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1.
Medicine (Baltimore) ; 100(14): e25367, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33832119

RESUMO

ABSTRACT: Carcinomatous meningitis (CM) is a critical issue for physicians. However, no study has reported a simple and useful diagnostic or predictive marker for CM.This study aimed to elucidate the potential markers for diagnosing CM derived from cerebrospinal fluid (CSF).We retrospectively enrolled 78 lung cancer patients with suspected CM during the clinical course, including 42 CM and 36 non-CM patients. We compared the clinical and CSF findings, including carcinoembryonic antigen (CEA), between CM and non-CM patients, and explored the diagnostic markers for early identification of CM as well as the contributing factors for mortality.On CSF analysis, with cutoff values of CEA ≥5 ng/ml, total protein (TP) in CSF ≥45 g/dl, and total cell count (TCC) ≥7 cells/µL, the sensitivity, specificity, and area under the curve (AUC) for CM were 85.7%, 84.6%, and 0.887 (95% CI: 0.758-1.0, P < .001); 80.5%, 69.4%, and 0.755 (95% CI: 0.646-0.865, P < .001); and 56.1%, 100%, and 0.817 (95% CI: 0.722-0.912, P < .001), respectively. TP levels in CSF ≥the patients' age had a sensitivity, specificity, and an AUC of 48.8%, 77.8%, and 0.633 (95% CI: 0.722-0.912, P = .045) for CM, respectively. Among CM patients, patients with 'TP in CSF (>patients' age)" (n = 19, P = .008) showed significantly shorter 90-day survival probability than the residual patients (n = 20). None of the CSF parameters could predict the risk of mortality on Cox regression analysis.The cutoff value of CEA ≥5 ng/ml in CSF is a simple and useful method with a high diagnostic value for CM diagnosis, but not a suitable predicting factor for mortality. 'TP in CSF >patients' age" might be a novel factor for assessing short-term mortality.


Assuntos
Antígeno Carcinoembrionário/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Neoplasias Pulmonares/patologia , Carcinomatose Meníngea/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/líquido cefalorraquidiano , Estudos de Casos e Controles , Contagem de Células/métodos , Feminino , Humanos , Neoplasias Pulmonares/complicações , Masculino , Carcinomatose Meníngea/líquido cefalorraquidiano , Carcinomatose Meníngea/mortalidade , Carcinomatose Meníngea/secundário , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Sensibilidade e Especificidade
3.
Turk Neurosurg ; 31(2): 282-289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33575999

RESUMO

AIM: To assess and compare the antioxidant capacities of high-grade gliomas (HGG) according to their grades and the presence of isocitrate dehydrogenase 1 (IDH1) mutation using tissue thiol level measurement. MATERIAL AND METHODS: Tissue thiol concentrations were measured in 41 HGG samples and 21 healthy brain tissues obtained from autopsy procedures, which were performed within the first 4 hours of death. All samples were stored at ?80°C, and a thiol quantification kit was used in evaluating tissue thiol levels. The Number Cruncher Statistical System was used for statistical analyses to detect the differences between the control group and the HGG group, which was also divided into subgroups according to their grade and IDH1 mutation presence. RESULTS: The tissue thiol levels of HGGs were found to be higher than the control group (p=0.001). Although the median thiol levels of Grade 4 gliomas were higher than those of Grade 3, no statistically significant difference was noted (p=0.076). When all tumors were compared according to the IDH1 mutation presence, IDH1-negative (IDH1-) HGGs had higher thiol contents than IDH1 mutant (IDH1+) HGGs (p=0.001). The thiol levels of Grade 4 IDH1- gliomas were statistically significantly higher than of Grade 3 gliomas (p=0.023), but no statistically significant difference between the thiol levels of Grade 3 and Grade 4 IDH1+ tumors was noted (p=0.459). CONCLUSION: We have demonstrated the higher thiol concentrations of HGGs, particularly IDH1- ones. The sulfhydryl contents of gliomas as an indicator of tumoral antioxidant capacity may be responsible for the treatment resistance of IDH1- gliomas, the mechanism of which is not clear. Thiols can be a novel target for treatment, considering the unsatisfactory results of current modalities for HGGs.


