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1.
Int Immunol ; 34(8): 421-434, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35689594

RESUMO

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.


Assuntos
Bacteriófagos , Camelídeos Americanos , Anticorpos de Domínio Único , Animais , Antígenos , Bacteriófagos/genética , Camelídeos Americanos/genética , Biblioteca Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
2.
Cancer Immunol Immunother ; 65(3): 341-54, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26880265

RESUMO

Induction of lymphopenia before adoptive transfer of T cells was followed by lymphopenia-induced proliferation (LIP) and generated a potent anti-tumor immune response in rodents and in a clinical setting. Previously, we reported that CD28 signaling is essential for the differentiation of functional effector cytotoxic T lymphocytes (CTLs) under lymphopenic conditions and sequential LIP of T cells. In this study, to clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited with interleukin 7 (IL7) receptor blockade at various stages, and the anti-tumor effect then assessed. We confirmed that IL7 signaling at the start of LIP is crucial for the anti-tumor immune response. In contrast, continuous IL7 signaling was not required for tumor regression, although LIP of naïve CD8+ T cells is usually regulated by IL7. The expansion and migration of CTLs in lymphopenic hosts depend on IL7 signaling during the induction phase. Here, we propose that IL7 signaling and subsequent LIP of T cells have distinct roles in the induction of T cell immunity during lymphopenia.


Assuntos
Interleucina-7/fisiologia , Neoplasias Pulmonares/imunologia , Linfopenia/imunologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/análise , Receptores de Interleucina-7/fisiologia , Linfócitos T Citotóxicos/imunologia
3.
Int Immunol ; 27(12): 609-20, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26152273

RESUMO

Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.


Assuntos
Anticorpos/metabolismo , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Memória Imunológica , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Anticorpos/genética , Diversidade de Anticorpos/genética , Diferenciação Celular , Células Cultivadas , Galinhas , Imunização Secundária , Ativação Linfocitária , Nitrofenóis/química , Nitrofenóis/imunologia , Fenilacetatos/química , Fenilacetatos/imunologia , Receptores de Antígenos de Linfócitos B/genética , Hipermutação Somática de Imunoglobulina , gama-Globulinas/química , gama-Globulinas/imunologia
4.
Surg Endosc ; 30(9): 4153-9, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26659227

RESUMO

BACKGROUND: Localization of colorectal tumors during laparoscopic surgery is generally performed by tattooing into the submucosal layer of the colon. However, faint and diffuse tattoos may lead to difficulties in recognizing cancer sites, resulting in inappropriate resection of the colon. We previously demonstrated that yttrium oxide nanoparticles doped with the rare earth ions (ytterbium and erbium) (YNP) showed strong near-infrared (NIR) emission under NIR excitation (1550 nm emission with 980 nm excitation). NIR light can penetrate deep tissues. In this study, we developed an NIR laparoscopy imaging system and demonstrated its use for accurate resection of the colon in swine. METHODS: The NIR laparoscopy system consisted of an NIR laparoscope, NIR excitation laser diode, and an NIR camera. Endo-clips coated with YNP (NIR clip), silicon rubber including YNP (NIR silicon mass), and YNP solution (NIR ink) were prepared as test NIR markers. We used a swine model to detect an assumed colon cancer site using NIR laparoscopy, followed by laparoscopic resection. The NIR markers were fixed at an assumed cancer site within the colon by endoscopy. An NIR laparoscope was then introduced into the abdominal cavity through a laparoscopy port. RESULTS: NIR emission from the markers in the swine colon was successfully recognized using the NIR laparoscopy imaging system. The position of the markers in the colon could be identified. Accurate resection of the colon was performed successfully by laparoscopic surgery under NIR fluorescence guidance. The presence of the NIR markers within the extirpated colon was confirmed, indicating resection of the appropriate site. CONCLUSIONS: NIR laparoscopic surgery is useful for colorectal cancer site recognition and accurate resection using laparoscopic surgery.


