de Macrófagos , Metaloproteinases da Matriz/metabolismo , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ureia/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Since 2004, bacterial screening of platelets has been required in the USA and is also done on a voluntary basis in many European countries. The German Red Cross blood donor services conducted a prospective multicentre study in order to investigate the prevalence of bacterially contaminated pool platelet concentrates and apheresis platelet concentrates. This substudy compares three different bacterial detection systems. STUDY DESIGN AND METHODS: Platelet concentrates were tested in parallel with BacT/ALERT, Scansystem and Pall eBDS (n = 6307) in pool platelets. Apheresis platelets were tested in parallel with BacT/ALERT and Pall eBDS (n = 4730). All initially positive results were evaluated by a standardized procedure including evaluation by a microbiology reference laboratory. RESULTS: One in 6307 pool platelets were confirmed positive by BacT/ALERT, whereas Pall eBDS and Scansystem failed to detect these samples. Only three samples were initially reactive with Pall eBDS without proof of any bacteria strains. The rate of false-positive results was substantially higher for BacT/ALERT (0.25%, 28 in 11,037 tested samples) than for eBDS (0.03%, 3 in 11 037 tested samples) or Scansystem (0.0%, 0 in 6307 tested samples). Three of 4730 apheresis platelets were confirmed positive by BacT/ALERT. These were negative with Pall eBDS. CONCLUSION: Sensitivity was best for BacT/ALERT, whereas specificity was enhanced for Pall eBDS and Scansystem. Scansystem required specially trained staff, whereas BacT/ALERT and Pall eBDS were easy, quick, user-friendly and objective methods.
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Bactérias/isolamento & purificação , Técnicas Bacteriológicas/instrumentação , Doadores de Sangue , Plaquetas/microbiologia , Transfusão de Plaquetas , Infecções Bacterianas/prevenção & controle , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.
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Criopreservação/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/análise , Bactérias/isolamento & purificação , Contagem de Células , Separação Celular/métodos , Sobrevivência Celular , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Dimetil Sulfóxido/química , Dimetil Sulfóxido/isolamento & purificação , Células Precursoras Eritroides/citologia , Sangue Fetal/microbiologia , Células-Tronco Hematopoéticas/química , Humanos , Contagem de Leucócitos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Albumina Sérica/química , Células-Tronco/citologiaRESUMO
In compliance with federal regulations, blood banks routinely use leukocyte depletion filters to eliminate contaminating leukocytes from blood products such as red blood cell and platelet concentrates. We developed and optimized conditions to elute leukocytes adsorbed to these filters; resulting in leukocyte suspensions which we termed Filter Buffy Coats (FBCs). These Filter Buffy Coats can replace standard buffy coats for various research applications. After optimizing both the filter elution medium as well as elution protocols, we compared commonly used leukocyte depletion filters from four different manufacturers. Relative fractions as well as total recoveries of leukocyte subsets, such as lymphocytes, monocytes and granulocytes, found in Filter Buffy Coats were identified and compared among the filters as well as to standard buffy coats and whole blood. Flow cytometric analysis of Filter Buffy Coats confirmed the presence of T- and B-lymphocytes, NK cells and monocytes. Furthermore, a significant quantity of CD34(+) hematopoietic stem or progenitor cells (HSC/HPC) was detected in Filter Buffy Coats prepared from different filters, thus making FBCs a valuable source for research on HSC/HPC. Colony assays revealed that most of these CD34(+) cells are functional. Using immunomagnetic cell sorting (MACS), we isolated a variety of leukocyte populations from FBC mononuclear cells (Filter-PBMCs) including T lymphocytes (CD4(+), CD8(+), CD3(+)), B lymphocytes (CD19(+)), NK cells (CD56(+)), HSC/HPC (CD34(+), CD133(+)) or dendritic cells (BDCA-4(+)). Functional properties of Filter-PBMCs, as well as of some of these isolated leukocyte populations, were confirmed using standard assays. In summary, Filter Buffy Coats are a valuable and convenient source of different peripheral leukocyte populations and can replace standard buffy coat preparations for research applications.
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Procedimentos de Redução de Leucócitos/instrumentação , Leucócitos Mononucleares/citologia , Antígenos CD/análise , Linfócitos B/química , Linfócitos B/citologia , Soluções Tampão , Contagem de Células , Separação Celular/instrumentação , Separação Celular/métodos , Ensaio de Unidades Formadoras de Colônias , Células Dendríticas/citologia , Células Dendríticas/imunologia , Citometria de Fluxo , Granulócitos/citologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Concentração de Íons de Hidrogênio , Separação Imunomagnética , Células Matadoras Naturais/química , Células Matadoras Naturais/citologia , Leucócitos Mononucleares/química , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Monócitos/química , Monócitos/citologia , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Adult stem cells from umbilical cord and cord blood are an interesting alternative to embryonic stem cells because such research is commonly recognized as ethical undisputed and many aspects are still insufficiently investigated. In the context of the STEMMAT research project (STEM = Stem Cell and MAT = Material) different aspects of stem cells from umbilical cord and cord blood are investigated, to improve basic science understanding and potentially leading someday to a clinical application.
