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1.
Nature ; 593(7859): 429-434, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34012082

RESUMO

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.


Assuntos
Sistemas CRISPR-Cas , LDL-Colesterol/sangue , Edição de Genes , Modelos Animais , Pró-Proteína Convertase 9/genética , Adenina/metabolismo , Animais , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Mutação com Perda de Função , Macaca fascicularis/sangue , Macaca fascicularis/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/metabolismo , Fatores de Tempo
2.
J Med Primatol ; 50(2): 120-127, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33665856

RESUMO

BACKGROUND: Primates represent a unique object for biomedical research, in particular in the field of physiology and pathology of the cardiovascular system. Echocardiography is the most important non-invasive method for the intravital study of the heart structure and function, intracardiac and systemic haemodynamics. The available data on reference values of echocardiographic parameters in primates are limited. METHODS: We determined and described 29 structural and functional parameters in echocardiographic examination using B-mode (two-dimensional scanning), M-mode (one-dimensional scanning) and in various Doppler modes together with blood pressure in 17 male cynomolgus macaques with an average age of 5.7 ± 0.6 years. We compared available literature data on reference values of echocardiography in this species. RESULTS AND CONCLUSIONS: Echocardiographic values in cynomolgus macaques depend on age, sex composition and the anaesthesia method. There is lack of presentation in the published studies of complete list of parameters that can be obtained by echocardiographic examination.


Assuntos
Pressão Sanguínea , Ecocardiografia , Macaca fascicularis/anatomia & histologia , Macaca fascicularis/sangue , Fatores Etários , Anestesia/métodos , Animais , Masculino , Fatores Sexuais
3.
J Med Primatol ; 49(6): 300-306, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32463113

RESUMO

BACKGROUND: Serum biochemistry and ultrasonography can be useful diagnostic tools in evaluating the general health condition of long-tailed macaques in rescue and rehabilitation centers. METHODS: This study was conducted to determine and correlate the serum biochemistry profile of 24 apparently healthy male and female rescued long-tailed macaques (LTM) with the body weight, crown-rump length, and ultrasonic histogram of liver parenchyma, gallbladder lumen, and renal cortices. RESULTS: There were no sex-related differences in serum biochemistry values of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, blood urea nitrogen, total cholesterol, and total protein. Creatinine was positively correlated with body weights and crown-rump length. Multiple weak positive and negative correlations of organ-specific serum parameters and mean ultrasonic histogram of liver, gallbladder, and kidneys were observed. CONCLUSION: This study established the correlations of serum biochemistry profile with ultrasonic histogram of liver, gallbladder, and kidneys and morphometry of rescued LTM.


Assuntos
Análise Química do Sangue , Vesícula Biliar/diagnóstico por imagem , Rim/diagnóstico por imagem , Fígado/diagnóstico por imagem , Macaca fascicularis/sangue , Soro/química , Ultrassonografia , Animais , Animais de Zoológico , Feminino , Masculino , Filipinas
4.
Bioanalysis ; 11(23): 2161-2173, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31701759

RESUMO

Aim: To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Materials & methods: Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. Results & conclusion: The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.


Assuntos
Anticorpos Monoclonais/sangue , Fator D do Complemento/análise , Macaca fascicularis/sangue , Polietilenoglicóis/química , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Cromatografia de Afinidade , Cromatografia Líquida , Fator D do Complemento/administração & dosagem , Fator D do Complemento/imunologia , Injeções Intravítreas , Espectrometria de Massas em Tandem
5.
HLA ; 94(6): 482-492, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31448567