Assuntos
Neoplasias do Colo/cirurgia , Érbio , Laparoscopia/métodos , Tatuagem/métodos , Itérbio , Ítrio , Animais , Neoplasias do Colo/diagnóstico por imagem , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/cirurgia , Fluorescência , Laparoscópios , Espectroscopia de Luz Próxima ao Infravermelho , Instrumentos Cirúrgicos , Suínos
5.
Biophys Physicobiol ; 21(1): e210004, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38803333

RESUMO

Cell migration plays an important role in the development and maintenance of multicellular organisms. Factors that induce cell migration and mechanisms controlling their expression are important for determining the mechanisms of factor-induced cell migration. Despite progress in the study of factor-induced cytotaxis, including chemotaxis and haptotaxis, precise control of the direction of cell migration over a wide area has not yet been achieved. Success in this area would update the cell migration assays, superior cell separation technologies, and artificial organs with high biocompatibility. The present study therefore sought to control the direction of cell migration over a wide area by adjusting the three-dimensional shape of the cell scaffold. The direction of cell migration was influenced by the shape of the cell scaffold, thereby optimizing cell adhesion and protrusion. Anisotropic arrangement of these three-dimensional shapes into a periodic structure induced unidirectional cell migration. Three factors were required for unidirectional cell migration: 1) the sizes of the anisotropic periodic structures had to be equal to or lower than the size of the spreading cells, 2) cell migration was restricted to a runway approximately the width of the cell, and 3) cells had to be prone to extension of long protrusions in one direction. Because the first two factors had been identified previously in studies of cell migration in one direction using two-dimensional shaped patterns, these three factors are likely important for the mechanism by which cell scaffold shapes regulate cell migration.

6.
Blood ; 117(9): 2640-8, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21220748

RESUMO

Dendritic cells (DCs) are known to regulate immune responses by inducing both central and peripheral tolerance. DCs play a vital role in negative selection of developing thymocytes by deleting T cells with high-affinity for self-peptide-major histocompatibility complexes. In the periphery, DCs mediate peripheral tolerance by promoting regulatory T-cell development, induction of T-cell unresponsiveness, and deletion of activated T cells. We studied whether allogeneic DCs, obtained from bone marrow cultured with either Flt3L (FLDCs) or granulocyte-macrophage colony-stimulating factor (GMDCs), could induce allospecific central and peripheral tolerance after IV injection; B cells were used as a control. The results showed that only FLDCs reached the thymus after injection and that these cells induced both central and peripheral tolerance to donor major histocompatibility complexes. For central tolerance, injection of FLDCs induced antigen-specific clonal deletion of both CD8 and CD4 single-positive thymocytes. For peripheral tolerance, injection of FLDCs induced donor-specific T-cell unresponsiveness and prolonged survival of donor-derived skin grafts. Tolerance induction by adoptive transfer of FLDCs could be a useful approach for promoting graft acceptance after organ transplantation.


Assuntos
Células Dendríticas/citologia , Células Dendríticas/imunologia , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Transplante de Pele/imunologia , Transferência Adotiva , Animais , Movimento Celular , Proliferação de Células , Células Clonais , Epitopos/imunologia , Injeções , Proteínas de Membrana/metabolismo , Camundongos , Células da Side Population/citologia , Células da Side Population/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Fatores de Tempo , Transplante Homólogo
7.
J Nanosci Nanotechnol ; 13(1): 229-35, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23646721

RESUMO

Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Meios de Cultura Livres de Soro , Camundongos , Células NIH 3T3
8.
J Mater Sci Mater Med ; 23(10): 2399-412, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22588504

RESUMO

The use of an "over 1000-nm near-infrared (NIR) in vivo fluorescence bioimaging" system based on lanthanide containing inorganic nanostructures emitting in the visible and NIR range under 980-nm excitation is proposed. It may overcome problems of currently used biomarkers including color fading, phototoxicity and scattering. Gd(2)O(3):Er(3+),Yb(3+) nanoparticles and nanorods showing upconversion and NIR emission are synthesized and their cytotoxic behavior is investigated by incubation with B-cell hybridomas and macrophages. Surface modification with PEG-b-PAAc provides the necessary chemical durability reducing the release of toxic Gd(3+) ions. NIR fluorescence microscopy is used to investigate the suitability of the nanostructures as NIR-NIR biomarkers. The in vitro uptake of bare and modified nanostructures by macrophages is investigated by confocal laser scanning microscopy. In vivo investigations revealed nanostructures in liver, lung, kidneys and spleen a few hours after injection into mice, while most of the nanostructures have been removed from the body after 24 h.


