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1.
Acta Cytol ; 68(2): 145-152, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38555634

RESUMO

INTRODUCTION: Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage. METHODS: We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis. RESULTS: Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens. CONCLUSION: Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.


Assuntos
Neoplasias da Mama , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/diagnóstico , Feminino , Biópsia por Agulha Fina/métodos , Biópsia Líquida , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Citodiagnóstico/métodos , Tumor Filoide/patologia , Tumor Filoide/genética , Tumor Filoide/diagnóstico , Fibroadenoma/patologia , Fibroadenoma/genética , Fibroadenoma/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Carcinoma Intraductal não Infiltrante/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/diagnóstico , Pessoa de Meia-Idade , Citologia
2.
Cancer Sci ; 115(2): 660-671, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38130032

RESUMO

REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin in vitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin in vivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.


Assuntos
Adenocarcinoma , Fenômenos Biológicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Platina/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Adenocarcinoma/tratamento farmacológico , Reparo do DNA/genética
3.
Pathol Int ; 72(1): 14-24, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34637584

RESUMO

REV7 is a multifunctional protein implicated in DNA damage tolerance, cell cycle control, and gene expression, and is involved in the carcinogenesis of various human tumors. It has been reported that REV7 expression is associated with ultraviolet-induced mutagenesis; however, the role of REV7 expression in skin cancers, including malignant melanomas, remains unclear. In the present study, we investigated the clinical and biological significance of REV7 in malignant melanoma. Levels of REV7 expression in human skin cancers were evaluated immunohistochemically. Positive expression of REV7 was frequently observed in malignant melanomas, as well as in squamous cell carcinomas and basal cell carcinomas. Enhanced immunoreactivity to REV7 was closely linked with cell proliferation assessed by Ki-67 labeling indexes in the three skin cancers, and was related with tumor thickness in malignant melanomas. REV7 depletion in malignant melanoma cells MEWO and G361 suppressed cell proliferation, migration, and invasion abilities. REV7 depletion also affected the expression of intracellular signaling molecules AKT and ERK in MEWO cells, resulting in downregulation of ERK signal activation. In addition, REV7 depletion facilitated sensitivity to cisplatin, but not to dacarbazine, in MEWO cells. Our results suggest that REV7 expression correlates with disease progression of malignant melanoma.


Assuntos
Proteínas Mad2/metabolismo , Melanoma , Neoplasias Cutâneas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Proliferação de Células , Criança , Pré-Escolar , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Adulto Jovem , Melanoma Maligno Cutâneo
4.
Intern Med ; 60(21): 3427-3433, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33967143

RESUMO

We herein report a rare case of cartilage-hair hypoplasia (CHH) complicated with liver cirrhosis. A 20-year-old Japanese man with CHH was found incidentally to have liver cirrhosis and an esophageal varix. This patient had been treated for infections due to immunodeficiency since early childhood. He ultimately died of liver failure at 31 years of age. An autopsy revealed an abnormality of the interlobular bile ducts and intrahepatic cholestasis. Liver cirrhosis was thought to have been caused by chronic intrahepatic cholestasis due to biliary duct hypoplasia and changes in the intestinal microbiome. Therefore, CHH may cause biliary cirrhosis due to multiple effects.


Assuntos
Colestase Intra-Hepática , Doença de Hirschsprung , Doenças da Imunodeficiência Primária , Adulto , Colestase Intra-Hepática/complicações , Colestase Intra-Hepática/diagnóstico , Evolução Fatal , Cabelo/anormalidades , Humanos , Cirrose Hepática/complicações , Masculino , Osteocondrodisplasias/congênito , Adulto Jovem
5.
Pathol Int ; 71(1): 15-23, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33112501