RESUMO

Compatibility tests to identify A, B, and O alleles are critical for establishing suitable donor-recipient matches among experimental animals. Using a qPCR-based SNP probe assay, we have identified A, B, AB, and indeterminate blood group phenotypes in cynomolgus and rhesus macaques. We have hypothesized, albeit without molecular confirmation, that the indeterminate phenotype represents homozygosity for the null O allele at the macaque ABO locus. The indeterminate phenotype represents the unsuccessful detection of either A or B alleles using primers targeting the A-specific and B-specific single nucleotide polymorphisms (SNPs) in a variable region of exon 7 of the ABO locus. These SNPs are associated with two functional sites, detected using two allele-specific probes in the qPCR assay where the codons leucine and methionine (at codon 266) and glycine and alanine (at codon 268) are required for the synthesis of the A and B transferases, respectively. While reference sequences for the A and B alleles exhibited no novel mutations in the functional exon, plasmid Sanger sequence analyses showed unique mutations within the diagnostic target sites in 10 macaques exhibiting the indeterminate phenotype. Eight of these indeterminate individuals exhibited SNPs at codon 268 that should prevent the syntheses of an A or B transferase. While the two other indeterminate samples had functional codons that were consistent with A or B alleles, mutations in either their probe- or primer-binding sites that altered their peptide sequences probably impeded their detection by our assay.


Assuntos
Sistema ABO de Grupos Sanguíneos , Macaca fascicularis , Macaca mulatta , Sistema ABO de Grupos Sanguíneos/sangue , Sistema ABO de Grupos Sanguíneos/genética , Alelos , Animais , Éxons/genética , Frequência do Gene , Genética Populacional , Teste de Histocompatibilidade/veterinária , Macaca fascicularis/sangue , Macaca fascicularis/genética , Macaca mulatta/sangue , Macaca mulatta/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/veterinária , Especificidade da Espécie
6.
Comp Med ; 69(3): 228-239, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31068244

RESUMO

In rheumatoid arthritis research, NHP models of collagen-induced arthritis are important because these species share many immunologic and pathologic features with humans. In addition, serum levels of various cytokines in patients with rheumatoid arthritis have been studied as immune markers for disease prediction, early diagnosis, and effective therapeutic management. The purpose of this study was to identify changes in cytokine levels that occur during the development of collagen-induced arthritis in female cynomolgus macaques (n = 8) and to assess the relationships between these changes and various disease parameters. Blood samples were collected weekly before (week 0) and after (weeks 1 through 7) immunization with type II collagen; clinicopathologic and cytokine data from those samples and other clinical parameters were used in correlation analysis. Serum levels of IFN γ, chemokine (C-C motif) ligand 2 (CCL2), and IL6 showed significant changes after generation of collagen-induced arthritis. IFNγ levels showed a strong negative correlation with body weight (an indicator of general body condition), and CCL2 and IL6 showed moderate negative correlation with body weight. Serum IL6 levels showed moderate positive correlation with the soft tissue swelling score and strong positive correlation with serum C-reactive protein levels in our NHP model of collagen-induced arthritis. In addition, serum levels of matrix metalloproteinase 3 increased significantly after inoculation with type II collagen and showed a moderate positive correlation with serum levels of C-reactive protein, IL6, and IL15. These results suggest close correlations between various cytokines and disease parameters in NHP models of rheumatoid arthritis. These cytokines therefore potentially could be used as markers for monitoring the efficacy of novel treatments in NHP models of rheumatoid arthritis.


Assuntos
Artrite Experimental/etiologia , Citocinas/sangue , Macaca fascicularis , Doenças dos Macacos/etiologia , Animais , Artrite Experimental/sangue , Artrite Experimental/diagnóstico por imagem , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/etiologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Colágeno Tipo II/imunologia , Feminino , Humanos , Macaca fascicularis/sangue , Macaca fascicularis/imunologia , Metaloproteinase 3 da Matriz/sangue , Doenças dos Macacos/sangue , Doenças dos Macacos/diagnóstico por imagem , Especificidade da Espécie
7.
Emerg Microbes Infect ; 8(1): 787-795, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31132935

RESUMO

Pteropine orthoreoviruses (PRV) are emerging bat-borne viruses with proven zoonotic transmission. We recently demonstrated human exposure to PRV in Singapore, which together with previous reports from Malaysia and Vietnam suggest that human infection of PRV may occur periodically in the region. This raises the question whether bats are the only sources of human infection. In this study, we screened 517 cynomolgus macaques caught in Singapore for evidence of exposure to PRV3M (also known as Melaka virus), which was first isolated from human patients in Melaka, Malaysia. We found that 67 serum samples were PRV3M positive by ELISA and 34 were also positive by virus neutralization assay. To investigate whether monkeys could act as hosts for PRV transmission, we experimentally infected cynomolgus macaques with PRV3M and housed these animals with uninfected monkeys. Although no clinical signs of infection were observed in infected animals, viral RNA was detected in nasal and rectal swabs and all infected macaques seroconverted. Additionally, one of the uninfected animals seroconverted, implying active shedding and transmission of PRV3M. We provide evidence that PRV exposure in the macaque population in Singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for PRVs.