Assuntos
Érbio/química , Gadolínio/química , Nanoestruturas , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Itérbio/química , Animais , Materiais Biocompatíveis , Linhagem Celular , Sobrevivência Celular , Érbio/farmacocinética , Gadolínio/farmacocinética , Técnicas In Vitro , Camundongos , Microscopia Eletrônica de Varredura , Difração de Pó , Propriedades de Superfície , Distribuição Tecidual , Itérbio/farmacocinética
9.
J Exp Med ; 195(3): 283-93, 2002 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-11828003

RESUMO

Examining the rate of in vivo T cell turnover (proliferation) in aged mice revealed a marked reduction in turnover at the level of memory-phenotype CD44(hi) CD8(+) cells relative to young mice. Based on adoptive transfer experiments, the reduced turnover of aged CD44(hi) CD8(+) cells reflected an inhibitory influence of the aged host environment. Aged CD44(hi) CD8(+) cells also showed poor in vivo responses to IL-15 and IL-15-inducing agents, but responded well to IL-15 in vitro. Two mechanisms could account for the reduced turnover of aged CD44(hi) CD8(+) cells in vivo. First, aging was associated with a prominent and selective increase in Bcl-2 expression in CD44(hi) CD8(+) cells. Hence, the reduced turnover of aged CD44(hi) CD8(+) cells may in part reflect the antiproliferative effect of enhanced Bcl-2 expression. Second, the impaired in vivo response of aged CD44(hi) CD8(+) cells to IL-15 correlated with increased serum levels of type I interferons (IFN-I) and was largely reversed by injection of anti-IFN-I antibody. Hence the selective reduction in the turnover of aged CD44(hi) CD8(+) cells in vivo may reflect the combined inhibitory effects of enhanced Bcl-2 expression and high IFN-I levels.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Transferência Adotiva , Animais , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Expressão Gênica , Genes bcl-2 , Homeostase/imunologia , Receptores de Hialuronatos/metabolismo , Técnicas In Vitro , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/genética , Interleucina-15/genética , Interleucina-15/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína bcl-X
10.
Adv Biosyst ; 4(10): e2000113, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32924291

RESUMO

The present study demonstrates unidirectional cell migration using a novel 3D microfabricated scaffold, as revealed by the uneven sorting of cells into an area of 1 mm × 1 mm. To induce unidirectional cell migration, it is important to determine the optimal arrangement of 3D edges, and thus, the anisotropic periodic structures of micropatterns are adjusted appropriately. The cells put forth protrusions directionally along the sharp edges of these micropatterns, and migrated in the protruding direction. There are three advantages to this novel system. First, the range of applications is wide, because this system effectively induces unidirectional migration as long as 3D shapes of the scaffolds are maintained. Second, this system can contribute to the field of cell biology as a novel taxis assay. Third, this system is highly applicable to the development of medical devices. In the present report, unique 3D microfabricated scaffolds that provoked unidirectional migration of NIH3T3 cells are described. The 3D scaffolds could provoke cells to accumulate in a single target location, or could provoke a dissipated cell distribution. Because the shapes are very simple, they could be applied to the surfaces of various medical devices. Their utilization as a cell separation technology is also anticipated.


