Efficacy of single intravenous injection of peramivir against influenza B virus infection in ferrets and cynomolgus macaques.

We evaluated the efficacy of a single intravenous dose peramivir for treatment of influenza B virus infection in ferrets and cynomolgus macaques in the present study. A single dose of peramivir (60 mg/kg of body weight) given to ferrets on 1 day postinfection with influenza B virus significantly reduced median area under the curve (AUC) virus titers (peramivir, 8.3 log(10) 50% tissue culture infective doses [TCID(50)s] · day/ml; control, 10.7 log(10) TCID(50)s · day/ml; P < 0.0001). Furthermore, nasal virus titers on day 2 postinfection in ferrets receiving a single injection of peramivir (30 mg/kg) and AUCs of the body temperature increase in ferrets receiving a single injection of peramivir (30 and 60 mg/kg) were lower than those in ferrets administered oral oseltamivir phosphate (30 and 60 mg/kg/day twice daily for 3 days). In macaques infected with influenza B virus, viral titers in the nasal swab fluid on days 2 and 3 postinfection and body temperature after a single injection of peramivir (30 mg/kg) were lower than those after oral administration of oseltamivir phosphate (30 mg/kg/day for 5 days). The two animal models used in the present study demonstrated that inhibition of viral replication at the early time point after infection was critical in reduction of AUCs of virus titers and interleukin-6 production, resulting in amelioration of symptoms. Our results shown in animal models suggest that the early treatment with a single intravenous injection of peramivir is clinically recommended to reduce symptoms effectively in influenza B virus infection.

Amelioration of pneumonia with Streptococcus pneumoniae infection by inoculation with a vaccine against highly pathogenic avian influenza virus in a non-human primate mixed infection model.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria. METHODS: H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles. RESULTS: Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae. Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant. CONCLUSIONS: Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.

MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque.

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as important animal models for biomedical research, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphism or diversity of the polygenetic Mafa class I A loci is limited in comparison to the more commonly studied rhesus macaque Mafa class I A loci. Therefore, in this paper, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-A1 among three native Southeast Asian populations (Indonesian, Vietnamese, and Filipino) and to investigate how the allele differences between macaques and humans might have evolved to affect their respective immune responses, we identified 83 Mafa-A loci-derived alleles by DNA sequencing of which 66 are newly described. Most alleles are unique to each population, but seven of the most frequent alleles were identical in sequence to some alleles in other macaque species. We also revealed (1) the large and dynamic genetic and structural differences and similarities in allelic variation by analyzing the population allele frequencies, Hardy-Weinberg's equilibrium, heterozygosity, nucleotide diversity profiles, and phylogeny, (2) the difference in genetic structure of populations by Wright's FST statistic and hierarchical analysis of molecular variance, and (3) the different demographic and selection pressures on the three populations by performing Tajima's D test of neutrality. The large level of diversity and polymorphism at the Mafa-A1 was less evident in the Filipino than in the Vietnam or the Indonesian populations, which may have important implications in animal capture, selection, and breeding for medical research.

Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells.

Conditions that influence the selective development or recruitment of connective tissue-type and mucosal-type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta-gonad-mesonephros-derived stromal cell line AGM-S1 differentiated into cobblestone (CS)-like cells by day 10-15. When replated onto fresh AGM-S1 with the addition of stem cell factor, interleukin-6, and Flt3 ligand, these CS-like cells displayed robust growth and generated almost 100% tryptase/chymase double-positive MCs within 3 weeks. At all time points, the percentage of tryptase-positive cells did not exceed that of chymase-positive cells. These ES-derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA-DR- and coexpressed a high-affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES-derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES-derived MCs have the phenotype of functionally mature connective tissue-type MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue-type MCs, which may be independent from the developmental pathway of mucosal-type MCs.

Cultivated corneal endothelial cell sheet transplantation in a primate model.

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis).

In nonhuman primates (NHPs), there have so far been few reports about nuclear transfer (NT), especially using adult somatic cells. The objective of this study was to determine the developmental competence of NT embryos derived from various somatic cells embryonic stem (ES), amniotic epithelial, cumulus, or fetal fibroblast cells] and the nuclear transfer method, such as electro fusion or piezo microinjection, activation with chemical reagent [ionomycine/6-dimethylaminopurine (DMAP), calcium ionophore A23187/DMAP, or cycloheximide (CHX)] and reprogramming time (1, 2, or 4 h; in this study, the duration from injection or fusion to activation was defined as the reprogramming time). Our results showed that a 1-h reprogramming and activation with ionomycin/DMAP are suitable for NT in monkeys. Developing cleaved embryos up to the six-cell stage was similar among all experiments. However, beyond the eight-cell stage, developmental rates were higher in NT embryos reconstructed with fetal fibroblast cells and amniotic epithelial cells, and we were able to produce NT blastocysts from these cells. Interestingly, electro fusion is sufficient for amniotic epithelial cells and piezo microinjection is better suited for fetal fibroblast cells to produce NT blastocysts, thus suggesting that the best method for somatic cell NT may be different between cell types.