Assuntos
Antioxidantes/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Isocitrato Desidrogenase , Mutação , Compostos de Sulfidrila/metabolismo , Adulto , Idoso , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Contagem de Células/métodos , Feminino , Glioma/genética , Glioma/patologia , Humanos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Adulto Jovem
4.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466261

RESUMO

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The aim of the present study was to validate a transgenic model for SST-expressing amacrine cells and to investigate the chronic effect of PACAP on the aging of SSTergic and dopaminergic cells of the retina. SST-tdTomato transgenic mice that were 6, 12 and 18 months old were treated intravitreally with 100 pmol of PACAP every 3 months. The density of SST and dopaminergic amacrine cells was assessed in whole-mounted retinas. Cells displaying the transgenic red fluorescence were identified as SST-immunopositive amacrine cells. By comparing the three age groups. PACAP treatment was shown to induce a moderate elevation of cell densities in both the SST and dopaminergic cell populations in the 12- and 18-month-old animals. By contrast, the control untreated and saline-treated retinas showed a minor cell loss. In conclusion, we report a reliable transgenic model for examining SSTergic amacrine cells. The fundamental novelty of this study is that PACAP could increase the cell density in matured retinal tissue, anticipating new therapeutic potential in age-related pathological processes.


Assuntos
Senescência Celular/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Retina/efeitos dos fármacos , Animais , Contagem de Células/métodos , Neurônios Dopaminérgicos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
Methods Mol Biol ; 2188: 51-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119846

RESUMO

Heterologous expression of recombinant ion channel subunits in mammalian cell lines allows for characterization of their functional properties and pharmacological regulation. In this chapter, we describe methods for thawing, refreezing, passaging, cell culture, and transfection of tsA201 cells suitable for electrophysiology and imaging experiments. Furthermore, we discuss the strengths and limitations of using these methods.


Assuntos
Canais Iônicos/genética , Transfecção/métodos , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Criopreservação/métodos , Expressão Gênica , Humanos , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Recombinantes/genética , Técnicas de Cultura de Tecidos/métodos
6.
Rev. cuba. invest. bioméd ; 39(3): e634, jul.-set. 2020. tab, graf
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1138935

RESUMO

Introducción: El carcinoma papilar de tiroides es la causa más frecuente de cáncer de naturaleza endocrina. Constituye la variante histológica de mejor pronóstico, sin embargo, en ocasiones es motivo de importantes dudas diagnósticas con otras variantes de evolución tórpida, lo que dificulta que un mayor número de pacientes se beneficie con un tratamiento individualizado y conservador. Por esta razón, se desarrollan estudios en los que cada vez más se añaden procedimientos morfométricos y densitométricos, los que permiten disminuir la subjetividad en el diagnóstico histopatológico. Objetivo: Determinar la densidad óptica nuclear en el carcinoma papilar de tiroides. Método: Se realizó un estudio morfométrico de serie de casos con 12 pacientes con carcinoma papilar de tiroides, atendidos en el Hospital Provincial Universitario Vladimir Ilich Lenin. Se seleccionaron 36 campos y se midieron 965 núcleos celulares, lo que constituyó la muestra del estudio. Se determinó la densidad óptica nuclear como indicador morfométrico del carcinoma papilar de tiroides. Resultados: El valor de la densidad óptica nuclear fue 1,14, considerado bajo. Conclusiones: Se determinó la densidad óptica nuclear del carcinoma papilar de tiroides en los casos estudiados, lo que puede contribuir a su diagnóstico histopatológico(AU)


Background: Papillary thyroid carcinoma is the most frequent cause of endocrine cancer. It is an histological variant with the best prognosis, however, sometimes it is a reason for significant diagnostic doubts with other variants of torpid evolution, which makes it difficult for a greater number of patients to benefit from an individualized and conservative treatment. For this reason, studies are developed in which more and more morphometric and densitometric procedures are added, which allow reducing the subjectivity in the histopathological diagnosis and could represent a tool of great value. Objective: To determine the nuclear optical density in papillary thyroid carcinoma. Method: A morphometric study of a series of cases was carried out with 12 patients with this histopathological diagnosis, attended at the Vladimir Ilich Lenin University Provincial Hospital. We chose 36 fields and 965 cell nuclei were measured, which constituted the study sample. Nuclear optical density was determined as a morphometric indicator of papillary thyroid carcinoma. Results: The value of nuclear optical density was 1, 14. It is considered low. Conclusion: Nuclear optical density of papillary thyroid carcinoma was determined in the studied cases that may contribute to histopathological diagnosis(AU)