Assuntos
Movimento Celular/fisiologia , Técnicas Citológicas/métodos , Microtecnologia/métodos , Alicerces Teciduais , Animais , Adesão Celular/fisiologia , Técnicas Citológicas/instrumentação , Desenho de Equipamento , Camundongos , Microtecnologia/instrumentação , Células NIH 3T3
11.
Int Immunol ; 20(12): 1507-15, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18829987

RESUMO

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Antígenos CD28/metabolismo , Proteína Adaptadora GRB2/metabolismo , Guanilato Ciclase/metabolismo , Isoenzimas/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/imunologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Células CHO , Fracionamento Celular , Cricetinae , Cricetulus , Proteína Adaptadora GRB2/imunologia , Guanilato Ciclase/imunologia , Humanos , Isoenzimas/imunologia , Células Jurkat , Mutagênese Sítio-Dirigida , Mutação , NF-kappa B/genética , NF-kappa B/imunologia , Fosforilação , Ligação Proteica , Proteína Quinase C/imunologia , Proteína Quinase C-theta , Transporte Proteico , Transdução de Sinais/imunologia , Transfecção
12.
Transpl Immunol ; 55: 101205, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30946889

RESUMO

Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.


Assuntos
Doenças da Medula Óssea , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Tecido Adiposo , Aloenxertos , Animais , Doenças da Medula Óssea/etiologia , Doenças da Medula Óssea/imunologia , Doenças da Medula Óssea/patologia , Doenças da Medula Óssea/terapia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/terapia , Células-Tronco Mesenquimais/patologia , Camundongos
13.
Sci Rep ; 8(1): 14559, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266961

RESUMO

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM+ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.


Assuntos
Linfócitos B/imunologia , Imunoglobulina M/imunologia , Animais , Células Produtoras de Anticorpos/citologia , Células Produtoras de Anticorpos/imunologia , Linfócitos B/citologia , Diferenciação Celular , Células Cultivadas , Galinhas , Imunoglobulina G/imunologia , Memória Imunológica , Ativação Linfocitária , Camundongos Endogâmicos C57BL , Linfócitos T/citologia , Linfócitos T/imunologia
14.
J Biomed Mater Res B Appl Biomater ; 106(3): 976-985, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28474403

RESUMO

Here, a new technology was developed to selectively produce areas of high and low surface Young's modulus on biomedical polymer films using micropatterns. First, an elastic polymer film was adhered to a striped micropattern to fabricate a micropattern-supported film. Next, the topography and Young's modulus of the film surface were mapped using atomic force microscopy. Contrasts between the concave and convex locations of the stripe pattern were obvious in the Young's modulus map, although the topographical map of the film surface appeared almost flat. The concave and convex locations of a polymer film supported by a different micropattern also contrasted clearly. The resulting Young's modulus map showed that the Young's modulus was higher at convex locations than at concave locations. Hence, regions of high and low stiffness can be locally generated based on the shape of the micropattern supporting the film. When cells were cultured on the micropattern-supported films, NIH3T3 fibroblasts preferentially accumulated in convex regions with high Young's moduli. These findings demonstrate that this new technology can regulate regions of high and low surface Young's modulus on a cellular scaffold with high planar resolution, as well as providing a method for directing cellular patterning. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 976-985, 2018.


Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Polímeros/química , Células 3T3 , Animais , Elasticidade , Fenômenos Mecânicos , Camundongos , Cloreto de Polivinila/química , Propriedades de Superfície , Alicerces Teciduais
15.
Biomater Sci ; 3(1): 59-64, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26214189

RESUMO

The use of near-infrared (NIR) light over 1000 nm (OTN-NIR or second NIR) is advantageous for bioimaging because it enables deep tissue penetration due to low scattering and autofluorescence. In this report, we describe the application of rare earth ion-doped ceramic nanoparticles to cancer-targeted NIR imaging using erbium and ytterbium ion-doped yttrium oxide nanoparticles (YNP) functionalized with streptavidin via bi-functional PEG (SA-YNP). YNP has NIR emission at 1550 nm, with NIR excitation at 980 nm (NIR-NIR imaging). Cancer-specific NIR-NIR imaging was demonstrated using SA-YNP and biotinylated antibodies on cancer cells and human colon cancer tissues. NIR-NIR imaging through porcine meat of 1 cm thickness was also demonstrated, supporting the possible application of deep tissue NIR-NIR bioimaging using YNP as a probe. Our results suggest that non-invasive imaging using YNP has great potential for general application in cancer imaging in living subjects.


