RESUMO
The demand for artificial blood vessels to treat vascular disease will continue to increase in the future. To expand the application of blood-compatible poly(2-methoxyethyl acrylate) (pMEA) to artificial blood vessels, control of the mechanical properties of pMEA is established using supramolecular cross-links based on inclusion complexation of acetylated cyclodextrin. The mechanical properties, such as Young's modulus and toughness, of these pMEA-based elastomers change with the amount of cross-links, maintaining tissue-like behavior (J-shaped stress-strain curve). Regardless of the cross-links, the pMEA-based elastomers exhibit low platelet adhesion properties (approximately 3% platelet adherence) compared with those of poly(ethylene terephthalate), which is one of the commercialized materials for artificial blood vessels. Contact angle measurements imply a shift of supramolecular cross-links in response to the surrounding environment. When immersed in water, hydrophobic supramolecular cross-links are buried within the interior of the materials, thereby exposing pMEA chains to the aqueous environment; this is why supramolecular cross-links do not affect the platelet adhesion properties. In addition, the elastomers exhibit stable adhesion to human umbilical vein endothelial cells. This report shows the potential of combining supramolecular cross-links and pMEA.
RESUMO
BACKGROUND: About twice as many boys as girls undergo growth hormone (GH) therapy in GH deficiency (GHD). However, this sex difference may not correctly reflect a real incidence. OBJECTIVES: We analyzed the evidence of a selection bias whereby more boys seek treatment at short stature clinics. SUBJECTS AND METHODS: The present study included 3,902 children who visited 17 short stature clinics with a height SD score of -2 SD or less. The percentage of children who underwent the GH stimulation test was compared between boys and girls, as was the percentage of children ultimately diagnosed with GHD. RESULTS: The children comprised 2,390 boys (61.3%) and 1,512 girls (38.7%), with a boy:girl ratio of 1.58:1. The percentage of children who underwent the GH stimulation test did not differ between boys (45.7%) and girls (49.8%). Among the children who underwent the GH stimulation test, the percentage diagnosed with GHD did not differ significantly between boys (22.0%) and girls (20.1%). The boy:girl ratio of children diagnosed with GHD was 1.59:1. CONCLUSIONS: The boy:girl ratio of children with short stature (1.58:1) did not differ significantly from that of children diagnosed with GHD (1.59:1). These results indicate that the predominance of boys in GHD does not reflect a real incidence, but rather a selection bias whereby a higher proportion of boys with short stature seek treatment at clinics. This difference arises because parents are more concerned about boys' height, and because boys reach adult height at an older age.
Assuntos
Instituições de Assistência Ambulatorial , Viés , Estatura/fisiologia , Hormônio do Crescimento Humano/deficiência , Criança , Feminino , Humanos , Japão , Masculino , Fatores SexuaisRESUMO
Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.
Assuntos
Anticorpos Antivirais/imunologia , Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Provírus/genética , Desaminase APOBEC-3G/metabolismo , Doadores de Sangue , Western Blotting , Linhagem Celular , Códon sem Sentido/genética , Feminino , Genoma Viral/genética , Infecções por HTLV-I/virologia , Humanos , Gravidez , Reação em Cadeia da Polimerase em Tempo Real/métodos , Testes Sorológicos/métodos , Carga Viral , Replicação Viral/genéticaRESUMO
BACKGROUND: Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS: Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS: A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION: This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection.
Assuntos
Vírus da Dengue/genética , Dengue/diagnóstico , Reação em Cadeia da Polimerase/métodos , Sorogrupo , Reação Transfusional , Segurança do Sangue , Dengue/prevenção & controle , Dengue/transmissão , Humanos , Reação em Cadeia da Polimerase Multiplex , RNA Viral/análise , RNA Viral/sangue , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.
Assuntos
DNA Viral/análise , Vírus Linfotrópico T Tipo 1 Humano/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Carga Viral/genética , Linhagem Celular Tumoral , DNA Viral/genética , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Humanos , Japão , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/virologia , Leucócitos Mononucleares/virologia , Provírus/genética , Integração Viral/genéticaRESUMO
BACKGROUND: Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, there are no therapies to prevent ATL development in high-risk asymptomatic carriers. To develop a therapy targeting HTLV-1-infected cells that are known to express CCR4 frequently, we tested whether truncated Pseudomonas exotoxin (PE38) fused to a CCR4 ligand, CCL17/thymus and activation-regulated chemokine (TARC), selectively eliminates such cells. RESULTS: Our data show that TARC-PE38 efficiently killed HTLV-1-infected cell lines. It also shrank HTLV-1-associated solid tumors in an infected-cell-engrafted mouse model. In HTLV-1-positive humanized mice, TARC-PE38 markedly inhibited the proliferation of HTLV-1-infected human CD4(+)CD25(+) or CD4(+)CD25(+)CCR4(+) cells and reduced the proviral loads (PVLs) in peripheral blood mononuclear cells (PBMCs). Importantly, TARC-PE38 significantly reduced the PVLs in PBMCs obtained from asymptomatic carriers. We show that the cytotoxicity of TARC-PE38 is mediated by the expression of the proprotein convertase, furin. The expression of furin was enhanced in HTLV-1-infected cells and correlated positively with PVLs in HTLV-1-infected individuals, suggesting that infected cells are more susceptible to TARC-PE38 than normal cells. CONCLUSIONS: TARC-PE38 robustly controls HTLV-1 infection by eliminating infected cells in both a CCR4- and furin-dependent manner, indicating the excellent therapeutic potential of TARC-PE38.