RESUMO

REV7 is involved in multiple biological processes including DNA damage tolerance, cell cycle regulation and gene expression, and is an accessory subunit of the mutation-prone DNA polymerase ζ. It has been reported that REV7 expression is associated with poor prognosis in several human cancers. The aim of this study is to investigate the significance of REV7 in lung carcinogenesis. Immunohistochemical analyses of surgically resected lung cancer specimens revealed that REV7 shows an increased expression in small cell lung carcinomas (SCLCs) when compared with other histological types of lung carcinoma. Association between REV7 expression levels and clinicopathological factors was investigated using SCLC cases with or without surgical resection. Our analyses revealed that high REV7 expression significantly correlated with tumor cell proliferation, assessed by Ki-67 labeling indices, and was negatively associated with distant metastasis and extensive-stage disease. No significant association was detected between REV7 expression and other factors, including prognosis or response to chemoradiotherapy in SCLC. Increase in REV7 expression in SCLC was confirmed using SCLC cell lines. In addition, siRNA-mediated depletion of REV7 activated the apoptotic pathway and suppressed cell growth in SCLC cells. These results suggest that REV7 plays an important role in tumor cell survival and proliferation in SCLC.


Assuntos
Proteínas Mad2/metabolismo , Carcinoma de Pequenas Células do Pulmão , Adulto , Idoso , Apoptose , Biomarcadores Tumorais , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Prognóstico , Estudos Retrospectivos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia
6.
Genes Cells ; 23(7): 590-598, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29767469

RESUMO

Osteoporosis is a global public health problem that is increasing along with an aging population. A major determinant of osteoporosis is high bone turnover, which results from osteoclast activation. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, a deficiency that leads to a psoriasis-like skin inflammation in mice. Although the expression of CD109 has been reported in mouse pre-osteoclast cells, its function in osteoclasts in vivo remains largely unknown. To investigate the physiological role of CD109 in bone metabolism, we analyzed bones from wild-type and CD109-deficient adult mice. Micro-computed tomography analysis of the femur (thigh bone) showed that bone volume was lower in CD109-deficient mice than in wild-type mice. Bone histomorphometric analysis showed not only a reduction in bone volume but also an increase in bone turnover in CD109-deficient mice as compared with wild-type mice. Additionally, we measured serum levels of several markers of bone turnover and found a significant increase in the N-terminal telopeptide of type I collagen, a bone resorption marker, as well as alkaline phosphatase, a bone formation marker, in CD109-deficient mice. These results indicate that CD109 deficiency induces a high-turnover, osteoporosis-like phenotype, which suggests that CD109 plays a role in bone metabolism in vivo.


Assuntos
Antígenos CD/metabolismo , Doenças Ósseas Metabólicas/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoporose/metabolismo , Animais , Biomarcadores/metabolismo , Doenças Ósseas Metabólicas/patologia , Reabsorção Óssea , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Colágeno Tipo I/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese , Fenótipo
7.
Nat Commun ; 8(1): 2226, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29263385

RESUMO

C-C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5 -/- ) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts.


Assuntos
Reabsorção Óssea/genética , Movimento Celular/genética , Osteoclastos/metabolismo , Osteogênese/genética , Osteoporose/genética , Receptores CCR5/genética , Animais , Adesão Celular/genética , Quinase 2 de Adesão Focal/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/ultraestrutura , Osteoporose/induzido quimicamente , Podossomos/ultraestrutura , Ligante RANK/toxicidade
8.
PLoS One ; 9(1): e83385, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24400073

RESUMO

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is expressed at high levels in some human tumors including squamous cell carcinomas. As CD109 is reportedly cleaved by furin and its soluble form is secreted into culture medium in vitro, we hypothesized that CD109 could serve as a tumor marker in vivo. In this study, we investigated CD109 as a novel serum tumor marker using transgenic mice that overexpress mouse CD109 (mCD109-TG mice) and tumor xenografted mice inoculated with human CD109 (hCD109)-overexpressing HEK293 cells. In sera and urine of mCD109-TG mice, mCD109 was detected using western blotting. In xenografted mice, hCD109 secreted from inoculated tumors was detected in sera, using western blotting and CD109 ELISA. Concentrations of tumor-secreted CD109 increased proportionally as tumors enlarged. Concentrations of secreted CD109 decreased notably by 17 h after tumor resection, and became undetectable 48 h after resection. The half-life of tumor-secreted CD109 was about 5.86±0.17 h. These results indicate that CD109 is present in serum as a soluble form, and suggest its potential as a novel tumor marker in patients with cancers that express CD109.