Assuntos
Cerâmica/química , Neoplasias do Colo/terapia , Érbio/química , Nanopartículas Metálicas/química , Metais Terras Raras/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Estreptavidina/química , Ítrio/química , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/química , Humanos , Nanopartículas Metálicas/administração & dosagem , Metais Terras Raras/administração & dosagem , Suínos
16.
Intern Med ; 54(19): 2513-6, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26424314

RESUMO

A 65-year-old man, who recently returned from Liberia, visited a clinic complaining of fever, and azithromycin was prescribed. The patient presented to a general hospital 5 days after the onset of symptoms, however, a blood smear examination failed to detect malaria. Contrary to the blood smear result, a rapid antigen test in our hospital was strongly-positive for falciparum malaria, indicating a high level of malarial antigen in the blood. Moreover, laboratory examinations on admission showed a tendency for improvement. We assumed that the administration of azithromycin partially treated malaria, thus complicating the blood smear diagnosis. We should be careful in prescribing azithromycin, which is widely used in clinics, to travelers returning from malaria-endemic countries.


Assuntos
Antibacterianos/administração & dosagem , Antimaláricos/uso terapêutico , Azitromicina/administração & dosagem , Febre/tratamento farmacológico , Malária Falciparum/diagnóstico , Idoso , Diagnóstico Tardio , Febre/etiologia , Humanos , Malária Falciparum/sangue , Malária Falciparum/complicações , Masculino , Padrões de Prática Médica , Viagem
17.
Nanoscale ; 5(23): 11339-61, 2013 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23938606

RESUMO

In recent years, significant progress was achieved in the field of nanomedicine and bioimaging, but the development of new biomarkers for reliable detection of diseases at an early stage, molecular imaging, targeting and therapy remains crucial. The disadvantages of commonly used organic dyes include photobleaching, autofluorescence, phototoxicity and scattering when UV (ultraviolet) or visible light is used for excitation. The limited penetration depth of the excitation light and the visible emission into and from the biological tissue is a further drawback with regard to in vivo bioimaging. Lanthanide containing inorganic nanostructures emitting in the near-infrared (NIR) range under NIR excitation may overcome those problems. Due to the outstanding optical and magnetic properties of lanthanide ions (Ln(3+)), nanoscopic host materials doped with Ln(3+), e.g. Y2O3:Er(3+),Yb(3+), are promising candidates for NIR-NIR bioimaging. Ln(3+)-doped gadolinium-based inorganic nanostructures, such as Gd2O3:Er(3+),Yb(3+), have a high potential as opto-magnetic markers allowing the combination of time-resolved optical imaging and magnetic resonance imaging (MRI) of high spatial resolution. Recent progress in our research on over-1000 nm NIR fluorescent nanoprobes for in vivo NIR-NIR bioimaging will be discussed in this review.


Assuntos
Elementos da Série dos Lantanídeos/química , Nanoestruturas/química , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/química , Meios de Contraste/toxicidade , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Humanos , Imageamento por Ressonância Magnética , Magnetismo , Nanoestruturas/toxicidade , Neoplasias/diagnóstico , Polímeros/química
18.
Acta Biomater ; 9(1): 4734-43, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22963845

RESUMO

Bioimaging is an important diagnostic tool in the investigation and visualization of biological phenomena in cells and in medicine. In this context, up-converting Gd(2)O(3):Er(3+),Yb(3+) nanostructures (nanoparticles, nanorods) have been synthesized by precipitation methods and hydrothermal synthesis. Independent of size and morphology, Gd(2)O(3):Er(3+),Yb(3+) powders show up-conversion (550 nm, 670 nm) and near-infrared emission (1.5 µm) upon 980 nm excitation, which makes these structures interesting for application as biomarkers. With regard to their potential application in bioimaging, cytotoxicity is an important aspect and is strongly affected by the physico-chemical properties of the investigated nanostructures. Therefore, the cytotoxic effect of bare and poly(ethylene glycol)-b-poly(acrylic acid) block co-polymer-modified nanostructures on non-phagocytic and phagocytic cells (B-cell hybridoma cells and macrophages) was investigated. The observed cytotoxic behavior in the case of macrophages incubated with bare nanostructures was assigned to the poor chemical durability of gadolinium oxide, but could be overcome by surface modification.