Assuntos
Quimiocina CCL17/farmacologia , Exotoxinas/farmacologia , Furina/genética , Furina/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/virologia , Receptores CCR4/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Adulto , Animais , Infecções Assintomáticas/terapia , Linhagem Celular Tumoral , Quimiocinas/genética , Modelos Animais de Doenças , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Humanos , Células Jurkat , Leucócitos Mononucleares/virologia , Camundongos , Provírus/efeitos dos fármacos , Provírus/fisiologia , Receptores CCR4/genética , Células U937RESUMO
We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas contra Influenza/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Animais , Ensaio de Amplificação de Sinal de DNA Ramificado/métodos , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos , Humanos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Masculino , Controle de Qualidade , Ratos , Ratos Wistar , Segurança , Regulação para Cima , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/efeitos adversos , Vacinas Virossomais/farmacologiaRESUMO
Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Carga Viral/métodos , Linhagem Celular Tumoral , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Japão , Provírus/genéticaRESUMO
Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, ß2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.
Assuntos
Vacinas contra Influenza/efeitos adversos , Segurança , Testes de Toxicidade/métodos , Transcriptoma/imunologia , Animais , Biomarcadores/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Masculino , Ratos , Estações do AnoRESUMO
Beta-amyloid protein (Abeta), a proteolytic byproduct of Alzheimer's amyloid precursor protein (APP), has been shown to play a central role in the development of Alzheimer's disease (AD). In addition, recent studies strongly suggest that other byproducts of proteolysis, such as C-terminal fragments of APP (APP-CTF), are also critically involved in the AD pathology. To explore this possibility, we investigated the histopathological changes induced by repeated low-dose intrahippocampal injection of a recombinant 105 amino acid C-terminal fragment of APP (CT105). First, we carried out a behavioral analysis by using the three-panel runway task, and found that the working memory was significantly impaired by CT105 exposure. Then, via propidium iodide staining, we encountered a number of cells exhibiting fragmented or shrank nuclei in the mossy fiber pathway (stratum lucidum and dentate hilus) in CT105-treated rats. These cells were positive for single-stranded DNA (ssDNA), an apoptosis-specific marker, and thus were considered to be apoptotic. Some of the ssDNA-positive cells were also positive for somatostatin. But neither ionized calcium-binding adapter molecule 1 (Iba1) nor S100beta occurred in ssDNA-positive cells. These findings suggest that CT105 induces apoptotic changes in cells of neuronal origin. Quantitative analysis showed that the densities of ssDNA-positive cells in the mossy fiber pathway were significantly higher in CT105-treated rats than in control animals. The present results suggest that CT105 causes dysfunction in the hippocampal mossy fiber system, and also provide some key to understand the relationship between APP-CTF and glutamatergic synaptic dysregulation in AD.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Apoptose/fisiologia , Fibras Musgosas Hipocampais/fisiologia , Neurônios/fisiologia , Fragmentos de Peptídeos/metabolismo , Precursor de Proteína beta-Amiloide/química , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , DNA de Cadeia Simples/análise , Imuno-Histoquímica , Masculino , Memória de Curto Prazo , Proteínas dos Microfilamentos , Fibras Musgosas Hipocampais/química , Fibras Musgosas Hipocampais/patologia , Fatores de Crescimento Neural/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/química , Fotomicrografia , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismoRESUMO
Most human behaviors (responses) are volitional, the frequency of which is changed based on stimulus presentations contingent upon the response, that is, operant behavior. It is considered that the findings on cognitive functions based on operant behaviors are more reliable in extrapolating the results to humans. Impairment of memory function, most notably the impairments of working memory and attention, is an important research focus to elucidate the mechanism underlying the core syndrome of Alzheimer's disease. Among various methods to measure working memory and attention, a delayed matching-to-sample paradigm utilizing operant chambers equipped with 3 levers and a choice reaction time paradigm have been proven to be very useful. Aside from these, adaptation to new environment is an important function for survival, and its impairment has been considered to be one of the factors inducing psychiatric disorders. Preclinical methods to measure the adaptation ability include a position reversal learning paradigm utilizing 2-lever operant chambers. Since the findings of studies on cognitive functions utilizing operant behaviors have been in good correlation with clinical findings, it would serve as a good strategy for elucidating the causes of such disorders as well as developing therapeutic agents.