Assuntos
Macaca fascicularis/virologia , Doenças dos Macacos/virologia , Orthoreovirus/fisiologia , Infecções por Reoviridae/transmissão , Infecções por Reoviridae/veterinária , Zoonoses/transmissão , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Feminino , Humanos , Macaca fascicularis/sangue , Doenças dos Macacos/sangue , Doenças dos Macacos/transmissão , Testes de Neutralização , Orthoreovirus/genética , Infecções por Reoviridae/sangue , Infecções por Reoviridae/virologia , Singapura , Zoonoses/sangue , Zoonoses/virologia
8.
Nucleic Acid Ther ; 29(2): 82-91, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30817230

RESUMO

Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKKRx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKKRx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.


Assuntos
Angioedemas Hereditários/terapia , Bradicinina/genética , Complemento C1s/genética , Pré-Calicreína/genética , Angioedemas Hereditários/sangue , Angioedemas Hereditários/genética , Animais , Animais Geneticamente Modificados/sangue , Bradicinina/sangue , Proteína Inibidora do Complemento C1/farmacologia , Complemento C1s/antagonistas & inibidores , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macaca fascicularis/sangue , Camundongos , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Pré-Calicreína/antagonistas & inibidores
9.
Lipids Health Dis ; 18(1): 55, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819205

RESUMO

BACKGROUND: Non-human primates (NHPs) are important models of medical research on obesity and cardiovascular diseases. As two of the most commonly used NHPs, cynomolgus macaque (CM) and African green monkey (AGM) own different capacities in lipid metabolism of which the mechanism is unknown. This study investigated the expression profiles of lipid metabolism-related microRNAs (miRNAs) in CM and AGM and their possible roles in controlling lipid metabolism-related gene expression. METHODS: By small RNA deep sequencing, the plasma miRNA expression patterns of CM and AGM were compared. The lipid metabolism-related miRNAs were validated through quantitative reverse-transcription (RT) polymerase chain reaction (PCR). Related-target genes were predicted by TargetScan and validated in Vero cells. RESULTS: Compared to CM, 85 miRNAs were upregulated with over 1.5-fold change in AGM of which 12 miRNAs were related to lipid metabolism. miR-122, miR-9, miR-185, miR-182 exhibited the greatest fold changes(fold changes are 51.2, 3.8, 3.7, 3.3 respectively; all P < 0.01). And 77 miRNAs were downregulated with over 1.5-fold change in AGM of which 3, miR-370, miR-26, miR-128 (fold changes are 9.3, 1.8, 1.7 respectively; all P < 0.05) were related to lipid metabolism. The lipid metabolism-related gene targets were predicted by TargetScan and confirmed in the Vero cells. CONCLUSION: We report for the first time a circulating lipid metabolism-related miRNA profile for CM and AGM, which may add to knowledge of differences between these two non-human primate species and miRNAs' roles in lipid metabolism.


Assuntos
Chlorocebus aethiops/genética , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Macaca fascicularis/genética , MicroRNAs/genética , Transportador 1 de Cassete de Ligação de ATP/sangue , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Carnitina O-Palmitoiltransferase/sangue , Carnitina O-Palmitoiltransferase/genética , Chlorocebus aethiops/sangue , Proteína 7 com Repetições F-Box-WD/sangue , Proteína 7 com Repetições F-Box-WD/genética , Ácido Graxo Sintase Tipo I/sangue , Ácido Graxo Sintase Tipo I/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Macaca fascicularis/sangue , MicroRNAs/sangue , Anotação de Sequência Molecular , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Receptores de Lipoproteínas/sangue , Receptores de Lipoproteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esterol O-Aciltransferase/sangue , Esterol O-Aciltransferase/genética , Células Vero
10.
Molecules ; 24(2)2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30634677