Assuntos
Gadolínio/química , Nanoestruturas , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Microscopia Eletrônica de Varredura , Difração de Pó
19.
Exp Clin Transplant ; 10(4): 375-85, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22758208

RESUMO

OBJECTIVES: Interleukin-6, a pleiotropic cytokine that functions in both innate and adaptive immune responses, has been implicated in allograft rejection. We analyzed the efficacy of anti interleukin-6 receptor monoclonal antibody in delaying allograft rejection in a murine model of a heart. MATERIALS AND METHODS: To investigate the role of interleukin-6 receptor signal transduction in acute and chronic allograft rejection, we blocked interleukin-6 receptor signaling to suppress the alloimmune response in C57BL/6 recipients of BALB/c cardiac allografts. RESULTS: Administration of a high-dose α-interleukin-6 receptor monoclonal antibody prevented the intragraft infiltration of inflammatory cells and lymphocytes and prolonged allograft survival during the peritransplant period. However, all allografts were rejected by 23.5 days after transplant. In contrast, cardiac allograft recipients treated with a cytotoxic T-lymphocyte antigen 4-immunoglobulin plus continued administration of low-dose α-interleukin-6 receptor monoclonal antibody showed long-term graft survival compared with cytotoxic T-lymphocyte antigen 4-immunoglobulin monotherapy. A histologic analysis revealed that graft fibrosis was prevented in cytotoxic T-lymphocyte antigen 4-immunoglobulin plus high-dose α-interleukin-6 receptor monoclonal antibody group, but not in the cytotoxic T-lymphocyte antigen 4-immunoglobulin alone group. This suggests that deterioration of graft function associated with chronic rejection could be prevented by blocking interleukin-6 receptor signaling. CONCLUSIONS: Disruption of interleukin-6 receptor signaling is an effective strategy for modulating proinflammatory immune responses and preventing chronic rejection.


Assuntos
Anticorpos Monoclonais/farmacologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração/imunologia , Imunossupressores/farmacologia , Miocárdio/imunologia , Receptores de Interleucina-6/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Abatacepte , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Doença Crônica , Feminino , Fibrose , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Transplante de Coração/efeitos adversos , Imunoconjugados/farmacologia , Inflamação/imunologia , Inflamação/prevenção & controle , Interleucina-6/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Receptores de Interleucina-6/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo
20.
Immunol Lett ; 128(1): 51-8, 2010 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-19914290

RESUMO

Although there is growing evidence that NKT cells play an important role in various immune responses through the invariant T cell receptor, other cell surface molecules responsible for their function are not fully understood. Here we study the role of ICOS, the third member of the CD28 family of costimulatory receptors, in in vivo and in vitro NKT cell responses. To establish its in vivo role in systems dependent on NKT cells, we examined the development of Con A-induced hepatitis in ICOS knockout (ICOS(-/-)) mice. We demonstrated that hepatic injury in ICOS(-/-) mice was greatly suppressed as evidenced by the reduced elevation of serum transaminases, reduced apoptosis of hepatocytes and mild histopathological changes. In investigating the cause of this defect, we first found that the NKT cell population is significantly reduced in the liver and spleen of ICOS(-/-) mice. We made and analyzed mixed bone marrow chimera mice with bone marrow cells from ICOS(+/+) and ICOS(-/-) mice, and demonstrated that the defect in ICOS-mediated costimulation results in a significant defect in the development of NKT cells, especially of Valpha14i NKT cells, in the thymus. When we examined the function of residual NKT cells in ICOS(-/-) mice, we found that their cytokine production following stimulation with alpha-galactosylceramide (alpha-GalCer) was strongly impaired. Based on these findings, we propose that ICOS-mediated costimulation may play a critical role in both the development and the optimal function of NKT cells, and that defective ICOS-mediated costimulation may result in impaired Con A-induced hepatitis in ICOS(-/-) mice.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Hepatite/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Células Cultivadas , Quimera , Feminino , Galactosilceramidas/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Ativação Linfocitária , Macrolídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/patologia
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