Search

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that lack therapeutic solutions. Here, we show that the molecular chaperone (N,N'-([cyclohexylmethylene]di-4,1-phenylene)bis(2-[1-pyrrolidinyl]acetamide)), designed via docking simulations, molecular dynamics simulations and quantum chemical calculations, slows down the progress of TSEs. In vitro, the designer molecular chaperone stabilizes the normal cellular prion protein, eradicates prions in infected cells, prevents the formation of drug-resistant strains and directly inhibits the interaction between prions and abnormal aggregates, as shown via real-time quaking-induced conversion and in vitro conversion NMR. Weekly intraperitoneal injection of the chaperone in prion-infected mice prolonged their survival, and weekly intravenous administration of the compound in macaques infected with bovine TSE slowed down the development of neurological and psychological symptoms and reduced the concentration of disease-associated biomarkers in the animals' cerebrospinal fluid. The de novo rational design of chaperone compounds could lead to therapeutics that can bind to different prion protein strains to ameliorate the pathology of TSEs.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)–derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

It remains unclear whether therapeutic drug monitoring (TDM) of pazopanib improves treatment outcomes in routine clinical practice. We did a prospective cohort study to evaluate the benefits of TDM for pazopanib therapy in real-world practice. Among 25 patients with pharmacokinetically guided dosing, only 5 (20%, 95% confidence interval 6.8-40.7%) discontinued treatment because of adverse events. However, 5 (41.7%, 95% confidence interval 15.2-72.3%) of historical controls including 12 patients not receiving such a strategy experienced adverse events leading to early termination. PK-guided dosing significantly increased median time-to-treatment discontinuation (252 vs 74 days, P = .012) with reduced toxicity and improved overall survival (not reached vs 313 days, P = .002) relative to conventional dosing in the control group. In conclusion, PK-guided dose adaptation through the use of TDM has the potential to improve treatment outcomes of pazopanib in routine clinical practice, warranting larger, randomized studies.

Macaca fascicularis is a highly advantageous model in which to study human cochlea with regard to both evolutionary proximity and physiological similarity of the auditory system. To better understand the properties of primate cochlear function, we analyzed the genes predominantly expressed in M. fascicularis cochlea. We compared the cochlear transcripts obtained from an adult male M. fascicularis by macaque and human GeneChip microarrays with those in multiple macaque and human tissues or cells and identified 344 genes with expression levels more than 2-fold greater than in the other tissues. These “cochlear signature genes” included 35 genes responsible for syndromic or nonsyndromic hereditary hearing loss. Gene set enrichment analysis revealed groups of genes categorized as “ear development” and “ear morphogenesis” in the top 20 gene ontology categories in the macaque and human arrays, respectively. This dataset will facilitate both the study of genes that contribute to primate cochlear function and provide insight to discover novel genes associated with hereditary hearing loss that have yet to be established using animal models.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Although the number of reports describing tumors in aged NHP has increased, spontaneous neoplasias in NHP are extremely rare, with the notable exception of prosimians, in which spontaneous hepatic neoplasms arise. In addition to radiography and ultrasonography, superparamagnetic iron oxide (SPIO)-enhanced MRI tends to be applied in human practice to non-invasively locate, identify, and size liver tumors and to define the border between neoplastic and normal tissues. Here we report a 13-y-old female cynomolgus monkey with anorexia and serologically normal liver enzymes. After fluid therapy, the condition remained in remission for several months. Later, however, a palpable mass was assessed by using ultrasonography, radiology, and SPIO-MRI; T2-weighted images revealed a clear border between a hepatocellular carcinoma and normal liver tissue. Findings at necropsy supported the imaging data. Serologic assessment after euthanasia revealed a positive reaction to an abnormal form of prothrombin (PIVKA-II). We recommend SPIO-MRI as a practical and useful for diagnosing hepatocellular neoplasias in NHP. This study is the first to demonstrate the applicability of SPIO-MRI for the identification of hepatocellular carcinoma in NHP.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger's disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