RESUMO

A rapid and sensitive method was established for arsenic (As) speciation based on high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method was validated for the quantification of four arsenic species, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in cynomolgus macaque plasma. Separation was achieved in just 3.7 min with an alkyl reverse phase column and highly aqueous mobile phase containing 20 mM citric acid and 5 mM sodium hexanesulfonate (pH = 4.3). The calibration curves were linear over the range of 5⁻500 ng·mL-1 (measured as As), with r > 0.99. The above method was validated for selectivity, precision, accuracy, matrix effect, recovery, carryover effect and stability, and applied in a comparative pharmacokinetic study of arsenic species in cynomolgus macaque samples following intravenous and intragastrical administration of arsenic trioxide solution (0.80 mg·kg-1; 0.61 mg·kg-1 of arsenic); in addition, the absolute oral bioavailability of the active ingredient AsIII of arsenic trioxide in cynomolgus macaque samples was derived as 60.9 ± 16.1%.


Assuntos
Trióxido de Arsênio/administração & dosagem , Trióxido de Arsênio/farmacocinética , Arsênio/análise , Macaca fascicularis/sangue , Administração Intravenosa , Animais , Arseniatos/análise , Arseniatos/sangue , Arsênio/sangue , Arsenicais/análise , Arsenicais/sangue , Arsenitos/análise , Arsenitos/sangue , Disponibilidade Biológica , Ácido Cacodílico/análise , Ácido Cacodílico/sangue , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas/métodos
11.
Gen Comp Endocrinol ; 269: 171-176, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30243886

RESUMO

AMH is regarded as a promising predictor for ovarian reserve in humans and non-human primate, and widely used in human medicine to predict ovarian response to gonadotropin, menopause and premature ovarian failure. However, large data set on the range of AMH levels in nonhuman primates is still scarce, which limited its applications largely. In this study, age-related AMH nomograms of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) were produced and compared. 219 rhesus and 529 cynomolgus monkeys ranging from infancy to adult were included. In total, the mean serum AMH levels in cynomolgus monkeys were higher than that of rhesus monkeys (14.6 ±â€¯5.3 ng/ml vs 9.5 ±â€¯6.0 ng/ml, P < 0.001). AMH was inversely correlated with age (r = -0.371, P < 0.001) in rhesus, while the negative correlation did not reach statistical significance in cynomolgus monkeys (r = -0.044, P = 0.156). The maximum mean AMH levels were attained at the subgroup of 4-11 yr and the lowest AMH levels were obtained at the subgroup of ≧12 yr in both primates, corresponding to their fertility potential. In rhesus monkeys, from 1 to 11 years old, AMH level remained stable (1-3 yr: ß = 2.784, P = 0.340; 4-11 yr: r = 0.100, P = 0.110) whereas from 12 yr onward, an inverse correlation between AMH and age (r = -0.450, P = 0.02) was observed. Similarly, AMH appeared stable from 1 to 3 yr (ß = -2.289, P = 0.429) and showed an inverse correlation with age (r = -0.521, P < 0.001) from 12 yr onward in cynomolgus monkeys, while a positive correlation was observed (r = 0.156, P = 0.001) from 4 to 11 yr. AMH levels were relatively stable across the menstrual cycle in both primates and no seasonal difference for AMH levels was observed in rhesus monkeys. Body mass index did not affect serum AMH levels in both primates. Our nomograms of serum AMH provide a reference guide on AMH longitudinal distribution by age for Macaca monkeys and might facilitate its applications.