Assuntos
Humanos , Neoplasias da Glândula Tireoide/ultraestrutura , Câncer Papilífero da Tireoide/diagnóstico , Prognóstico , Contagem de Células/métodos , Câncer Papilífero da Tireoide/secundário
7.
Anticancer Res ; 40(8): 4701-4706, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727795

RESUMO

BACKGROUND/AIM: Bovine mastitis is caused by the invasion and propagation of pathogenic microorganisms into the udder and mammary gland tissues of cattle. In this study, the therapeutic effect of a low-molecular-weight whey protein (LMW-WP) on bovine mastitis was evaluated. MATERIALS AND METHODS: LMW-WP was orally, intraperitoneally, and vaginally administered to bovine with mastitis. The number of somatic cells in milk was measured 24 h before the administration of LMW-WP. The effect of LMW-WP on cytokine production was measured with a microarray that evaluates the expression of cytokines. RESULTS: In the group that received 1,000 mg intraperitoneally, the somatic cell count was reduced to less than 400,000 at the shipment standard value in three of the four udders, indicating 75% efficacy. The group that received 1,000 mg by vaginal administration showed 67% efficacy. It was confirmed that LMW-WP increased the production of cytokines such as IL-5, IL-6, IL-9, IL-12, MCP-1, and VEGF in mouse macrophage cells, but it did not show any antibacterial activity. CONCLUSION: LMW-WP may be an effective therapeutic agent for bovine mastitis.


Assuntos
Macrófagos/efeitos dos fármacos , Mastite Bovina/tratamento farmacológico , Proteínas do Soro do Leite/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Contagem de Células/métodos , Linhagem Celular , Citocinas/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/microbiologia , Camundongos , Leite/metabolismo , Células RAW 264.7
8.
Sci Rep ; 10(1): 12226, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699281

RESUMO

Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples, by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network, a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments, we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users, thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future, the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis.


Assuntos
Contagem de Células/métodos , Separação Celular/métodos , Neoplasias/diagnóstico , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Algoritmos , Linhagem Celular Tumoral , Células HCT116 , Humanos , Aprendizado de Máquina
9.
Sci Rep ; 10(1): 10181, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576883

RESUMO

Circulating tumor cells (CTCs) are derivatives of solid cancerous lesions that detach from the tumor mass and enter the blood circulation. CTCs are considered to be the precursors of metastasis in several cancer types. They are present in the blood of cancer patients as single cells or clusters, with the latter being associated with a higher metastatic potential. Methods to eliminate CTCs from the bloodstream are currently lacking. Here, we took advantage of the lower shear stress-resistance of cancer cells compared to blood cells, and developed a device that can eliminate cancer cells without blood damage. The device consists of an axial pump and a coupled rotating throttle, controllable to prevent local blood flow impairment, yet maintaining a constant shear performance. When processing cancer cells through our device, we observe cancer cell-cluster disruption and viability reduction of single cancer cells, without noticeable effects on human blood cells. When injecting cancer cell-containing samples into tumor-free recipient mice, processed samples fail to generate metastasis. Together, our data show that a selective disruption of cancer cells is possible while preserving blood cells, paving the way towards the development of novel, implantable tools for CTC disruption and metastasis prevention.


Assuntos
Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Animais , Contagem de Células/métodos , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Metástase Neoplásica/patologia
10.
Rev. cuba. oftalmol ; 33(2): e739, tab
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1139068

RESUMO

RESUMEN Objetivo: Relacionar la severidad del edema corneal poscirugía de catarata en pacientes con córnea guttata, considerados sin riesgo o bajo riesgo de edema posquirúrgico según densidad celular, con los parámetros de la microscopia endotelial. Métodos: Se realizó un estudio descriptivo, prospectivo de 42 pacientes con córnea guttata, considerados sin riesgo o bajo riesgo de edema posquirúrgico según la densidad celular, quienes fueron sometidos a la cirugía de catarata por el mismo cirujano en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer", desde abril del año 2016 a diciembre de 2017. Se determinó la severidad del edema corneal y se relacionó con el grado de córnea guttata, así como con los parámetros de la microscopia endotelial. Resultados: El 52,4 por ciento fueron mayores de 70 años y el 64,3 por ciento mujeres. No hubo edema en el 54,8 por ciento en las primeras 24 horas, y fue en el 26,2 por ciento leve y en el 19,0 por ciento mínimo, donde predominó la córnea guttata grado 3 y presentaban un bajo riesgo prequirúrgico. Sin embargo, no hubo diferencias en relación con el polimegatismo y el pleomorfismo. Conclusiones: La severidad del edema corneal en pacientes con córnea guttata posfacoemulsificación a los siete días no se asocia con el polimegatismo, ni con el polimorfismo prequirúrgico, pero sí con el conteo endotelial prequirúrgico(AU)