Although the number of reports describing tumors in aged NHP has increased, spontaneous neoplasias in NHP are extremely rare, with the notable exception of prosimians, in which spontaneous hepatic neoplasms arise. In addition to radiography and ultrasonography, superparamagnetic iron oxide (SPIO)-enhanced MRI tends to be applied in human practice to non-invasively locate, identify, and size liver tumors and to define the border between neoplastic and normal tissues. Here we report a 13-y-old female cynomolgus monkey with anorexia and serologically normal liver enzymes. After fluid therapy, the condition remained in remission for several months. Later, however, a palpable mass was assessed by using ultrasonography, radiology, and SPIO-MRI; T2-weighted images revealed a clear border between a hepatocellular carcinoma and normal liver tissue. Findings at necropsy supported the imaging data. Serologic assessment after euthanasia revealed a positive reaction to an abnormal form of prothrombin (PIVKA-II). We recommend SPIO-MRI as a practical and useful for diagnosing hepatocellular neoplasias in NHP. This study is the first to demonstrate the applicability of SPIO-MRI for the identification of hepatocellular carcinoma in NHP.

We investigate the laser-induced spin precession dynamics in ferromagnetic $\mathrm{N}{\mathrm{i}}_{0.8}\mathrm{F}{\mathrm{e}}_{0.2}$ thin films by using a time-resolved magneto-optical Kerr effect (TR-MOKE) measurement with two pump pulses. By changing the time interval between the pump pulses, the amplitude of the TR-MOKE signal is strongly modulated according to the phase of the TR-MOKE signal at the time when the second pulse arrives. The change in the oscillation amplitude can be explained with the temporal evolution of the spin precession induced by laser heating, as shown with a numerical simulation. We conclude that the ultrafast control of coherent spin precession dynamics is possible by multiple pump pulses via thermal excitation.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPScin macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt–Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method forin vitroamplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPScby protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPCsubstrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSccontained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPScin the CSF by serial PMCA, and the CSF levels of PrPSctended to increase with disease progression. In addition, PrPScwas detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPScin non-human primate models of CJD.

Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

Aim. When determining therapeutic strategy, it is important to diagnose small intestinal lesions in Crohn's disease (CD) precisely and to evaluate mucosal healing as well as clinical remission in CD. The purpose of this study was to compare findings from computed tomographic enteroclysis/enterography (CTE) with those from the mucosal surface and to determine whether the state of mucosal healing can be determined by CTE.Materials and Methods. Of the patients who underwent CTE for CD, 39 patients were examined whose mucosal findings could be confirmed by colonoscopy, capsule endoscopy, balloon endoscopy, or with the resected surgical specimens.Results. According to the CTE findings, patients were determined to be in the active CD group (n=31) or inactive CD group (n=8). The proportion of previous surgery, clinical remission, stenosis, and CDAI score all showed significant difference between groups. Mucosal findings showed an association with ulcer in 93.6% of active group patients but in only 12.5% of inactive group patients (P<0.0001), whereas mucosal healing was found in 62.5% of inactive group patients but in only 3.2% of active group patients (P<0.0001).Conclusion. CTE appeared to be a useful diagnostic method for assessment of mucosal healing in Crohn's disease.

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger’s disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

Protease-resistant, misfolded isoforms (PrP(Sc)) of a normal cellular prion protein (PrP(C)) in the bodily fluids, including blood, urine, and saliva, are expected to be useful diagnostic markers of prion diseases, and nonhuman primate models are suited for performing valid diagnostic tests for human Creutzfeldt-Jakob disease (CJD). We developed an effective amplification method for PrP(Sc) derived from macaques infected with the atypical L-type bovine spongiform encephalopathy (L-BSE) prion by using mouse brain homogenate as a substrate in the presence of polyanions and L-arginine ethylester. This method was highly sensitive and detected PrP(Sc) in infected brain homogenate diluted up to 10(10) by sequential amplification. This method in combination with PrP(Sc) precipitation by sodium phosphotungstic acid is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF), saliva, urine, and plasma of macaques that have been intracerebrally inoculated with the L-BSE prion. Furthermore, PrP(Sc) was detectable in the saliva or urine samples as well as CSF samples obtained at the preclinical phases of the disease. Thus, our novel method may be useful for furthering the understanding of bodily fluid leakage of PrP(Sc) in nonhuman primate models.