Assuntos
Antígenos CD/sangue , Proteínas de Neoplasias/sangue , Neoplasias/sangue , Animais , Antígenos CD/genética , Antígenos CD/urina , Modelos Animais de Doenças , Feminino , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/urina , Neoplasias/genética , Neoplasias/patologia , Carga Tumoral
9.
Pathog Dis ; 70(1): 28-39, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23929604

RESUMO

Spinal tuberculosis is a condition characterized by massive resorption of the spinal vertebrae due to the infection with Mycobacterium tuberculosis (Mtb). However, the pathogenesis of spinal tuberculosis has not been established because it was almost completely eradicated by the establishment of antibiotic treatment in the mid-20th century. In this study, we investigated the inflammatory responses of human multinucleated osteoclasts infected with virulent Mtb strain. We found that the intracellular Mtb infection of multinuclear osteoclasts resulted in the rapid growth of Mtb and an osteolytic response, rather than inflammation. In response to Mtb infection, the mononuclear osteoclast precursors produced proinflammatory cytokines including tumor necrosis factor (TNF)-α, an intrinsic characteristic they share with macrophages. In contrast, highly fused multinucleated osteoclasts incapacitated the production of these cytokines. Instead, the intracellular Mtb inside multinuclear osteoclasts escaped from the endosome/phagosome, leading to a different pattern of osteoclast activation, with the production of chemokines such as CCL5, CCL17, CCL20, CCL22, CCL24, and CCL25. Moreover, intracellular infection with an avirulent Mtb strain resulted in diminished production of these chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation of cytokines and chemokines.


Assuntos
Quimiocinas/imunologia , Citocinas/imunologia , Mycobacterium tuberculosis/imunologia , Osteoclastos/imunologia , Fagossomos/imunologia , Tuberculose da Coluna Vertebral/imunologia , Tuberculose da Coluna Vertebral/microbiologia , Endossomos/imunologia , Endossomos/microbiologia , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Ligantes , Osteoclastos/microbiologia , Fagossomos/microbiologia , Fator de Necrose Tumoral alfa/imunologia
10.
Int J Mol Sci ; 14(1): 1323-34, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-23306154

RESUMO

Silicon quantum dots (Si-QDs) have great potential for biomedical applications, including their use as biological fluorescent markers and carriers for drug delivery systems. Biologically inert Si-QDs are less toxic than conventional cadmium-based QDs, and can modify the surface of the Si-QD with covalent bond. We synthesized water-soluble alminoprofen-conjugated Si-QDs (Ap-Si). Alminoprofen is a non-steroid anti-inflammatory drug (NSAID) used as an analgesic for rheumatism. Our results showed that the "silicon drug" is less toxic than the control Si-QD and the original drug. These phenomena indicate that the condensed surface integration of ligand/receptor-type drugs might reduce the adverse interaction between the cells and drug molecules. In addition, the medicinal effect of the Si-QDs (i.e., the inhibition of COX-2 enzyme) was maintained compared to that of the original drug. The same drug effect is related to the integration ratio of original drugs, which might control the binding interaction between COX-2 and the silicon drug. We conclude that drug conjugation with biocompatible Si-QDs is a potential method for functional pharmaceutical drug development.


Assuntos
Anti-Inflamatórios não Esteroides/química , Propionatos/química , Pontos Quânticos , Silício/química , Anti-Inflamatórios não Esteroides/farmacologia , Ácido Araquidônico/metabolismo , Biocatálise/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprosta/metabolismo , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Células Hep G2 , Humanos , Cinética , Microscopia Eletrônica de Transmissão , Propionatos/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier
11.
J Cell Sci ; 126(Pt 4): 1032-45, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23264747