ABSTRACT Objective: Determine the relationship between the severity of corneal edema in cornea guttata patients undergoing cataract surgery considered to be at no risk or low risk for postsurgical edema in terms of cell density, and endothelial microscopy parameters. Methods: A descriptive prospective study was conducted of 42 cornea guttata patients considered to be at no risk or low risk for postsurgical edema in terms of cell density, who underwent cataract surgery performed by the same surgeon at Ramón Pando Ferrer Cuban Institute of Ophthalmology from April 2016 to December 2017. Corneal edema severity was determined and related to cornea guttata grade and endothelial microscopy parameters. Results: Of the patients studied, 52.4 percent were aged over 70 years and 64.3 percent were women. Edema was not observed in 54.8 percent in the first 24 hours, whereas it was mild in 26.2 percent and minimum in 19.0 percent. Grade 3 cornea guttata and low presurgical risk prevailed. However, no differences were found in relation to polymegethism and pleomorphism. Conclusions: At seven days, corneal edema severity in cornea guttata patients undergoing phacoemulsification is not associated to polymegethism or presurgical polymorphism, but it as associated to the presurgical endothelial count(AU)


Assuntos
Humanos , Feminino , Idoso , Catarata/etiologia , Edema da Córnea , Contagem de Células/métodos , Facoemulsificação/métodos , Estudos Observacionais como Assunto , Parâmetros de Referência/efeitos adversos , Epidemiologia Descritiva , Estudos Prospectivos , Microscopia/métodos
11.
PLoS Comput Biol ; 16(5): e1007611, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379821

RESUMO

Modeling cancer cells is essential to better understand the dynamic nature of brain tumors and glioma cells, including their invasion of normal brain. Our goal is to study how the morphology of the glioma cell influences the formation of patterns of collective behavior such as flocks (cells moving in the same direction) or streams (cells moving in opposite direction) referred to as oncostream. We have observed experimentally that the presence of oncostreams correlates with tumor progression. We propose an original agent-based model that considers each cell as an ellipsoid. We show that stretching cells from round to ellipsoid increases stream formation. A systematic numerical investigation of the model was implemented in [Formula: see text]. We deduce a phase diagram identifying key regimes for the dynamics (e.g. formation of flocks, streams, scattering). Moreover, we study the effect of cellular density and show that, in contrast to classical models of flocking, increasing cellular density reduces the formation of flocks. We observe similar patterns in [Formula: see text] with the noticeable difference that stream formation is more ubiquitous compared to flock formation.


Assuntos
Neoplasias Encefálicas/patologia , Biologia Computacional/métodos , Glioma/patologia , Contagem de Células/métodos , Movimento Celular/fisiologia , Forma Celular/fisiologia , Humanos , Modelos Biológicos , Modelos Teóricos , Simulação de Dinâmica Molecular
12.
Cancer Chemother Pharmacol ; 85(5): 979-993, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32314030

RESUMO

PURPOSE: Following promising responses to the DNA methyltransferase (DNMT) inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) combined with tetrahydrouridine (THU) in phase 1 testing, we initiated a non-randomized phase 2 study to assess response to this combination in patients with advanced solid tumor types for which tumor suppressor gene methylation is potentially prognostic. To obtain pharmacodynamic evidence for DNMT inhibition by FdCyd, we developed a novel method for detecting expression of tumor suppressor protein p16/INK4A in circulating tumor cells (CTCs). METHODS: Patients in histology-specific strata (breast, head and neck [H&N], or non-small cell lung cancers [NSCLC] or urothelial transitional cell carcinoma) were administered FdCyd (100 mg/m2) and THU (350 mg/m2) intravenously 5 days/week for 2 weeks, in 28-day cycles, and progression-free survival (PFS) rate and objective response rate (ORR) were evaluated. Blood specimens were collected for CTC analysis. RESULTS: Ninety-three eligible patients were enrolled (29 breast, 21 H&N, 25 NSCLC, and 18 urothelial). There were three partial responses. All strata were terminated early due to insufficient responses (H&N, NSCLC) or slow accrual (breast, urothelial). However, the preliminary 4-month PFS rate (42%) in the urothelial stratum exceeded the predefined goal-though the ORR (5.6%) did not. An increase in the proportion of p16-expressing cytokeratin-positive CTCs was detected in 69% of patients evaluable for clinical and CTC response, but was not significantly associated with clinical response. CONCLUSION: Further study of FdCyd + THU is potentially warranted in urothelial carcinoma but not NSCLC or breast or H&N cancer. Increase in the proportion of p16-expressing cytokeratin-positive CTCs is a pharmacodynamic marker of FdCyd target engagement.