Assuntos
Envelhecimento/sangue , Hormônio Antimülleriano/sangue , Macaca fascicularis/sangue , Macaca mulatta/sangue , Nomogramas , Animais , Feminino , Humanos , Modelos Lineares , Estações do Ano
12.
Toxicol Sci ; 164(2): 613-626, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29846725

RESUMO

ISIS 104838, a 2'-O-methoxyethyl (2'-MOE)-modified antisense oligonucleotide (ASO), causes a moderate, reproducible, dose-dependent, but selflimiting decrease in platelet (PLT) counts in monkeys and humans. To determine the etiology of PLT decrease in cynomolgus monkeys, a 12-week repeat dose toxicology study in 5 cynomolgus monkeys given subcutaneous injections of ISIS 104838 (30-60 mg/kg/week). Monkeys were also injected intravenously with 111Indium(In)-oxine-labeled PLTs to investigate PLT sequestration. In response to continued dosing, PLT counts were decreased by 50%-90% by day 30 in all monkeys. PLT decreases were accompanied by 2- to 4.5-fold increases in immunoglobulin M(IgM), which were typified by a 2- to 5-fold increase in antiplatelet factor 4 (antiPF4) IgM and antiPLT IgM, respectively. Monocyte chemotactic protein 1 increased upon dosing of ISIS 104838, concomitant with a 2- to 6-fold increase in monocyte-derived extracellular vesicles (EVs), indicating monocyte activation but not PLT activation. Despite a 2- to 3-fold increase in von Willebrand factor antigen in all monkeys following ASO administration, only 2 monkeys showed a 2- to 4-fold increase in endothelial EVs. Additionally, a ∼60 - 80%% increase in PLT sequestration in liver and spleen was also observed. Collectively, these results suggest the overall increase in total IgM, antiPLT IgM and/or antiPF4 IgM, in concert with monocyte activation contributed to increased PLT sequestration in spleen and liver, leading to decreased PLTs in peripheral blood.


Assuntos
Plaquetas/efeitos dos fármacos , Macaca fascicularis/sangue , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Plaquetas/citologia , Quimiocina CCL2/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Selectina-P/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacocinética , Contagem de Plaquetas , Baço/efeitos dos fármacos , Baço/metabolismo , Fator de von Willebrand/metabolismo
13.
J Vis Exp ; (132)2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29553521

RESUMO

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification. A fixed volume (200 µL) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step. Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/µL, for most of the examined miRNAs. This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.


Assuntos
Macaca fascicularis/sangue , Macaca fascicularis/genética , MicroRNAs/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Perfilação da Expressão Gênica
14.
Virol J ; 14(1): 210, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29100534

RESUMO

BACKGROUND: In 1980, smallpox disease was eradicated from nature and Variola virus, the etiological agent of smallpox, was confined to two laboratories, one located in Russia (Moscow) later moved to VECTOR (Novosibirsk, Siberia) and one in the United States (CDC Atlanta). Vaccinations among the general public ceased shortly after the successful eradication campaign, resulting in an increasingly immunologically susceptible population. Because of the possibility of intentional reintroduction of Variola virus and the emergence of other pathogenic poxviruses, there is a great need for the development of medical countermeasures to treat poxvirus disease. It is highly likely that the U.S. FDA "animal rule" will be necessary for regulatory approval of these interventions. Therefore, relevant animal models and the associated supporting assays will require development to stand up to regulatory scrutiny. METHODS: An optimized real time PCR assay for the detection of orthopoxviruses has been developed by researchers at the United States Army Research Institute of Infectious Diseases (USAMRIID). To support animal studies that will be used to support approval of medical countermeasures by the U.S. FDA, the assay was designed to quantitate poxvirus genomic DNA in a nonhuman primate (cynomolgus macaque) blood matrix as a measurement of viremia. This manuscript describes the validation of the process, including DNA extraction from whole blood anticoagulated with EDTA, for obtaining and quantitating monkeypox genomes by evaluating precision, accuracy, the standard curve, specificity, robustness and stability of the assay and/or components of the assay. RESULTS: The assay had a lower limit of quantitation of 50 genome copies/5 uL sample, upper limit of quantitation of 5 × 107 GC/5uL sample and a limit of detection of 2.5 genome copies /5uL sample. The assay was specific for orthopoxvirus. Matrix effects were detected and suggest the presence of PCR inhibitor(s) that was co-extracted with the target DNA. CONCLUSIONS: The assay has been validated for the purpose of quantitating monkeypox viral load in blood from cynomolgus macaques. This assay has and will continue to support submissions to the FDA for approval of antiviral therapeutics for smallpox.