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.

CT enteroclysis（CTE）とは，水や等張性緩下剤等（Niflec®，Elental®等）の陰性造影剤を小腸チューブから注入し，全小腸を十分に拡張させた後Dynamic CTを撮影して小腸病変の診断を行う検査法である．海外ではクローン病に対する報告が多数なされており，腸管の炎症，狭窄や瘻孔に対して高い確率での検出が可能とされている．症例は30歳代男性．2006年に小腸大腸型クローン病と診断され，5－アミノサリチルサン（5-ASA）製剤内服と成分栄養剤で加療されていた．2008年，臀部痛が出現し，近医の造影CTで仙骨前面に膿瘍を認めたが保存的加療にて症状は軽快し，以後の受診歴はない．2009年4月，鮮血便を頻回に認め，上部・下部消化管内視鏡検査では出血源が同定されなかったことから小腸出血が疑われた．出血は絶食のみで自然止血したが，出血源の同定目的で当院へ転院となった．陰性造影剤としてNiflec®を用いてCTEを施行したところ，回腸の壁肥厚，狭窄および直腸との内瘻の形成が疑われ手術適応と判断し，当院外科で手術が行われた．術中所見でCTEにて検出された回腸直腸瘻が実際に確認された．従来クローン病における内瘻の診断には小腸X線造影検査が用いられてきたが，瘻孔を明確に描出するのは難しく，術中所見ではじめて瘻孔形成部位が確定することも少なくなかった．本症例では内瘻の存在および局在の同定にCTEが安全に実施でき，手術適応を判断する上で有用であったことから，文献的考察を加えて報告する．

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using 18F-labeled BoHc/A–positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [18F] radioactivity or the traditional [111In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of 18F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.

The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi- scid, IL-2Rγnull (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.

Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.

Sendai virus (SeV) vectors can introduce foreign genes efficiently and stably into primate embryonic stem (ES) cells. For the application of these cells, the control of transgene expression is important. Cynomolgus ES cells transduced with a SeV vector expressing the green fluorescent protein (GFP) gene were propagated in Knockout serum replacement (KSR)-supplemented medium, used widely for the serum-free culture of ES cells, and growth and transgene expression were evaluated. The SeV vector-mediated GFP expression was suppressed in the KSR-supplemented medium, although it was stable in regular fetal bovine serum (FBS)-supplemented medium. Propagation in the KSR-supplemented medium eventually resulted in a complete suppression of GFP expression and eradication of the SeV genome. The inhibitory effect of KSR on the transduction was attributable to the positive selection of untransduced ES cells in addition to the removal of the SeV vector from transduced cells. KSR also reduced the efficiency of the transduction. SeV vector-mediated transgene expression in ES cells was suppressed in the KSR-supplemented medium. Although the suppression is limited in specified cells such as ES cells, these findings will help elucidate how to control transgene expression.

A patency capsule (PC) is used to safely perform capsule endoscopy. When the PC is not excreted within the defined time frame, radiography often cannot localize the PC. Computed tomography (CT) localizes a PC more definitively than radiography. We evaluated the localization of PCs using low-dose (LD)-CT.Forty-nine patients received a PC and 33 did not excrete the PC within the defined time frame and underwent radiography and LDCT with a 90% exposure reduction.LDCT localized the PC in 31 patients (93.9%), whereas radiography localized it in 7 (21.2%), indicating a significantly higher detection rate with LDCT (P0.0001). PC retention in the small intestine was confirmed by LDCT in 4 patients. Retention was not observed during capsule endoscopy in the patients with confirmed patency of the gastrointestinal tract. In 21 patients who underwent LDCT with the conventional photographing area, the effective radiographic exposure dose associated with LDCT was 1.43±1.08 mSv. Conversely, in the 12 patients who underwent LDCT with a reduced photographing area, the effective dose was reduced to 0.62±0.27 mSv (P0.05).LDCT with a reduced exposure dose can definitively localize a PC. Therefore, this method may allow capsule endoscopy to be performed for more diseases.

