RESUMO
Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was > 4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.
Assuntos
Diferenciação Celular/fisiologia , Fenótipo , Placenta/citologia , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Proliferação de Células/fisiologia , Feminino , Macaca , Placentação/fisiologia , GravidezRESUMO
Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/patologia , Sequência de Aminoácidos , Animais , Autofagia , Callithrix , Diferenciação Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Estresse do Retículo Endoplasmático , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutagênese Sítio-Dirigida , Neuritos/fisiologia , Doença de Parkinson/genética , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and loss of large motor neurons in the spinal cord and brain stem. While much research has focused on mechanisms of motor neuron cell death in the spinal cord, degenerative processes in skeletal muscle and neuromuscular junctions (NMJs) are also observed early in disease development. Although recent studies support the potential therapeutic benefits of targeting the skeletal muscle in ALS, relatively little is known about inflammation and glial responses in skeletal muscle and near NMJs, or how these responses contribute to motor neuron survival, neuromuscular innervation, or motor dysfunction in ALS. We recently showed that human mesenchymal stem cells modified to release glial cell line-derived neurotrophic factor (hMSC-GDNF) extend survival and protect NMJs and motor neurons in SOD1(G93A) rats when delivered to limb muscles. In this study, we evaluate inflammatory and glial responses near NMJs in the limb muscle collected from a rat model of familial ALS (SOD1(G93A) transgenic rats) during disease progression and following hMSC-GDNF transplantation. Muscle samples were collected from pre-symptomatic, symptomatic, and end-stage animals. A significant increase in the expression of microglial inflammatory markers (CD11b and CD68) occurred in the skeletal muscle of symptomatic and end-stage SOD1(G93A) rats. Inflammation was confirmed by ELISA for inflammatory cytokines interleukin-1 ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in muscle homogenates of SOD1(G93A) rats. Next, we observed active glial responses in the muscle of SOD1(G93A) rats, specifically near intramuscular axons and NMJs. Interestingly, strong expression of activated glial markers, glial fibrillary acidic protein (GFAP) and nestin, was observed in the areas adjacent to NMJs. Finally, we determined whether ex vivo trophic factor delivery influences inflammation and terminal Schwann cell (TSC) response during ALS. We found that intramuscular transplantation of hMSC-GDNF tended to exhibit less inflammation and significantly maintained TSC association with NMJs. Understanding cellular responses near NMJs is important to identify suitable cellular and molecular targets for novel treatment of ALS and other neuromuscular diseases.
Assuntos
Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/patologia , Inflamação/etiologia , Macrófagos/fisiologia , Músculo Esquelético/patologia , Neuroglia/fisiologia , Esclerose Lateral Amiotrófica/genética , Animais , Antígenos CD/metabolismo , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Inflamação/genética , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/patologia , Ratos , Ratos Transgênicos , Receptores Colinérgicos/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patologia , Superóxido Dismutase/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The LuCED Lung Test comprises an automated 3-dimensional morphologic analysis of epithelial cells in sputum. For each cell, 594 morphology-based features are measured to drive algorithmic classifiers that quantitatively assess whether neoplastic cells are present. The current interim clinical study involves sputum samples from patients with known benign and malignant outcomes to assess the feasibility of LuCED as an adjunctive test after suspicious low-dose computed tomography (LDCT) results or as an independent screening test for lung cancer. METHODS: Sputum samples were fixed, enriched for epithelial cells, and analyzed with a 3-dimensional cell scanner called Cell-CT. Candidate abnormal cells were identified by the classifiers for manual review. The sensitivity, specificity, and negative and positive predictive values were calculated for the detection of neoplastic cases. RESULTS: A total of 91 sputum samples from patients with confirmed lung cancer (49 patients) and patients with no known malignancy (42 patients) were evaluated. After cytology review, sensitivity in the positive group was 91.8%, and specificity was 95.2%. Specificity was not 100% because there were 2 cases in which abnormal cells were identified by the Cell-CT that were confirmed as such at the time of manual cytology review. However, at the time of last follow-up, malignancy had not been detected in these 2 cases. Modeling in a population with a 1% prevalence of lung cancer, the positive and negative predictive values would be 95.4% and 99.9%, respectively. CONCLUSIONS: LuCED testing is highly sensitive and specific for the detection of lung cancer and has potential value as an adjunctive test after suspicious LDCT findings or as a primary screening test in which LuCED-positive cases would be triaged to diagnostic CT. Further prospective studies currently are underway to evaluate its full usefulness.