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Ética em Pesquisa , Sangue Fetal/citologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/ética , Feminino , Humanos , Gravidez , Pesquisa/normas , Doadores de TecidosRESUMO
Environmental pollutants activated by UV sunlight may have contributed to the recent decline in frog populations and the concomitant increase in malformations in the USA and abroad. UV radiation is able to mutate DNA and to initiate photosensitization processes that generate mutagenic and biologically disruptive oxygen transients. We have examined water from selected sites in Minnesota and Vermont using singlet molecular oxygen (1O(2)), detected by its phosphorescence and free radicals detected by spin trapping, as markers for photosensitization. Water from a pond in Minnesota with malformed frogs, which also causes malformations in the laboratory, photosensitized more 1O(2), even though it absorbed less UV light compared to water from a site that did not cause malformations. This suggested that unknown natural or pollutant agents were present, and that photosensitization may be involved. Although UV irradiation of the two Minnesota water samples in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of the DMPO/*OH, DMPO/*H(e(aq)-) and DMPO/*C(unknown) adducts there were no qualitative or quantitative differences between them. We also examined water samples from several sites in Vermont, and compared them by measuring the quantum yield of 1O(2) photosensitization. While all the Vermont samples produced a small amount of 1O(2), there was no clear correlation with the incidence of frog malformations. However, the samples differed strongly in absorption spectra and the ability to quench 1O(2). These factors may determine how much UV light is absorbed and converted into chemical reactions. Our results show that photochemical characterization of 1O(2) photosensitization is possible in untreated natural water samples. Photosensitization falls into the category of global factors that may be closely associated with the effects of UV irradiation of the Earth's environments. Thus, photosensitization might be an important component in global amphibian malformation and decline. The observation of 1O(2) emission directly from natural water may also provide new opportunities to investigate the involvement of 1O(2) in other complex environmental processes.
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Anuros/anormalidades , Água Doce/análise , Oxigênio Singlete/efeitos da radiação , Raios Ultravioleta , Poluição Química da Água/análise , Animais , Óxidos N-Cíclicos/química , Meio Ambiente , Medições Luminescentes , Minnesota , Fotoquímica , Oxigênio Singlete/química , Detecção de Spin , VermontRESUMO
BACKGROUND: Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS: During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS: Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS: Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.
Assuntos
Deformidades Congênitas dos Membros , Rana pipiens , Animais , Deformidades Congênitas dos Membros/etiologia , Estados UnidosRESUMO
BACKGROUND: As an essential hospital facility, the central oxygen supply system should be designed with features allowing backup and/or redundancy in the event of system failure. As part of an organized institutional review of The Cleveland Clinic Foundation hospital inpatient central oxygen supply system, we undertook a survey of all hospitals in two Ohio cities to determine the characteristics of hospital central supply systems. METHODS: The questionnaire was developed and completed by structured telephone interview during calls placed to managers of facilities engineering departments in 35 hospitals in the greater Cleveland and Columbus, Ohio, metropolitan areas. To encourage candid responses to the telephone interview, respondents were assured that institutional names would not be presented in published reports. The questionnaire addressed the type of primary and reserve oxygen sources in the hospital, whether a backup system exists, and if so, in what configuration. The questionnaire also addressed whether any unplanned interruption or other problem (such as contamination of the piped-in oxygen supply) had ever occurred in the facility. RESULTS: Of the 35 eligible hospitals, responses were available from 32 (91.4%). The mean number of beds in the hospitals responding was 397 +/- 251 (standard deviation), and the original construction dates of the responding hospitals ranged from 1887 to 1982. All 32 responding institutions reported a reserve system, described as a liquid reservoir in 72% (23/32), manifolded cylinders in 16% (5/32), and both in 13% (4/32). Twenty-six (81%) of those responding reported having the reserve supply liquid or manifolded gas cylinders at the same location as the primary liquid vessel. The supply lines of these contiguous primary and reserve containers were reported to join proximal to entering the hospital structure, so at each of these 26 hospitals the primary and reserve systems depend on a single length of pipe. Only 4 (13%) of the hospitals have manifolded cylinders in addition to the primary and reserve liquid supplies. These manifolds are in different locations from the primary and reserve, have physically separate feed lines, and represent the only true examples of redundant piped-in oxygen supplies recorded during the survey. Of the 32 hospitals surveyed, 5 (16%) reported having experienced mishaps with the bulk liquid supply. CONCLUSIONS: (1) Not surprisingly, most of the hospitals in these two urban areas use bulk liquid oxygen systems (with primary and reserve liquid reservoirs) as the main central supply source, with some providing manifolded cylinders as backup. (2) Mishaps regarding the main supply line from the bulk oxygen reservoir were reported by 16% (5/32) of responding institutions. (3) In this context, the fact that most main and reserve tanks were contiguous and fed through a single line to the hospital facility suggests ongoing risk for interruption of an oxygen supply by line mishaps (e.g., street repair). (4) Contingency planning to lessen the risk of an interrupted supply should involve back-up systems with physically separated feed lines, as well as tanks of manifolded cylinders along the course of the main hospital oxygen circuit line.