Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts.     

Development of an IgG-Fc fusion COVID-19 subunit vaccine,AKS-452

AKS-452 is a biologically-engineered vaccine comprising an Fc fusion protein of the SARS-CoV-2 viral spike protein receptor binding domain antigen (Ag) and human IgG1 Fc (SP/RBD-Fc) in clinical development for the induction and augmentation of neutralizing IgG titers against SARS-CoV-2 viral infection to address the COVID-19 pandemic. The Fc moiety is designed to enhance immunogenicity by increasing uptake via Fc-receptors (FcγR) on Ag-presenting cells (APCs) and prolonging exposure due to neonatal Fc receptor (FcRn) recycling. AKS-452 induced approximately 20-fold greater neutralizing IgG titers in mice relative to those induced by SP/RBD without the Fc moiety and induced comparable long-term neutralizing titers with a single dose vs. two doses. To further enhance immunogenicity, AKS-452 was evaluated in formulations containing a panel of adjuvants in which the water-in-oil adjuvant, Montanide™ ISA 720, enhanced neutralizing IgG titers by approximately 7-fold after one and two doses in mice, including the neutralization of live SARS-CoV-2 virus infection of VERO-E6 cells. Furthermore, ISA 720-adjuvanted AKS-452 was immunogenic in rabbits and non-human primates (NHPs) and protected from infection and clinical symptoms with live SARS-CoV-2 virus in NHPs (USA-WA1/2020 viral strain) and the K18 human ACE2-trangenic (K18-huACE2-Tg) mouse (South African B.1.351 viral variant). These preclinical studies support the initiation of Phase I clinical studies with adjuvanted AKS-452 with the expectation that this room-temperature stable, Fc-fusion subunit vaccine can be rapidly and inexpensively manufactured to provide billions of doses per year especially in regions where the cold-chain is difficult to maintain.     

Decreased Bone Mass and Strength in Ovariectomized Cynomolgus Monkeys (Macaca fascicularis)

Agents for prevention or treatment of osteoporosis must now be tested in a large animal species that exhibits bone remodeling. Ovariectomized, nonhuman primates provide one such model, and they consistently develop osteopenia accompanied by high bone turnover rates. The goal of this study was to further characterize this model, and particularly to determine the effect of ovariectomy on bone strength in vertebrae and femoral necks. Longitudinal evaluations of spinal bone mass and serum markers of bone turnover were performed in 19 sham-ovariectomized (SHAM) and 18 ovariectomized (OVX), domestically reared cynomolgus monkeys, aged >9 years. OVX monkeys lost bone relative to both baseline values and SHAM controls. Serum markers of bone turnover were increased by OVX. After 72 weeks, both vertebral bone compressive strength and femoral neck breaking strength were significantly decreased in OVX animals compared with SHAM. Ovariectomized cynomolgus monkeys, like postmenopausal women, develop accelerated bone loss, increased bone turnover, and reduced bone strength, and provide a suitable large animal model for efficacy studies with agents for prevention or treatment of osteoporosis. Received: 24 June 1996 / Accepted: 3 September 1996     

Varicella in a gorilla

Naturally occurring varicella was observed in a young gorilla in captivity. The isolate was demonstrated to be varicella-zoster virus by restriction enzyme analysis.     

Abrogation of IL-3 Requirements and Stimulation of Hematopoietic Cell Proliferationin Vitroandin Vivoby Carboxylic Acids

ABSTRACT: Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the β-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced γ globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observedin vivoin mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce γ globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.     

GABAergic elements in the neuronal circuits of the monkey neostriatum: a light and electron microscopic immunocytochemical study

An antibody raised in rabbits against a GABA-BSA conjugate was used together with the PAP technique to label elements in the neostriatum of three Old World monkeys. Light microscopy revealed numerous immunoreactive medium-size neurons of various staining intensities, some of which had indented nuclei, as well as an occasional large cell. The neuropil showed a plexus of fine processes with frequent puncta. Ultrastructurally, the medium-size GABA-positive neurons were of two types: one with smooth nuclei and scanty cytoplasm, similar to spiny I cells, the other with invaginated nuclear envelopes and more abundant perikaryon, resembling the aspiny type. Correspondingly, labeled dendrites were either spiny or varicose. Some stained axons were myelinated, and the boutons had either large and ovoid, or small and pleomorphic vesicles. All of these boutons formed symmetric synapses, the former type with GABA-positive dendritic shafts but also with unlabeled dendrites; the latter type usually with GABA-negative elements, either dendrites, dendritic spines, or somata. Synapses were also observed between unreactive boutons and immunostained dendrites. Terminals with densely packed, small round vesicles that established asymmetric synapses with spines were always GABA-negative. Glial elements were consistently unlabeled, save for some astroglial endfeet. These findings provide positive evidence for the existence of two classes of GABAergic striatal neurons corresponding to a long-axoned spiny I type and an aspiny interneuron. Furthermore, the simultaneous labeling of GABA-immunoreactive presynaptic and postsynaptic profiles offers possible morphologic bases for the various kinds of intrastriatal inhibitory processes, including the feedforward, feedback, and "autaptic" types.     