RESUMO

Chemokines have recently been reported to be involved in pathological bone destruction. However, the physiological roles of chemokines in bone metabolism in vivo have not been well documented. We analyzed the bone phenotypes in Cx3cr1-deficient mice. The mice exhibited slight but significant increases in trabecular and cortical thickness, reduced numbers of osteoclasts and increased rates of osteoid formation. Although the morphometric parameters showed marginal differences, the Cx3cr1-deficient bones showed an elevated expression of Osterix/SP7, which encodes an essential transcriptional factor for osteoblasts, whereas the gene Osteocalcin/Bglap, which encodes a late marker, was downregulated. The levels of transcripts for various osteoclastic markers, such as receptor activator of NF-κB (RANK)/TNFRSF11A, receptor activator of NF-κB ligand (RANKL)/TNFSF11, tartrate-resistant acid phosphatase 5b (TRAP5B)/ACP5B, Cathepsin K(CTSK), MMP3 and MMP13, were significantly decreased in the Cx3cr1-deficient bones. Cultured Cx3cr1-deficient osteoblastic cells showed inverse temporal patterns of osteoblastic marker expression and reduced calcium deposition. Furthermore, in vitro studies and immunofluorescence staining against CX3CR1 and CX3CL1 suggested a role for the CX3CR1-CX3CL1 axis in an early stage of osteoblast differentiation, possibly through their trans and cis interactions. Cultured Cx3cr1-deficient pre-osteoclasts showed impaired differentiation, mainly due to a deficiency of the CD115(+)CD11b(lo) osteoclastogenic population of myeloid-lineage precursors. The treatment of bone-marrow-derived osteoclastic cultures with recombinant CX3CL1 at different time points suggested that the CX3CR1-CX3CL1 axis favors the maintenance of osteoclastic precursors, but not differentiated osteoclasts. These observations uncovered novel roles of the CX3CR1-CX3CL1 axis in the differentiation of both osteoblasts and osteoclasts.


Assuntos
Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Citometria de Fluxo , Homeostase/genética , Homeostase/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Quimiocinas/genética
12.
Bone ; 51(3): 447-58, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22766096

RESUMO

Confocal immunofluorescence tiling imaging revealed the spatio-temporal distributions of osterix and sclerostin in femurs from 3-day-old, 2-week-old and 4-week-old rats to be reciprocally exclusive at the tissue level. Further quantitative three-dimensional immuno fluorescence morphometry demonstrated the increasing distribution of sclerostin in the osteocytic lacuno-canalicular system specifically in diaphysis, which paralleled the cooperative participation and depletion of osterix and ß-catenin in adjacent periosteum cells. Treating MC3T3-E1 cells with BIO (a GSK3 inhibitor) induced the stabilization of ß-catenin and nuclear translocation of osterix, and negatively regulated osteocalcin/BGLAP and Dmp1. These results collectively demonstrate that the increasing distribution of sclerostin in diaphyseal cortical bone appears to be involved in the attenuation of osterix and ß-catenin in adjacent periosteum cells, thus possibly contributing to osteoblast maturation and reducing the osteoblast formation at this bone site. Our confocal microscopy-based imaging analyses provide a comprehensive and detailed view of the spatio-temporal distribution of sclerostin, ß-catenin and osterix at the tissue to subcellular level in a coherent manner, and uncovered their spatio-temporal cooperation in postnatal bone development, thus providing evidence that they link skeletogenic growth and functional bone development.


Assuntos
Desenvolvimento Ósseo , Proteínas Morfogenéticas Ósseas/metabolismo , Imunofluorescência/métodos , Imageamento Tridimensional/métodos , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo/efeitos dos fármacos , Contagem de Células , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diáfises/citologia , Diáfises/efeitos dos fármacos , Diáfises/crescimento & desenvolvimento , Diáfises/metabolismo , Fêmur/citologia , Fêmur/efeitos dos fármacos , Fêmur/crescimento & desenvolvimento , Fêmur/metabolismo , Marcadores Genéticos , Indóis/farmacologia , Masculino , Camundongos , Microscopia Confocal , Osteócitos/citologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Oximas/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Fator de Transcrição Sp7 , Fatores de Tempo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
13.
Arch Toxicol ; 85(7): 707-20, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21445587

RESUMO

With the development of nanotechnology, nanometer-sized products smaller than several 100 nm have been applied for all areas of science and technology. The nanometer-sized products, including carbon nanotubes, fullerene derivatives, and nanocrystals made of various materials, are widely employed as novel tools in various fields, not only in material engineering, electronics, plastics, automobile, aviation, and aerospace industries, but also even in cellular biology, molecular biology, and basic and clinical medical fields. In particular, nanocrystal quantum dots (QDs) have been widely used in biological and medical studies because of their far brighter photoemission and photostability. The physical and chemical properties of QDs have been circumstantially investigated, but little is known about the potential harmful effects of QDs on human health. In addition to the physical and chemical properties of the QDs, their toxicity and biological behavior are generally regulated by three other conditions: (1) the QD core material itself, (2) the surface modifications of the QD, and (3) the external environmental condition of the QDs. We herein report on the in vitro and in vivo toxicity and biological behavior of nanocrystals such as QDs. Accumulating evidence suggests that the QD-capping material, rather than the core metalloid complex, is responsible for the majority of their toxicity and biological activity. For example, molecules covered with a toxic agent showed cytotoxicity, whereas QDs conjugated with biomolecules retained the biological effects of the conjugate.


