MazF, an endoribonuclease encoded by Escherichia coli, specifically cleaves the ACA (adenine-cytosine-adenine) sequence of single-stranded RNAs. Conditional expression of MazF under the control of the HIV-1 LTR promoter rendered CD4(+) T cells resistant to HIV-1 replication without affecting cell growth. To investigate the safety, persistence and efficacy of MazF-modified CD4(+) T cells in a nonhuman primate model in vivo, rhesus macaques were infected with a pathogenic simian/human immunodeficiency virus (SHIV) and transplanted with autologous MazF-modified CD4(+) T cells. MazF-modified CD4(+) T cells were clearly detected throughout the experimental period of more than 6 months. The CD4(+) T cell count values increased in all four rhesus macaques. Moreover, the transplantation of the MazF-modified CD4(+) T cells was not immunogenic, and did not elicit cellular or humoral immune responses. These data suggest that the autologous transplantation of MazF-modified CD4(+) T cells in the presence of SHIV is effective, safe and not immunogenic, indicating that this is an attractive strategy for HIV-1 gene therapy.
A bone marrow (BM) response induced by cardiopulmonary bypass (CPB) as a systemic inflammatory reaction has previously been postulated but not clarified. Newly released polymorphonuclear leukocytes (PMNs) and monocytes from the BM are known to be immature, indicating their greater potential to damage tissue. The present study aimed to examine the kinetics of BM-derived leukocytes associated with CPB in a nonhuman primate model.
Mutant forms of transthyretin (TTR) cause the most common type of autosomal-dominant hereditary systemic amyloidosis. In addition, wild-type TTR causes senile systemic amyloidosis, a sporadic disease seen in the elderly. Although spontaneous development of TTR amyloidosis had not been reported in animals other than humans, we recently determined that two aged vervet monkeys (Chlorocebus pygerythrus) spontaneously developed systemic TTR amyloidosis. In this study here, we first determined that aged vervet monkeys developed TTR amyloidosis and showed cardiac dysfunction but other primates did not. We also found that vervet monkeys had the TTR Ile122 allele, which is well known as a frequent mutation-causing human TTR amyloidosis. Furthermore, we generated recombinant monkey TTRs and determined that the vervet monkey TTR had lower tetrameric stability and formed more amyloid fibrils than did cynomolgus monkey TTR, which had the Val122 allele. We thus propose that the Ile122 allele has an important role in TTR amyloidosis in the aged vervet monkey and that this monkey can serve as a valid pathological model of the human disease. Finally, from the viewpoint of molecular evolution of TTR in primates, we determined that human TTR mutations causing the leptomeningeal phenotype of TTR amyloidosis tended to occur in amino acid residues that showed no diversity throughout primate evolution. Those findings may be valuable for understanding the genotype-phenotype correlation in this inherited human disease.
It has been shown that infection of GB virus B (GBV-B), which is closely related to hepatitis C virus, develops acute self-resolving hepatitis in tamarins. In this study we sought to examine longitudinally the dynamics of viral and immunological status following GBV-B infection of marmosets and tamarins. Surprisingly, two of four marmosets but not tamarins experimentally challenged with GBV-B developed long-term chronic infection with fluctuated viremia, recurrent increase of alanine aminotransferase and plateaued titers of the antiviral antibodies, which was comparable to chronic hepatitis C in humans. Moreover, one of the chronically infected marmosets developed an acute exacerbation of chronic hepatitis as revealed by biochemical, histological, and immunopathological analyses. Of note, periodical analyses of the viral genomes in these marmosets indicated frequent and selective non-synonymous mutations, suggesting efficient evasion of the virus from antiviral immune pressure. These results demonstrated for the first time that GBV-B could induce chronic hepatitis C-like disease in marmosets and that the outcome of the viral infection and disease progression may depend on the differences between species and individuals.
MazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys.
Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.
In this study, diffusion tensor MRI was used to examine the restoration of the cerebral white matter of macaque monkeys after unilateral cerebral multiple microinfarctions. Post-stroke, the monkeys showed deficits in several neurological functions, including motor functions, but most of the deficits resolved within 6 weeks. Very interestingly, the fractional anisotropy (a value determined by diffusion tensor MRI), of the monkeys affected motor pathways dropped transiently, indicating a damage in the neural tracts. However, it returned to normal levels within 6 weeks after the stroke, concomitant with the gradual recovery of motor functions at subacute phase.
Newborn neurons are continuously produced in the hippocampus, which may be involved in several cognitive functions, including learning and memory, throughout life. However, both hippocampus-dependent cognitive functions and the level of adult neurogenesis are gradually attenuated as aging progresses. Few studies have explored the relationship between adult neurogenesis and cognitive functions, especially in primates. In this study, we evaluated learning performance and hippocampal neurogenesis utilizing young and aged cynomolgus monkeys. Significant attenuations in learning performance and adult neurogenesis were detected in aged monkeys. Interestingly, there was a positive correlation between learning performance and the level of neurogenesis. Our findings suggest that cognitive functions and adult neurogenesis may have some interdependent relationships during aging.
Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation.
A phase 1 clinical trial evaluating the safety of gene therapy for patients with wet age-related macular degeneration (AMD) or retinoblastoma has been completed without problems. The efficacy of gene therapy for Lebers congenital amaurosis (LCA) was reported by three groups. Gene therapy may thus hold promise as a therapeutic method for the treatment of intractable ocular diseases. However, it will first be important to precisely evaluate the efficiency and safety of alternative gene transfer vectors in a preclinical study using large animals. In the present study, we evaluated the acute local (ophthalmic) and systemic toxicity of our simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vectors carrying human pigment epithelium-derived factor (SIV-hPEDF) for transferring genes into nonhuman primate retinas. Transient inflammation and elevation of intraocular pressure were observed in some animals, but these effects were not dose dependent. Electroretinograms (ERGs), including multifocal ERGs, revealed no remarkable change in retinal function. Histopathologically, SIV-hPEDF administration resulted in a certain degree of inflammatory reaction and no apparent structural destruction in retinal tissue. Regarding systemic toxicity, none of the animals died, and none showed any serious side effects during the experimental course. No vector leakage was detected in serum or urine samples. We thus propose that SIVagm-mediated stable gene transfer might be useful and safe for ocular gene transfer in a clinical setting.
Lacunar-type stroke accounts for approximately a quarter of all ischemic strokes, and is the most common cause of vascular dementia. Despite its importance, there are few specific treatments for lacunar stroke, probably due largely to a lack of animal models. In this study, we developed a stroke model in a higher primate, the Macaque monkey. This was achieved by occluding the deep subcortical penetrating arteries with agarose spheres of mean diameters around 50 microm, and the appropriateness of this model as a lacunar-type stroke was verified by MRI. We observed widespread gliosis in the ipsilateral white matter (WM) of the stroke monkey. We also analyzed the expression of neurotrophins in the activated glial cells, and found that their expression of BDNF was stimulated in the affected WM following ischemic injury. Our results support the idea that WM glial cells play an active role in protecting and promoting the regeneration of nerve fibers in the affected WM of the ischemic brain, by producing BDNF. These findings may be useful for the development of new therapeutic strategies aimed at preventing or treating stroke.
Abstract Gene therapy may hold promise as a therapeutic approach for the treatment of intractable ocular diseases, including retinitis pigmentosa (RP). Gene transfer vectors that are able to show long-lasting transgene expression in vivo are highly desirable to treat RP; however, there is a dearth of information regarding long-term transgene expression in the eyes of large animals. We previously reported that the simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vector showed efficient, stable, and safe retinal gene transfer, resulting in significant prevention of retinal degeneration by gene transfer of a neurotrophic factor, human pigment epithelium-derived factor (hPEDF), in rodents. Before applying this strategy in a clinical setting, we here assessed the long-lasting transgene expression of our third-generation SIVagm-based lentiviral vectors in the retinal tissue of nonhuman primates. Approximately 20-50 mul of SIV-EGFP (enhanced green fluorescent protein) or SIV-hPEDF was injected into the subretinal space via a glass capillary tube. To detect EGFP expression in the retina, we used a fluorescence fundus camera at various time points after gene transfer. Human PEDF expression was assessed by immunohistochemical analysis, Western blot assay, and enzyme-linked immunosorbent assay. The retinas demonstrated frequent EGFP expression that was preserved for at least 4 years without significant decline. The expression of hPEDF was stable, and occurred mainly in the retinal pigment epithelium. The secreted protein was detected in vitreous and aqueous humor. We thus propose that SIVagm-mediated stable gene transfer might be significantly useful for ocular gene transfer in a clinical setting.
In the dentate gyrus of the hippocampus, new neurons are generated from neural stem/progenitor cells (NPCs) throughout life. As aging progresses, the rate of neurogenesis decreases exponentially, which might be responsible, in part, for age-dependent cognitive decline in animals and humans. However, few studies have analyzed the alterations in NPCs during aging, especially in primates. Here, we labeled NPCs by triple immunostaining for FABP7, Sox2, and GFAP and found that their numbers decreased in aged macaque monkeys (>20 years old), but not in aged mice. Importantly, we observed marked morphological alterations of the NPCs in only the aged monkeys. In the aged monkey hippocampus, the processes of the NPCs were short and ran horizontally rather than vertically. Despite these alterations, the proliferation rate of the NPCs in aged monkeys was similar to that in young monkeys. Thus, morphological alterations do not affect the proliferation rate of NPCs, but may be involved in the maintenance of NPCs in aged primates, including elderly humans.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.