AIM: To investigate the utility of photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) to detect gastric/colorectal tumors. METHODS: This prospective single-center study investigated inter-subject variability in patients with early-stage gastric/colorectal tumor indicated for endoscopic resection. Subjects were patients with gastric or colorectal tumors who had undergone endoscopic resection between November 2012 and November 2013. Selection criteria included age 20-80 years, either sex, and provision of informed consent. Patients were orally administered 20 mg/kg of 5-ALA enteric-coated capsules (SBI ALApromo Co., Tokyo, Japan). Administration of 5-ALA was followed by endoscopic resection of gastric or colorectal tumors, and the resected specimens were examined using a video autofluorescence processor and a fluorescence endoscope (SAFE-3000 and EB-1970AK, respectively; Pentax, Tokyo, Japan). The primary endpoint was the presence of fluorescence in tumors. Endoscopic, macroscopic, and histopathologic findings of tumors were assessed. We also evaluated adverse events of the present procedure as a secondary endpoint and examined each patient for the presence of known adverse effects of 5-ALA, namely, hematocytopenia, liver dysfunction, hypotension, nausea, and photosensitivity. RESULTS: We enrolled 10 patients (7 men, 3 women) (n = 13 lesions: 10 gastric/3 colorectal tumors). Fluorescence was detected in 7/13 (53.8%) lesions. No significant differences in sex (male: 55.6% vs female: 50.5%, P = 1.00), age (67.1 ± 1.9 years vs 65.0 ± 2.0 years, P = 0.45), tumor color (reddish: 60.0% vs discolored: 33.3%, P = 0.56), tumor diameter (15.0 ± 2.1 mm vs 14.2 ± 2.3 mm, P = 0.80), macroscopic type (protruded: 70.0% vs depressed 0%, P = 0.07), histologic type (differentiated type: 58.3% vs 0%, P = 0.46), invasion depth (mucosal layer: 55.6% vs submucosal layer: 33.3%, P = 1.00), lymphatic invasion (present: 33.3% vs absent: 50.0%, P = 1.00), venous invasion (present: 0% vs absent: 54.5%, P = 1.00) or procedure time of endoscopic resection (36.3 ± 8.3 min vs 36.7 ± 9.0 min, P = 0.98) were observed between the patients with and without fluorescence. Fluorescence detection rate tended to be high for elevated lesions. Liver dysfunction developed in 4/10 (40.0%) patients. The extent of the liver dysfunction was a slight increase in transaminases and total bilirubin levels, which spontaneously improved in the patients. None of the patients developed photosensitivity. CONCLUSION: Results of this preliminary study suggest the utility of PDD using 5-ALA for screening of gastric and colorectal cancers.

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.

Background. Although directed differentiation of human embryonic stem (ES) cells would enable a ready supply of cells and tissues required for transplantation therapy, the methodology is limited. We have developed a novel method for hematopoietic development from primate ES cells. We first cultured cynomolgus monkey ES cells in vitro and transplanted the cells in vivo into fetal sheep liver, generating sheep with cynomolgus hematopoiesis. Methods. Cynomolgus ES cells were induced to mesodermal cells on murine stromal OP9 cells with multiple cytokines for 6 days. The cells (average 4.8×10 7 cells) were transplanted into fetal sheep in the liver (n=4) after the first trimester (day 55-73, full term 147 days). The animals were delivered at full term, and two of them were intraperitoneally administered with human stem-cell factor (SCF). Results. Cynomolgus hematopoietic progenitor cells were detected in bone marrow at a level of 1% to 2% in all four sheep up to 17 months posttransplant. No teratoma was found in the lambs. After SCF administration, the fractions of cynomolgus hematopoiesis increased by several-fold (up to 13%). Cynomolgus cells were also detected in the circulation, albeit at low levels (