Assuntos
Nanopartículas/química , Nanopartículas/toxicidade , Pontos Quânticos , Animais , Humanos , Metaloides/química , Metaloides/toxicidade , Nanomedicina/tendências , Propriedades de Superfície , Transferência de Tecnologia
14.
Nanotechnology ; 21(33): 335103, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20660952

RESUMO

Quantum dots (QDs) are well known for their potential application in biosensing, ex vivo live-cell imaging and in vivo animal targeting. The brain is a challenging organ for drug delivery, because the blood brain barrier (BBB) functions as a gatekeeper guarding the body from exogenous substances. Here, we evaluated the distribution of bioconjugated QDs, i.e., captopril-conjugated QDs (QDs-cap) following intraperitoneal injection into male ICR mice as a model system for determining the tissue localization of QDs, employing ICP-MS and confocal microscopy coupled with spectrometric analysis. We have demonstrated that intraperitoneally administered QDs-cap were delivered via systemic blood circulation into liver, spleen, kidney and brain at 6 h after injection. QDs-cap were located predominantly inside the blood vessels in the liver, kidney and brain, but a few were distributed in the parenchyma, especially noteworthy in the brain. Careful studies on acute as well as chronic toxicity of QDs in the brain are required prior to clinical application to humans.


Assuntos
Encéfalo/metabolismo , Captopril/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Pontos Quânticos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Captopril/química , Imuno-Histoquímica , Injeções Intraperitoneais , Rim/metabolismo , Laminina , Fígado/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Microscopia de Fluorescência , Distribuição Tecidual
15.
J Biol Chem ; 285(37): 28826-37, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20571024

RESUMO

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1(-/-) mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1(-/-) mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1(-/-) mice. Osteoclastogenesis induced from cultured Ccr1(-/-) bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1(-/-) osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1(-/-) mice. The co-culture of wild-type osteoclast precursors with Ccr1(-/-) osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.


Assuntos
Reabsorção Óssea/metabolismo , Comunicação Celular , Quimiocinas/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Receptores CCR1/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Densidade Óssea/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Células Cultivadas , Quimiocinas/genética , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/patologia , Osteoclastos/patologia , Receptores CCR1/genética
16.
Int J Mol Sci ; 10(6): 2722-2732, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19582225

RESUMO

Mars is a CO(2)-abundant planet, whereas early Earth is thought to be also CO(2)-abundant. In addition, water was also discovered on Mars in 2008. From the facts and theory, we assumed that soda fountains were present on both planets, and this affected amino acid synthesis. Here, using a supercritical CO(2)/liquid H(2)O (10:1) system which mimicked crust soda fountains, we demonstrate production of amino acids from hydroxylamine (nitrogen source) and keto acids (oxylic acid sources). In this research, several amino acids were detected with an amino acid analyzer. Moreover, alanine polymers were detected with LC-MS. Our research lights up a new pathway in the study of life's origin.


Assuntos
Aminoácidos/síntese química , Dióxido de Carbono/química , Água/química , Aminoácidos/química , Hidroxilamina/química , Cetoácidos/química , Peptídeos/química , Temperatura
17.
IEEE Trans Nanobioscience ; 8(1): 51-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19304501