Assuntos
Detecção Precoce de Câncer/métodos , Imageamento Tridimensional , Neoplasias Pulmonares/patologia , Escarro/citologia , Escarro/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Idoso , Automação , Estudos de Coortes , Citodiagnóstico/métodos , Células Epiteliais/diagnóstico por imagem , Células Epiteliais/patologia , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Sensibilidade e Especificidade , Análise e Desempenho de TarefasRESUMO
The war against cancer has yielded important advances in the early diagnosis and treatment of certain cancer types, but the poor detection rate and 5-year survival rate for lung cancer has changed little over the past 40 years. Early detection through emerging lung cancer screening programs promise the most reliable means of improving mortality. Sputum cytology has been tried without success because sputum contains few malignant cells that are difficult for cytologists to detect. However, research has shown that sputum contains diagnostic malignant cells and could serve as a means of lung cancer detection if those cells could be detected and correctly characterized. Recently, the National Lung Screening Trial reported that screening using 3 consecutive low-dose x-ray computed tomography scans provides a 20% reduction in lung cancer mortality compared with chest x-ray. However, this reduction in mortality comes with an unacceptable false-positive rate that increases patient risks and the overall cost of lung cancer screening. The LuCED test for detection of early lung cancer is reviewed in the current article. LuCED is based on patient sputum that is enriched for bronchial epithelial cells. The enriched sample is then processed on the Cell-CT, which images cells in 3 dimensions with submicron resolution. Algorithms are applied to the 3-dimensional cell images to extract morphometric features that drive a classifier to identify cells that have abnormal characteristics. The final status of these candidate abnormal cells is established by the pathologist's manual review. LuCED promotes accurate cell classification that could enable the cost-effective detection of lung cancer.
Assuntos
Interpretação de Imagem Assistida por Computador , Imageamento Tridimensional/métodos , Neoplasias Pulmonares/patologia , Escarro/citologia , Escarro/diagnóstico por imagem , Criopreservação/métodos , Citodiagnóstico/métodos , Detecção Precoce de Câncer/métodos , Células Epiteliais/diagnóstico por imagem , Células Epiteliais/patologia , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Masculino , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Tomografia Computadorizada por Raios X/métodosRESUMO
Skeletal muscle progenitor cells (SMPCs) are considered one of the most valuable cells for cell-based therapy targeting skeletal muscle. However, an efficient protocol for isolating and maintaining human myogenic progenitors in vitro has not been fully established. In this study, we demonstrate that human myogenic progenitors can be expanded and proliferated from human fetal muscles. Human SMPCs were prepared from fetal hind limb muscles and induced to proliferate as free-floating spheres termed myospheres in the medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Both myogenic progenitors and myoblast populations from human fetal muscles were effectively propagated in myospheres and passaged by a mechanical chopping. After expanding these spheres in culture, we tested whether myogenic progenitor cells can differentiate into multinucleated myotubes. The myospheres were dissociated, plated down on coverslips and cultured in the medium for terminal differentiation. We could confirm that the plated cells formed well-developed, multinucleated myotubes. This culture method using myospheres is an effective protocol to isolate and maintain SMPCs from human fetal skeletal muscles in culture.
Assuntos
Feto/citologia , Músculo Esquelético/citologia , Células-Tronco/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Músculo Esquelético/metabolismo , Células-Tronco/metabolismoRESUMO
Optical projection tomography (OPT) requires the depth of field (DOF) of the lens to cover at least half of the sample. There is a trade-off between obtaining high resolution with a high-NA lens and obtaining large DOF with a low-NA lens. The DOF of a high-NA objective lens can be extended by scanning its focal plane through the sample. We call this extended DOF image a "pseudoprojection." Images reconstructed from these pseudoprojections have isometric resolution, which can be the same as the lateral resolution of the high-NA objective. The focal scanning method produces an over 10× improvement in OPT resolution.
Assuntos
Técnicas Citológicas/métodos , Microscopia de Fluorescência/métodos , Tomografia Óptica/métodos , Algoritmos , Animais , Linhagem Celular , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Cervo Muntjac , Coloração e RotulagemRESUMO
An optical projection tomography microscope (OPTM) can improve axial resolution by viewing a sample from different perspectives. Here, we report a dual-mode OPTM that can generate 3D images of single cancer cells in both absorption mode and polarization mode. Cancer cells were labeled with hematoxylin for absorption imaging and nanorods for polarization imaging. Absorption images can provide morphologic information, and polarization images can provide molecular information. The combination of molecular detection and 3D cytological cell analysis may help with early cancer diagnosis.
Assuntos
Ouro , Hematoxilina , Aumento da Imagem/instrumentação , Neoplasias Pulmonares/patologia , Microscopia/instrumentação , Nanoestruturas , Tomografia/instrumentação , Linhagem Celular Tumoral , Meios de Contraste , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Aumento da Imagem/métodos , Nanoestruturas/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 microm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.