Hematopoietic stem cells in bone marrow can be mobilized into peripheral blood by cytokine administration. Cytokine-mobilized peripheral blood stem cells are of great use in clinical applications. We previously established a modified procedure for the collection of cytokine-mobilized peripheral blood cells from rhesus monkeys (Macaca mulata) using a commercially available apparatus originally developed for human subjects. In this study, we examined the efficacy and safety of this method with even smaller macaques, cynomolgus monkeys (Macaca fascicularis), which are equivalent to human newborns in body weight (mean = 3.3 kg). Using the manufacturer's unmodified protocol (n=6), one monkey died of cardiac failure and three developed severe anemia. In contrast, using our modified procedure (n=6), no such complication was observed in any animal. In addition, the harvested nuclear cell, mononuclear cell and CD34(+) cell counts were significantly higher with the modified method. The modified method should allow safe and efficient collection of cytokine-mobilized peripheral blood cells from non-human primates as small as human newborns in a non-invasive manner.

The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2–30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (

It has recently been reported that bone marrow cells can efficiently engraft without marrow conditioning when implanted directly into the bone marrow cavity (intra-bone marrow transplantation, iBMT) in mice. We have successfully examined the efficacy of autologous iBMT in a cynomolgus monkey model in conjuction with an in vivo expansion of transplanted cells by a selective amplifier transgene (Ueda et al., 2004) and provide here the detailed parameters of our iBMT method. We injected retrovirally-marked autologous CD34+ cells directly into the non-conditioned marrow cavity of the femur and humerus after gently irrigating the cavity with saline. This transplant procedure was safely performed without pulmonary embolism. Gene-marked cells were not detectable in the peripheral blood at one hour and one day after iBMT as assessed by sensitive PCR, indicating that iBMT hardly generated a systemic delivery of transplanted cells. On the other hand, 2 to 30% of clonogenic hematopoietic colonies produced from the implanted marrow were gene-marked at 6–12 months after iBMT. Our iBMT method for non-human primates is thus discussed in terms of long-lived hematopoietic stem/progenitor cells, bone marrow niche and long-term engraftment after iBMT without myeloablative conditioning.

We evaluated 9.4-T magnetic resonance (MR) microimaging in assessing normal and defective bone development in mouse embryos. For this purpose, we performed 9.4-T MR microimaging on developing bones in normal embryos, and also in Runx2/Cbfa1-/- embryos with severely defective bone development. MR images were compared with the histological and histochemical features of these fetal bones. MR microimaging delineate successfully the normal long bone development in embryos. The T1- and T2-weighted MR microimaging demonstrated chondrocyte maturation in different regions of growing cartilage, such as epiphysis, physis, hypertrophic cartilage, and zone of provisional calcification. These developmental changes were detectable in as early as E14.5 embryos. The MR microimaging clearly demonstrated defective bone development in Runx2/Cbfa1-/- embryos. The femur from E18.5 homozygous Runx2/Cbfa1-/- embryos lacked MR signal intensity patterns including the hypertrophic cartilage, which are characteristic of the bone from the age-matched Runx2/Cbfa1+/+ embryos. Interestingly, however, the tibia from the same mutants was associated with MR signal patterns indicative of hypertrophic cartilage but not of the primary spongiosa and ossifying perichondrium, suggesting that bone development is differently regulated in these two long bones. On the other hand, the bones from heterozygous Runx2/Cbfa1+/- embryos exhibited an MR phenotype intermediate between the Runx2/Cbfa1+/+ and Runx2/Cbfa1-/- embryos; the primary spongiosa and ossifying perichondrium formation occurred normally even in the absence of preceding organized maturation of chondrocytes, a phenotype that was not detected by histological examinations. We concluded that MR microimaging is useful in assessing the bone development.