RESUMO

Fluorescent nanocrystal quantum dots (QDs) are widely used as novel tools in various biological fields including cellular biology, molecular biology, and even in basic and clinical medical fields, due to their far brighter photoemission and photostability. Although many amounts of biological studies, including in vivo experiments, were circumstantially investigated, there is no informative report that investigates whether the QDs affect the mammalian immune system. This study investigated the immune response and biological behavior of QDs in vitro and in vivo. The immune response to QDs by both lymphocytes and kinds of macrophages in vitro and in vivo was investigated. Co-culture of QDs with immune cells showed that apparently normal production of cytokines and chemokines in both mouse CD4+ lymphocytes and peritoneal F4/80+ macrophages (PM phi). In addition, the bionanocomplex of QDs with enhanced-green-fluorescent-protein (eGFP)-encoding nucleotides successfully induced the expression of eGFP protein by PM phi. However, direct injection of QD+nucleotides bionanocomplex aqueous solution into the peritoneal cavity of mice resulted in the inflammation with the infiltration of inflammatory cells into the peritoneal cavity. Furthermore, QD+nucleotides bionanocomplex (but not QD bionanocomplex without nucleotides), induced the production of both proinflammatory cytokines and chemokines by PM phi in vitro. These results indicated that QDs covered with nucleotides caused the peritoneal inflammation in vivo via activation of PM phi and probably nonimmune cells. Taken together, these data indicated that QDs affect the proliferation of immune cells, but not in immune response including cytokine production. We propose here that all nanotechnology researchers should confirm the biological responses of their nanoscale products, because the biological response against nanoscale products can be occurred by not only in immune cells but also other nonimmune cells.


Assuntos
Imunidade Inata/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Macrófagos/imunologia , Pontos Quânticos , Animais , Células Cultivadas , Teste de Materiais , Camundongos , Camundongos Endogâmicos BALB C
18.
J Autoimmun ; 31(1): 79-89, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18501296

RESUMO

The elevation of serum anti-neutrophil cytoplasmic autoantibodies (ANCA) is significantly associated with the progression of some patients with systemic vasculitis. Especially, myeloperoxidase-specific ANCA (MPO-ANCA) play a pivotal role in the progression of systemic vasculitis including crescentic glomerulonephritis. Here we demonstrated that MPO-ANCA-activated neutrophils allow the local environment to differentiate Th(17) cells through IL-6, IL-17A, and IL-23 production. We found a variety of elevated serum cytokines, especially IL-17A, in ANCA-mediated systemic vasculitis mice. Furthermore, activated peritoneal neutrophils in vitro also produced IL-17A and IL-23 in response to MPO-ANCA. Co-stimulation of fungal mannoprotein and complements significantly enhanced the MPO-ANCA-mediated IL-17A expression, but F(ab)'(2) fragments of MPO-ANCA diminished the cytokine response. These results suggest that the activated neutrophils produce IL-17A and IL-23 in response to MPO-ANCA via their Fc-region and classical complement pathway, which initiate the first steps of chronic autoimmune inflammation by allowing the local environment to develop Th(17)-mediated autoimmunity.


Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Via Clássica do Complemento/imunologia , Interleucina-17/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Anticitoplasma de Neutrófilos/sangue , Anticorpos Anticitoplasma de Neutrófilos/farmacologia , Autoanticorpos/imunologia , Células Cultivadas , Via Clássica do Complemento/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Peroxidase/imunologia , Vasculite/imunologia , Vasculite/metabolismo
19.
Nanotechnology ; 19(49): 495102, 2008 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21730662

RESUMO

Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

20.
Nanotechnology ; 19(41): 415102, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21832637

RESUMO

Semiconductor quantum dots (QDs) hold some advantages over conventional organic fluorescent dyes. Due to these advantages, they are becoming increasingly popular in the field of bioimaging. However, recent work suggests that cadmium based QDs affect cellular activity. As a substitute for cadmium based QDs, we have developed photoluminescent stable silicon quantum dots (Si-QDs) with a passive-oxidation technique. Si-QDs (size: 6.5 ± 1.5 nm) emit green light, and they have been used as biological labels for living cell imaging. In order to determine the minimum concentration for cytotoxicity, we investigated the response of HeLa cells. We have shown that the toxicity of Si-QDs was not observed at 112 µg ml(-1) and that Si-QDs were less toxic than CdSe-QDs at high concentration in mitochondrial assays and with lactate dehydrogenase (LDH) assays. Especially under UV exposure, Si-QDs were more than ten times safer than CdSe-QDs. We suggest that one mechanism for the cytotoxicity is that Si-QDs can generate oxygen radicals and these radicals are associated with membrane damages. This work has demonstrated the suitability of Si-QDs for bioimaging in lower concentration, and their cytotoxicity and one toxicity mechanism at high concentration.

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