Transient suppression of PPARgamma directed ES cells into an osteoblastic lineage.

Osteoblasts and adipocytes are believed to share a common progenitor. Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the switching of these two cell lineages. Here, we demonstrated the differentiation of ES cells into an osteoblastic lineage using siRNA against PPARgamma without the addition of any osteogenic factors. We found that PPARgamma-siRNA downregulated the expression of aP2 mRNA and lipid accumulation, whereas it upregulated the expression of osteocalcin and calcium deposition. These results suggested that ES cells were directed into an osteoblastic lineage. Therefore, transient suppression using PPARgamma-siRNA may be a novel tool to induce differentiation of ES cells into osteoblasts.

Monkey embryonic stem cells differentiate into adipocytes in vitro.

Production of functional adipocytes is important in adipocyte research and regenerative medicine. In this paper, we describe the differentiation of monkey embryonic stem (ES) cells into insulin-responsive adipocytes. Treatment of embryoid body (EB) outgrowth with adipogenic hormones induced the expression of adipocyte-specific genes, such as PPARgamma, C/EBPalpha, aP2, insulin receptor, and GLUT4. Expression of adipocytokines, leptin and adiponectin, was also detected. Furthermore, translocation of GLUT4 was observed by insulin stimulation in differentiated adipocytes. These results suggested that monkey ES cells can be a useful tool for studying adipogenesis in primate.

Molecular cloning of BNP from heart and its immunohistochemical localization in the hypothalamus of monkey.

Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus.

Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intra-cardially administered microglia in normal mouse brain.

Although the therapeutic use of microglia has received some attention for the treatment of brain diseases, few non-invasive techniques exist for monitoring the cells after administration. Here, we present a technique using magnetic resonance imaging (MRI) to track microglia injected intra-cardially. We labeled microglia expressing enhanced green fluorescent protein with superparamagnetic iron oxide (Resovist) using the hemagglutinating virus of Japan-envelope vector. We injected labeled microglia into the left ventricle of the heart of mice. After monitoring exogenously administered microglia in the mouse brain in vivo using T(2)*-weighted MRI at a magnetic field of 7T, we compared the MR images with histochemical localization of exogenous microglia in vitro. MRI revealed clear signal changes attributable to Resovist-containing microglia in the mouse brain. Histochemistry demonstrated the presence of exogenous microglia in the brain at the same locations shown by MRI. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for brain disease.

Generation of green fluorescent protein-expressing monkey embryonic stem cells.

Monkey embryonic stem (ES) cells are a useful tool for studying early human development and evaluating the efficacy of stem cell therapy. Monkey ES cells show closer similarity to human ES cells than their mouse counterparts regarding morphology, cell surface markers, and the maintenance of pluripotency, including the leukemia inhibitory factor requirement. The generation of genetically modified monkey ES cells with a biomarker such as green fluorescent protein, which allows noninvasive monitoring of progeny ES cells, is invaluable for the development of cell transplantation therapy and the study of differentiation mechanisms in primates. Here, we describe the generation of green fluorescent protein-expressing monkey ES cells using a conventional electroporation method.

Acidic fibroblast growth factor promotes hepatic differentiation of monkey embryonic stem cells.

Embryonic stem (ES) cells can replicate indefinitely and differentiate into all cell types, including hepatocytes. Research using primate ES cells is considered to be important for studies of potential cell therapies. Recently, we established cynomolgus monkey ES cells designated as CMK6. The CMK6 cell line is a useful tool for investigating the mechanism of differentiation in primate ES cells and developing cell therapies, because of its biological similarity to human ES cells. To examine whether cynomolgus monkey ES cells differentiate into hepatocytes, CMK6 cells were cultured with or without acidic fibroblast growth factor (aFGF). Evaluation of the hepatic differentiation was performed by analysis of the mRNA expression in early hepatic marker genes using the reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression of albumin (ALB) was also studied by immunocytochemistry. RT-PCR analyses revealed mRNA expressions of alpha-fetoprotein, transthyretin, and ALB in the presence of aFGF at 3 wk of differentiation, whereas no mRNA expression of these genes was detected in cells without aFGF. The protein expression of ALB in the presence of aFGF at 3 wk of differentiation was also confirmed by immunocytochemistry. However, tyrosine aminotransferase, which is a mature hepatic marker, was not detected in the presence or absence of aFGF at any stage of differentiation. These results suggested that aFGF successfully promoted in vitro differentiation of cynomolgus monkey ES cells to an early hepatic lineage.

Osteoblastic differentiation of monkey embryonic stem cells in vitro.

Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification.

Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells.

BACKGROUND: To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. METHODS: Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. RESULTS: A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (approximately 1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. CONCLUSIONS: Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they "leak" into an improper space such as the thoracic cavity.

Effect of ovarian hormones on periodical changes in immune resistance associated with estrous cycle in the beagle bitch.