Assuntos
Genoma Viral , Macaca fascicularis/sangue , Vírus da Varíola dos Macacos/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , DNA Viral/sangue , DNA Viral/isolamento & purificação , Vírus da Varíola dos Macacos/genética , Sensibilidade e Especificidade , Estados Unidos , United States Food and Drug Administration , Viremia/sangue
15.
Alzheimers Dement ; 13(12): 1397-1409, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28475854

RESUMO

INTRODUCTION: Although amyloid ß peptide (Aß) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role. METHODS: Multidisciplinary methods were used to demonstrate immune adherence capture of Aß by erythrocytes and its deficiency in Alzheimer's disease (AD). RESULTS: Aß was shown to be subject to immune adherence at every step in the pathway. Aß dose-dependently activated serum complement. Complement-opsonized Aß was captured by erythrocytes via CR1. Erythrocytes, Aß, and hepatic Kupffer cells were colocalized in the human liver. Significant deficits in erythrocyte Aß levels were found in AD and mild cognitive impairment patients. DISCUSSION: CR1 polymorphisms elevate AD risk, and >80% of human CR1 is vested in erythrocytes to subserve immune adherence. The present results suggest that this pathway is pathophysiologically relevant in AD.


Assuntos
Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/metabolismo , Disfunção Cognitiva/sangue , Eritrócitos/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Complemento/fisiologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/farmacologia , Animais , Estudos de Casos e Controles , Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Macaca fascicularis/sangue , Masculino , Testes de Estado Mental e Demência , Microscopia Eletrônica , Pessoa de Meia-Idade , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Complemento/genética
16.
Vet Clin Pathol ; 46(2): 371-379, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28403556

RESUMO

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.


Assuntos
Cromatografia Líquida/veterinária , Espectrometria de Massas em Tandem/veterinária , Vitamina D/análogos & derivados , 25-Hidroxivitamina D 2/sangue , Animais , Calcifediol/sangue , Cromatografia Líquida/métodos , Cães/sangue , Feminino , Macaca fascicularis/sangue , Masculino , Camundongos/sangue , Camundongos Endogâmicos C57BL/sangue , Ratos/sangue , Ratos Sprague-Dawley/sangue , Espectrometria de Massas em Tandem/métodos , Vitamina D/sangue
17.
J Vet Med Sci ; 79(5): 881-888, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28381665

RESUMO

Cynomolgus monkeys are closely related to humans phylogenetically, and this has resulted in their widespread use as a preclinical model. Hematological data with regard to these monkeys are thus important. Although reference values for blood components and sex hormones have been established for cynomolgus monkeys, those for arterial blood gases have not. The arterial blood gases quickly reflect respiratory and circulatory dynamics, and are thus useful for animal management and safe general anesthesia and surgical operations. Furthermore, since O2 is transported by RBC, CBC and blood gases are closely related. The present study aimed to establish reference values for arterial blood gases and CBC in cynomolgus monkeys over a wide age range. Blood gases and CBC of arterial blood, collected from 41 female and 21 male anesthetized monkeys, were measured. Age correlated with RBC, HGB and HCT in the CBC. Values differed significantly between males and females in pCO2, CO2 concentration, MCV and MCH. The pH of blood was equivalent to that of humans and pCO2 was more stable, whereas MCV and MCH were lower than those in humans. Erythrocytes were smaller and less pigmented than in other Macaca species. Several relationships between gender and age, and blood gases and CBC were identified in cynomolgus monkeys. In conclusion, these reference values will be useful as markers for veterinary applications and in the care and maintenance of these animals.


Assuntos
Contagem de Células Sanguíneas/veterinária , Gasometria/veterinária , Macaca fascicularis/sangue , Envelhecimento/sangue , Animais , Feminino , Masculino , Valores de Referência
18.
Sci Rep ; 6: 35989, 2016 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-27786258