Assuntos
Técnicas Citológicas/métodos , Microscopia de Fluorescência/métodos , Tomografia Óptica/métodos , Animais , Linhagem Celular , Feminino , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Metáfase/fisiologia , Cervo Muntjac , Coloração e Rotulagem/métodosRESUMO
CONTEXT: Appalachia has high rates of tobacco use and related health problems, and despite significant impediments to alcohol use, alcohol abuse is common. Adolescents are exposed to sophisticated tobacco and alcohol advertising. Prevention messages, therefore, should reflect research concerning culturally influenced attitudes toward tobacco and alcohol use. METHODS: With 4 grants from the National Institutes of Health, 34 focus groups occurred between 1999 and 2003 in 17 rural Appalachian jurisdictions in 7 states. These jurisdictions ranged between 4 and 8 on the Rural-Urban Continuum Codes of the Economic Research Service of the US Department of Agriculture. Of the focus groups, 25 sought the perspectives of women in Appalachia, and 9, opinions of adolescents. FINDINGS: The family represented the key context where residents of Appalachia learn about tobacco and alcohol use. Experimentation with tobacco and alcohol frequently commenced by early adolescence and initially occurred in the context of the family home. Reasons to abstain from tobacco and alcohol included a variety of reasons related to family circumstances. Adults generally displayed a greater degree of tolerance for adolescent alcohol use than tobacco use. Tobacco growing represents an economic mainstay in many communities, a fact that contributes to the acceptance of its use, and many coal miners use smokeless tobacco since they cannot light up in the mines. The production and distribution of homemade alcohol was not a significant issue in alcohol use in the mountains even though it appeared not to have entirely disappeared. CONCLUSIONS: Though cultural factors support tobacco and alcohol use in Appalachia, risk awareness is common. Messages tailored to cultural themes may decrease prevalence.
Assuntos
Consumo de Bebidas Alcoólicas/epidemiologia , Atitude , Cultura , Fumar/epidemiologia , Adolescente , Região dos Apalaches/epidemiologia , Criança , Feminino , Grupos Focais , Humanos , MasculinoRESUMO
Currently, little is known about the role of nurses' aides (NAs) in rural long-term care facilities or their impact on the process of death and dying in rural healthcare environments. Focus groups with NAs were held in 6 rural counties located in 5 states to assess attitudes and perceptions about end-of-life care and training needs. Key informants from 8 states and the District of Columbia added to the understandings. Nurses' aides (N = 63) and key informants (N = 21) worked in a variety of rural settings that provide end-of-life care (ie, nursing homes, hospitals, hospices, home healthcare agencies). Five themes about the needs of rural NAs around end-of-life care were identified in the focus groups, and 4 themes emerged from key informant interviews. A prototype computer-based training module on communication about end-of-life issues was developed, tested, and found useful and compelling.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Assistentes de Enfermagem , População Rural , Assistência Terminal , Adulto , Feminino , Grupos Focais , Humanos , Masculino , Pessoa de Meia-Idade , Estados UnidosRESUMO
Adolescents who live in tobacco-growing areas use tobacco at earlier ages and more frequently than other youth. These adolescents, like all tobacco users, have many health risks. To be successful, cessation efforts targeting these youth must reflect the cultural, social, and economic import of tobacco in their communities. Six focus groups with girls aged 12 to 14 who lived in tobacco-growing communities in Appalachian Ohio, Tennessee, and Virginia and 20 interviews with key informants were conducted. Barriers identified by informants included community norms around tobacco use, family use of tobacco, school practices and policies, peer influences, youth attitudes, and logistical difficulties with cessation program efforts. Key findings indicated: (1) the social community in tobacco-growing communities is a significant influence in tobacco use; (2) family is important among young people in tobacco-growing communities and influences cessation positively and negatively; (3) parental smoking was an influence to smoke (4) some parents condone and even facilitate tobacco use by their children, but others actively discourage use; and (5) concern for the health of younger brothers and sisters elicits a strongly protective reaction from youth in discussions of health risks related to secondhand smoke. Youth in tobacco-growing regions have many similarities to others, but they also have unique cultural characteristics pertinent in the development and delivery of tobacco cessation programs.
Assuntos
Comportamento do Adolescente/psicologia , Conhecimentos, Atitudes e Prática em Saúde , Abandono do Hábito de Fumar/psicologia , Fumar/psicologia , Conformidade Social , Adolescente , Região dos Apalaches/epidemiologia , Criança , Família/psicologia , Feminino , Grupos Focais , Humanos , Entrevistas como Assunto , Relações Pais-Filho , Grupo Associado , Fumar/etnologia , Abandono do Hábito de Fumar/etnologia , Prevenção do Hábito de Fumar , Poluição por Fumaça de Tabaco/prevenção & controleRESUMO
Since the 1980s, theorists have posited that health education that reflects the cultural realities of communities that health educators targeted for behavioral interventions would be more successful than interventions that are not culturally sensitive. Between 1997 and 2002, 52 focus groups of youth, women, and men were conducted in the Appalachian portions of 10 states to discern cultural themes relevant to health education in Appalachia. Groups occurred within the context of 5 studies funded by institutes within the National Institutes of Health. Findings suggest that an emphasis on family shows immense promise as a culturally sensitive approach to health education. Interventions that use the central role of women in the health of their families may be useful. The study results also suggest that one-on-one approaches to health education may prove a promising technique, attacks on individuals and institutions are not useful strategies, and a preference for realism or "the facts" may be a good way to present information.