Antiarrhythmic actions of tedisamil: Studies in rats and primates

Tedisamil is a new bradycardic agent, previously shown to block transient outward and delayed rectifier potassium currents in cardiac tissue [1,2]. In the present study tedisamil caused bradycardia and Q-Tc widening in rats and primates. Q-Tc widening is indicative of class III antiarrhythmic actions. In keeping with this, tedisamil had antiarrhythmic activity against electrical and ischemia-induced arrhythmias in rats. In rats, 0.5–4 mg/kg IV tedisamil caused parallel and dose-related increases in action-potential duration, Q-Tc interval, and refractory period; and decreases in maximum ventricular following frequency. In primates after 0.5–2.0 mg/kg IV, findings were similar for indices of Q-T widening and decreases in maximum ventricular following frequency. Tedisamil did not change QRS width, nor did it increase threshold currents for capture of ventricles, nor for fibrillo-flutter at doses below 4 mg/kg in rats. These findings were consistent with the lack of significant sodium-channel blockade. However, upon increasing the dose to 4 mg/kg, ventricular fibrillo-flutter could not be induced in rats by electrical stimulation; instead, only ventricular tachycardias with slow rates occurred. Ischemia-induced ventricular fibrillation was reduced in a dose-related manner by tedisamil in rats. The overall incidence of ischemia-induced ventricular tachycardia was not markedly reduced, but rates during tachycardic episodes were lower. When pacing was used to overcome tedisamil-induced bradycardia, antiarrhythmic actions during ischemia were more pronounced. These findings are consistent with the hypothesis that tedisamil increased refractoriness, which resulted in extended path lengths for reentry circuits and slower rates during episodes of ventricular tachycardia. High doses of tedisamil increased path lengths so much that the multiple reentry circuits of fibrillation could no longer occur. The limited study in primates suggests similar mechanisms could occur in humans.     

The origin of human chromosome 2 analyzed by comparative chromosome mapping with a DNA microlibrary

Fluorescencein situ hybridization (FISH) of microlibraries established from distinct chromosome subregions can test the evolutionary conservation of chromosome bands as well as chromosomal rearrangements that occurred during primate evolution and will help to clarify phylogenetic relationships. We used a DNA library established by microdissection and microcloning from the entire long arm of human chromosome 2 for fluorescencein situ hybridization and comparative mapping of the chromosomes of human, great apes (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus) and Old World monkeys (Macaca fuscata andCercopithecus aethiops). Inversions were found in the pericentric region of the primate chromosome 2p homologs in great apes, and the hybridization pattern demonstrates the known phylogenetically derived telomere fusion in the line that leads to human chromosome 2. The hybridization of the 2q microlibrary to chromosomes of Old World monkeys gave a different pattern from that in the gorilla and the orang-utan, but a pattern similar to that of chimpanzees. This suggests convergence of chromosomal rearrangements in different phylogenetic lines.     

Human chromosome 16 conservation in primates

A study was made of the organization of the chromosome orthologous to HSA16 in primates using a panel of 8 BAC probes spanning human chromosome 16. The probes were used in FISH experiments on great apes and on representatives of the Old World monkeys, New World monkeys, and lemurs. The domestic cat was used as an outgroup. The results indicate that 16p and 16q were separate chromosomes in a primate ancestor. They fused in a Catarrhini ancestor giving rise to the present day form found in HSA, great apes, and Old World monkeys. Several rearrangements were found in New World monkeys.     

Memory Deficits in Aged Cebus Monkeys and Facilitation With Central Cholinomimetics

Cebus monkeys of 3 different age groups were trained to perform an automated behavioral task (delayed response), intended to measure recent memory ability. In in initial study, the aged monkeys (18 years and older) exhibit prprogressively greater performance impairments (relative to young monkeys) as they were required to remember the location of a visual stimulus for increasingly longer durations (0 to 20 sec). This deficits replicated previously published results from aged Rhesus monkeys and appeared similar to the primary memory deficits reported in elderly humans and demented patients. In subsequent studies, the effects of three different cholinomimetics were evaluated for their ability to improve the aged monkey's performance on this task. Each monkey was tested under several acute doses of the cholinergic precursor, choline, the anticholinesterase, physostigmine, and the cholinergic muscarinic receptor agonist, arecoline. The results revealed clear differences in the ability of these drugs to improve performance on this task. Choline exerted no apparent effects in the aged monkeys at any dose tested. Physostigmine clearly enhanced performance in certain aged monkeys, but the optimal dose varied dramatically between subjects, replicating previously published results with aged Rhesus monkeys and humans. Arecoline produced clear improvement within a restricted dose range, with little variation in optimal dose between subjects. In addition to demonstrating differences in the effects of different cholinomimetics on memory performance in aged primates, these data also suggest a possible rationale for future investigations. Assuming that each of these drugs primarily affected cholinergic function in the manner conventionally attributed, these data suggest that, within the cholinergic system, the more directly one stimulates the receptor, the more one might expect robust and consistent effects on memory performance in aged subjects.