RESUMO

No study has reported an animal model of uterus transplantation (UTx) using cynomolgus macaques. We aimed to establish a surgical technique of allogeneic UTx assuming the recovery of a uterus from a deceased donor in cynomolgus macaques. Four allogeneic UTxs were performed in female cynomolgus macaques. Donor surgeries comprised en bloc recovery of organs with iliac vessels on both sides, and/or abdominal aorta/vena cava after sufficient perfusion from one femoral artery or external iliac artery. Before perfusion, 150 mL of whole blood was obtained from the donor for subsequent blood transfusion to the recipient. Four uterine grafts were orthotopically transplanted to recipients. End-to-side anastomosis was performed to the iliac vessels on one side in case 1 and iliac vessels on both sides in case 2; aorto-aorto/cavo-caval anastomosis was performed in cases 3 and 4. Arterial blood flow of the uterine grafts was determined by intraoperative indocyanine green (ICG) angiography. ICG angiography results showed sufficient blood flow to all uterine grafts, and anaemia did not progress. Under appropriate immune suppression, all recipients survived for more than 90 days post-transplantation, without any surgical complications. We describe a surgical technique for allogeneic UTx in cynomolgus macaques.


Assuntos
Macaca fascicularis/cirurgia , Útero/transplante , Animais , Feminino , Hemoglobinas/metabolismo , Imunossupressão , Macaca fascicularis/sangue , Modelos Anatômicos , Modelos Animais , Doadores de Tecidos , Transplante Homólogo/métodos , Útero/irrigação sanguínea , Útero/diagnóstico por imagem
19.
J Am Assoc Lab Anim Sci ; 55(5): 534-40, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27657707

RESUMO

To evaluate the effects of transportation on oxidative stress in cynomolgus monkeys, we measured serum levels of reduced glutathione (GSH), malondialdehyde, and protein carbonyl (PC) and the activities of total antioxidant capacity (TAOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase in cynomolgus macaques before transportation (day 0), on the day of arrival (day 1), and on days 7, 14, and 21 after transportation. Compared with that on day 0, TAOC and catalase activities on days 1, 7, and 14 after transportation were significantly decreased, reached their nadirs on day 7, and increased thereafter to reach their pretransportation levels by day 21 after transportation. Compared with day 0 levels, mean SOD activity and GSH concentration were decreased significantly on day 1; they thereafter increased to reach their pretransportation measures by day 7 after transportation. In contrast, PC and malondialdehyde concentrations in serum and the activity of GSH-Px were increased on day 1 compared with day 0 and thereafter decreased to reach their pretransportation levels by day 14 after transportation. In summary, GSH, TAOC, catalase, and SOD levels decreased and malondialdehyde, PC, and GSH-Px concentrations increased in cynomolgus macaques after transportation. These results suggest that transportation might imbalance oxidant and antioxidant levels to create excess oxidative stress in cynomolgus macaques. Therefore, cynomolgus macaques should have at least 21 d to recover after transportation and regain their healthy status.


Assuntos
Antioxidantes/metabolismo , Macaca fascicularis/fisiologia , Estresse Oxidativo/fisiologia , Transportes , Criação de Animais Domésticos , Animais , Catalase , Glutationa , Glutationa Peroxidase , Macaca fascicularis/sangue , Malondialdeído , Superóxido Dismutase
20.
J Am Assoc Lab Anim Sci ; 55(5): 558-64, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27657710

RESUMO

In this study, a metabolomics approach based on nuclear magnetic resonance spectroscopy and pertinent multivariate data analyses was used to evaluate the effect of ketamine on metabolic markers in cynomolgus macaques. Principal component analysis and orthogonal projection to latent structure with discriminant analysis showed that ketamine (10 mg/kg) induced metabolic perturbations. Compared with the control group, ketamine-treated macaques had lower serum levels of α-glucose, myoinositol, lactate and succinate and lower urine levels of pyruvate and lactate. In contrast, the levels of leucine in serum and arginine in urine were significantly higher in the ketamine group. Our results also demonstrated that a single injection of ketamine influenced the major energy and amino acid metabolic pathways in cynomolgus macaques. Our study suggests that these influences should be considered in the design of experiments and the interpretation related blood and urine data from ketamine-sedated cynomolgus macaques.


Assuntos
Analgésicos/farmacologia , Ketamina/farmacologia , Macaca fascicularis/sangue , Macaca fascicularis/urina , Metabolômica , Animais , Análise Discriminante , Análise de Componente Principal
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