Assuntos
Carcinoma de Células de Transição , Inibidor p16 de Quinase Dependente de Ciclina/análise , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Células Neoplásicas Circulantes/patologia , Neoplasias Urológicas , Administração Intravenosa , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Contagem de Células/métodos , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/farmacocinética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Farmacogenética , Tetra-Hidrouridina/administração & dosagem , Tetra-Hidrouridina/efeitos adversos , Tetra-Hidrouridina/farmacocinética , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/patologia
13.
PLoS One ; 15(4): e0231473, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315325

RESUMO

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.


Assuntos
Túnica Conjuntiva/metabolismo , Epitélio/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Antígenos HLA-DR/metabolismo , Soro/metabolismo , Síndrome de Sjogren/metabolismo , Adulto , Idoso , Contagem de Células/métodos , Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas/uso terapêutico , Síndrome de Sjogren/tratamento farmacológico
14.
Rev. cuba. oftalmol ; 33(1): e692, ene.-mar. 2020. tab
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1126725

RESUMO

RESUMEN Objetivo: Identificar las características morfológicas del epitelio, el estroma y el endotelio corneal, así como la densidad celular de este último mediante el empleo de la microscopia confocal de la córnea en pacientes diabéticos. Métodos: Se realizó un estudio descriptivo, comparativo, en 90 ojos; 60 de ellos pertenecientes a pacientes diabéticos (30 tipo 1 y 30 tipo 2) y 30 ojos a pacientes supuestamente sanos. El estudio se realizó en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer" entre enero del año 2012 y enero de 2017. Resultados: Predominó el sexo masculino con 66,7 por ciento en los pacientes con diabetes mellitus tipo 1; el sexo femenino en los pacientes con diabetes mellitus tipo 2 (60 por ciento) y aparentemente sanos (56,7 por ciento). En los pacientes con diabetes mellitus tipo 1 fueron más frecuentes las edades entre 45 y 54 años (33,3 por ciento) y entre 55 y 66 años en los pacientes con diabetes mellitus tipo 2 y aparentemente sanos con 60 y 40 por ciento respectivamente. La morfología del epitelio y el estroma corneal fue normal en el 86,7 y 87,3 por ciento respectivamente. Predominaron las alteraciones de la morfología endotelial en pacientes diabéticos tipo 1 (73,3 por ciento), así como el polimegatismo y el pleomorfismo (73,3 y 56,7 por ciento respectivamente) y la densidad celular más baja (2 222,76 células /mm2). Conclusiones: La ausencia de alteraciones morfológicas del epitelio y el estroma corneal y la presencia de polimegatismo y de pleomorfismo fueron los hallazgos más frecuentes(AU)


ABSTRACT Objective: Identify the morphological characteristics of the corneal epithelium, stroma and endothelium, as well as the cell density of the endothelium by means of confocal microscopy of the cornea in diabetic patients. Methods: A descriptive comparative study was conducted of 90 eyes: 60 from diabetic patients (30 type 1 and 30 type 2) and 30 from supposedly healthy patients, at Ramón Pando Ferrer Cuban Institute of Ophthalmology from January 2012 to January 2017. Results: A predominance was found of the male sex (66.7 percent) among patients with diabetes mellitus type 1 and of the female sex among patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (56.7 percent). The most common age ranges were 45-54 years for patients with diabetes mellitus type 1 (33.3 percent) and 55-66 years for patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (40 percent). Morphology of the corneal epithelium and stroma was normal in 86.7 percent and 87.3 percent, respectively. In type 1 diabetic patients there was a predominance of endothelial morphological alterations (73.3 percent), polymegethism and pleomorphism (73.3 percent and 56.7 percent, respectively) and the lowest cell density (2 222.76 cells /mm2). Conclusions: Absence of morphological alterations of the corneal epithelium and stroma, as well as the presence of polymegethism and pleomorphism were the most common findings(AU)


Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Contagem de Células/métodos , Córnea/anormalidades , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 2/etiologia , Epidemiologia Descritiva , Microscopia Confocal/métodos
15.
J Vis Exp ; (156)2020 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-32090991

RESUMO

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.