In bitches, the onset of pyometra, an infection of the uterus, characteristically occurs in the first half of the diestrous stage in the estrous cycle, in which the blood concentration of progesterone peaks and that of estradiol-17beta is lowest. To investigate the immunological mechanisms governing stage-specific onset of pyometra, peripheral blood mononuclear cells (PBMNCs) were collected from beagle bitches during different stages of the estrous cycle and examined using various immunological assays. When we examined the proliferative response of PBMNCs to PYO-252, that is a clone of Escherichia coli isolated from the uterus of a dog afflicted with pyometra, the response of PBMNCs significantly decreased in the first half (day 10) of diestrus, but increased in proestrus/estrus. No significant differences were observed in the responses to concanavaline A between stages of the cycle. Throughout the estrous cycle, canine PBMNCs did not respond to lipopolysaccharide derived from E. coli. The response of PBMNCs collected in anestrus to PYO-252 was significantly enhanced upon the addition of estradiol-17beta to the culture. In contrast, these responses were significantly suppressed in the presence of progesterone. Progesterone progenitor or metabolite molecules, which have a low affinity for the progesterone receptor, did not affect proliferative responses. Expression of gamma interferon (IFNgamma) in response to PYO-252 was also significantly enhanced by estradiol-17beta, but suppressed by progesterone. This evidence suggests that in the first half of the diestrous stage, suppressed activity of cellular immunity results from increasing progesterone concentration and minimal estrogen release. This marked decrease of immune resistance allows the expansion of E. coli, which enter the uterine cavity through the loosened cervical canal during estrus, leading to pyometra onset.

Monkey embryonic stem cell lines expressing green fluorescent protein.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Expression of estrogen receptor alpha and beta genes in the mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle.

Estrogen receptor alpha (ERalpha) and ERbeta mRNA levels were measured in the mediobasal hypothalamus, the anterior pituitary and the ovary of beagle bitches at various stages of the estrous cycle. With polymerase chain reaction analysis we detected ERbeta gene transcripts in all tissue samples. The levels of hypothalamic and pituitary ERalpha and beta mRNAs increased from mid anestrus to proestrus and declined thereafter. In the ovary, ERalpha mRNA levels increased from proestrus to diestrus and were positively correlated with plasma progesterone levels (r=0.62, P<0.01), whereas ERbeta mRNA levels increased from mid anestrus to proestrus and were positively correlated with plasma estradiol-17beta levels (r=0.73, P<0.001). These results suggest that the rise in hypothalamic and pituitary ERalpha and beta mRNAs is associated with termination of anestrus, and that increases in ovarian ERalpha and beta mRNAs may be involved in initiating development of the follicle or corpora lutea.

Enhancement of aromatase gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

The relationships among expression of cytochrome p450 aromatase (p450arom) mRNA in the mediobasal hypothalamus (MBH), ovarian aromatase activity, and estrogen secretion were examined throughout the estrous cycle in beagle bitches. Using polymerase chain reaction (PCR) analysis we were able to detect p450arom gene transcripts in the canine MBH. The level of hypothalamic p450arom mRNA increased during the progression of anestrus and declined thereafter. Ovarian p450arom activity, as measured by a (3)H2O assay, were low in anestrus, increased in proestrus, and declined thereafter. Ovarian p450arom activity and plasma estradiol-17beta levels were positively correlated (r=0.77, P<0.05). These results suggest that enhancement of hypothalamic p450arom gene expression is associated with termination of anestrus.

Clobazam shows a different antiepileptic action profile from clonazepam and zonisamide in Ihara epileptic rats.

PURPOSE: Clobazam (CLB, 1,5-benzodiazepine, 1,5-BZP) has been reported to show unique antiepileptic action profile distinguishing from standard 1,4-BZPs. To further elucidate the action profile of CLB, its effects on the abnormal circling fits (ACFs) and generalized tonic-clonic convulsions (GTCs) in Ihara epileptic rats (IERs), a genetically epileptic mutant, were examined in comparison with conventional antiepileptic drugs (AEDs), a 1,4-BZP, clonazepam (CZP) and a non-BZP, zonisamide (ZNS). METHODS: The incidence of ACFs or GTCs in IERs was recorded automatically by the computer-assisted behavior monitoring system (COBAS N-IV) before, during and after the drug treatment period for 5 days in each. The drugs were orally administered twice daily. The daily and total incidences of ACFs or GTCs were calculated every each period in each dose group. The incidences of various behaviors such as feeding, gnawing and scratching recorded simultaneously were used for evaluating the behavioral activity (BA). RESULTS: CLB (30 and 60 mg/kg) prevented the appearance of ACFs and GTCs without affecting BAs. CZP (1 and 3 mg/kg) suppressed the occurrence of ACFs but induced no effect on the incidence of GTCs. Furthermore, it inhibited BAs at the same doses. ZNS (15 mg/kg) suppressed GTCs but little ACFs without affecting BA. CONCLUSION: CLB exhibited a different action profile from CZP and ZNS in a novel epileptic mutant, IERs, and was expected to be a useful AED superior to 1,4-BZPs in clinical practice.

Detection of a unique genotype of monkey B virus (Cercopithecine herpesvirus 1) indigenous to native Japanese macaques (Macaca fuscata).

The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting the co-evolution of BV and macaques.