Fas ligand (FasL) cDNAs were cloned and sequenced from cynomolgus, rhesus, and pig-tailed monkeys. The 840-bp cDNAs were identical among these three species of monkeys except for one nucleotide. The deduced 280 amino acids were completely identical and displayed 97% homology with human FasL (hFasL). Recombinant soluble FasL obtained from COS cells transfected with cynomolgus monkey FasL (cm-FasL) cDNA induced apoptosis in cells displaying human or cynomolgus monkey Fas-expressing cells. Several anti-human FasL monoclonal antibodies (mAbs) were able to neutralize the cytotoxic activity of monkey FasL, and a combination of mAbs was selected to obtain the most sensitive detection of monkey soluble FasL (sFasL) under sandwich enzyme-linked immunosorbent assay (ELISA). Plasma from normal monkey did not contain detectable levels of sFasL, whereas plasma from monkeys acutely infected with simian immunodeficiency virus (SIV) displayed increased levels of sFasL.

We previously developed “selective amplifier genes (SAGs)” which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34+ cells than the GCR-type SAG. One mutant Mpl-type SAG (ΔGCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Background The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning. Methods Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34+ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells. Results Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes. Conclusions Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p

In order to establish the optimum fueling in a natural gas fueled dual fuel engine, tests were made for some operational parameters and their combination on the engine performances and the exhaust emissions. The results show that the gas oil quantity should be increased and gas oil injection timing should be advanced to suppress unburned hydrocarbon emission at middle and low output range, while the quantity should be reduced and the timing should be retarded to avoid onset of knock at high loads. The unburned hydrocarbon emission and the thermal efficiency are improved at the same load avoiding too lean natural gas premixture by restriction of intake charge air. However the improvement is limited because the ignition and initial combustion of pilot diesel fuel is deteriorated when the cylinder pressure is excessively lowered by throttling. The increase in pilot gas oil amount is effective for low-load operation and the adequate combination of throttle control and equivalence ratio ensures low hydrocarbon emission and the thermal efficiency comparable to diesel operation.

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.

The quantitative and phenotypic changes of peripheral blood monocytes during the acute stage of simian immunodeficiency virus infection were investigated. We inoculated intravenously three cynomolgus monkeys (Macaca fascicularis) with 100 TCID50 of SIVmac239 and collected whole blood twice a week until 35 days postinoculation. We found that the relative number of monocytes in peripheral blood leukocytes significantly increased at 7-17 days postinoculation. This increase was concomitant with the peak of primary SIV antigenemia. To determine if the monocytes observed during the acute stage were phenotypically altered, they were periodically examined for the expression of surface markers (i.e., CD11b, CD14, CD16, CD29, D32, CD56, CD62L, CD64, CD80, and MHC-II-DR) by flow cytometry. The results showed that the expression levels of CD14 and CD56 on most of the monocytes were remarkably reduced at 7-17 days postinoculation, and a new subpopulation, CD14lowCD16+CD80+ monocytes, was clearly detected at 10 days postinoculation. These results indicate that the phenotypic alteration of peripheral blood monocytes occurs during the primary SIV infection.

We found that most peripheral CD4 cells co-express a low density of CD8 alpha antigen in African green monkeys (AGM). Further, the cell surface expression of CD4 and the expression of CD4 mRNA underwent a decrease when purified CD4CD8low cells were cultured with mitogen and IL-2. These observations suggest that AGM CD4 cells are subject to loss of CD4 expression after lymphocyte activation. Part of the peripheral CD8 fraction exhibited a significant helper activity which suggested the phenotypic conversion in helper T cells from CD4+ to CD4- in vivo. Simian immunodeficiency virus (SIV) grew well in CD4 panning cells following SIV infection. In contrast, CD4CD8low cells were resistant to SIV infection after their conversion to CD4- cells.