Assuntos
Adesão Celular , Contagem de Células/métodos , Linfonodos/patologia , Metástase Linfática/patologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Experimentais/patologia , Ratos Wistar
16.
BMC Res Notes ; 13(1): 57, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019595

RESUMO

OBJECTIVE: Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. RESULTS: The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.


Assuntos
Contagem de Células/métodos , Células/citologia , Adesão Celular , Proliferação de Células , Fluoresceínas/metabolismo , Fluorescência , Células HEK293 , Humanos , Succinimidas/metabolismo
18.
Sci Rep ; 10(1): 2122, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034190

RESUMO

Although macrophages are important players in the injury/repair processes in animal models of acute kidney injury (AKI), their roles in human AKI remains uncertain owing to a paucity of human biopsy studies. We investigated the role of macrophages in 72 cases of biopsy-proven acute tubular necrosis (ATN) and six cases of healthy kidney. Macrophages were identified by CD68 and CD163 immunohistochemistry and analyzed for their effect on renal outcomes. CD163+ M2 macrophages outnumbered CD68+ cells in the healthy kidneys, suggesting that CD163+ macrophages are resident macrophages. The infiltration of both subtypes of macrophages increased significantly in ATN. The density of the CD68+ macrophages was significantly higher in advanced-stage AKI, whereas CD163+ M2 macrophages was not. Eighty percent of patients exhibited renal functional recovery during follow-up. Older age and a higher density of CD163+ macrophages predicted non-recovery, whereas the AKI stage, tubular injury score, and density of CD68+ cells did not. The density of CD163+ M2 macrophages was an independent predictor of low eGFR at 3 months in advanced-stage AKI. This is the first human study demonstrating the possible role of macrophages in the injury and repair phases of AKI.


Assuntos
Necrose Tubular Aguda/patologia , Rim/patologia , Macrófagos/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Contagem de Células/métodos , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Imuno-Histoquímica/métodos , Rim/metabolismo , Necrose Tubular Aguda/metabolismo , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Estudos Retrospectivos
19.
Am J Hematol ; 95(5): 456-464, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31990387

RESUMO

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.


Assuntos
Anemia Falciforme/terapia , Contagem de Células/métodos , Ativação do Complemento/genética , Hemólise/fisiologia , Hidroxiureia/uso terapêutico , Adolescente , Adulto , Feminino , Humanos , Hidroxiureia/farmacologia , Pessoa de Meia-Idade , Adulto Jovem
20.
Exp Cell Res ; 389(1): 111877, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31991124

RESUMO

Reversible electroporation is a temporary permeabilization of cell membrane through the formation of transient pores created by short high voltage electric pulses. This method has numerous applications in biology and biotechnology and has become an important technique in molecular medicine. Reversible electroporation is usually used to transfer macromolecules into the cells. However, the delivery of large molecules such as proteins into cells without loss of cell viability remains a challenge. In our study, we investigated whether electroporation can be used for this purpose. The study was performed with the primary mouse splenocytes and Jurkat cell line. The electroporation efficacy was evaluated by flow cytometry. We used the reversible electroporation for intracellular marker detection investigating antibody and fluorescein-conjugated dextran transfer efficiency, cell viability and metabolic activity. We have found that reversible electroporation parameters can be optimized for efficient transfer of large molecules such as antibodies/proteins into live cells without a significant loss of cell viability. We conclude that a well-established and relatively easy method of reversible electroporation can be adjusted to detect intracellular biomarkers in viable cells. This is a new approach on how electroporation could be utilised in medicine and biological research to detect rare subpopulations of cells that produce specific markers and to keep cells viable. This would allow the use of these rare subpopulations of isolated cells for further research and personalized medicine.


Assuntos
Biomarcadores/análise , Eletricidade , Eletroporação , Citometria de Fluxo/métodos , Animais , Biomarcadores/metabolismo , Contagem de Células/métodos , Permeabilidade da Membrana Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Estimulação Elétrica , Eletricidade/efeitos adversos , Eletroporação/métodos , Feminino , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL
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