Generation of transgenic non-human primates with germline transmission.

The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.

Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset (Callithrix jacchus).

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

Comparison of 30 immunity-related genes from the common marmoset with orthologues from human and mouse.

In the evolution of primates, the common marmoset belongs to the new world monkey family and is distinct from the great ape family (which includes humans). In this study, we predicted the amino acid sequences of 30 immunity-related genes from the common marmoset and compared them with those from human and mouse. The domain composition of each orthologous protein was analyzed by the SMART tool and was found to be the same among the three species. A BLAST search revealed that the common marmoset and human proteins were 86% identical on average, whereas the conservation between the common marmoset and mouse or between the human and mouse was only 60%. This indicates that the common marmoset and human proteins are closely related and are similarly divergent from the mouse. We divided the 30 proteins into two categories based on the degree of conservation between the common marmoset and mouse amino acid sequences. One group included 19 proteins and had a relatively high level of conservation (68% identical), whereas the other 11 proteins were less conserved (45% identical). This suggests that these immunity-related genes do not evolve at a uniform rate. Interestingly, however, ligand/receptor pairs such as interleukin-6 and interleukin-6 receptor appear to have evolved simultaneously.

Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies.

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.

Neurobehavioral protection by single dose l-deprenyl against MPTP-induced parkinsonism in common marmosets.

OBJECTIVE: Establishment of preclinical method evaluating behavioral protective actions of drugs for Parkinson's disease was attempted using l-deprenyl (DEP) as a reference drug in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated common marmosets. MATERIALS AND METHODS: Fifteen marmosets received MPTP at 2 mg/kg, subcutaneously (s.c.) per day for three consecutive days. To these marmosets, intragastric (i.g.) administration of DEP at 10 mg/kg was pretreated 2 h before each MPTP administration in DEP3 group and pretreated only in the first MPTP administration day in DEP1 group. As a control, distilled water (DW) was pretreated before each MPTP administration (n = 5 for each of three groups). RESULTS: In DW group, decreased daily activity counts and increased dysfunction scores were persistently observed for 3 weeks after MPTP. In DEP groups, the similar changes of both levels to those in DW group were temporally observed after MPTP for several days and then the values recovered to the pre-MPTP levels. The results of autoradiography performed after above behavioral observations indicated that markedly lower bindings of [(11)C]PE2I (ligand for dopamine transporters) were observed at the striatum of DW group marmoset as compared with the striatum of additionally prepared MPTP-free marmoset (n = 5). The bindings in DEP groups were almost the same as in the MPTP-free marmoset brains. CONCLUSION: The present preclinical methods using continuous recording of activity of marmosets in their living cages and autoradiography using dopamine transporter ligand might be sensitive for detecting protective actions of drugs for Parkinson's disease.

Helicobacter callitrichis sp. nov., a novel Helicobacter species isolated from the feces of the common marmoset (Callithrix jacchus).

A slowly growing microaerophilic Helicobacter species was isolated from the feces of the common marmoset (Callithrix jacchus). This bacterium possessed a pair of nonsheathed bipolar flagella, was positive for oxidase, catalase and alkaline phosphatase activities, but was negative for gamma-glutamyltranspeptidase and urease activity and for nitrate reduction. The bacterium was susceptible to nalidixic acid and resistant to cephalotine and did not hydrolyze hippurate. On the basis of phenotypic characteristics, 16S rRNA gene sequence analysis and whole-cell protein profiles, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter callitrichis sp. nov. is proposed; the type strain of the new species is R-204(T) (GenBank accession number AY192526).

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells.

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus).

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.

Immunolocalization of steroidogenic enzymes P450scc, 3betaHSD, P450c17, and P450arom in Göttingen miniature pig testes.

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in Göttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.

Hematopoietic activity of common marmoset CD34 cells isolated by a novel monoclonal antibody MA24.

OBJECTIVE: We focused on a small New World monkey, the common marmoset (Callithrix jacchus), to establish a nonhuman primate model of the treatment of hematological disorders. In this study, we developed the first monoclonal antibodies (MAbs) against marmoset CD34 and tested the in vitro and in vivo hemopoietic activity of cell populations isolated using one of these MAbs. METHODS AND RESULTS: Marmoset cDNA encoding a human CD34 homologue was cloned from bone marrow (BM)-derived RNA using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The amino acid sequence of the marmoset CD34 had 81% homology with the human sequence. Five mouse MAbs were raised against marmoset CD34 transfectant. One representative MAb, MA24 (IgM), reacted with approximately 0.5 to 1% of BM mononuclear cells (MNCs), where the colony-forming unit granulocyte/macrophage (CFU-GM) was enriched approximately 11- to 75-fold as compared with the whole BM MNCs. Multilineage differentiation of marmoset CD34+ cells in NOD/SCID mice was confirmed by flow cytometry 1 month after xenotransplantation. CONCLUSION: These results demonstrated that MA24 is useful for the analysis and enrichment of hematopoietic progenitor cells in the marmoset model for preclinical experiments.

Morphological evidence for two types of Mammalian vomeronasal system.

The vomeronasal (VN) systems of rodents and opossums are of the segregated type, i.e alpha-subtype G protein Gi2- or Go-expressing VN neurons, which are sensory cells, project discretely to the rostral or caudal region of the accessory olfactory bulb (AOB). Although this zone-specific projection is believed to be a common feature for processing pheromones in mammals, we previously found a uniform-type VN system in goat in which only Gi2-expressing VN axons terminate at the AOB. In most mammals, it remains unclear whether their VN systems are of the segregated or uniform type. Therefore, we investigated morphologically the VN systems of different mammalian species (dog, horse, musk shrew and common marmoset). Consequently, all VN axons of the examined animals were positively stained with immunohistochemistry for Gi2 in the same way as that in the goat. On the other hand, we observed immunoreactivities against Go in the olfactory axons, but not in the VN axons. These results suggest that many mammals have uniform-type VN systems, and at least two types of VN systems exist in terrestrial mammals. This morphological evidence will help us determine the processing function of VN systems.

Canine X-linked muscular dystrophy in Japan (CXMDJ).

The purpose of this study was to develop a strain of canine X-linked muscular dystrophy (CXMD), a model of Duchenne muscular dystrophy, in Japan. A female beagle was artificially inseminated with frozen-thawed spermatozoa derived from an affected golden retriever. Subsequently, two carrier female dogs (G1 carriers) and four normal male littermates were produced. Thereafter, the two G1 carriers were mated with beagle sires. As a result, each bitch whelped three times, and out of 54 pups, 17 affected male descendants, and 11 carrier female descendants (G2 carriers) were detected. One G2 carrier was then mated with a beagle sire and 15 pups in two whelpings were produced, including five affected males and four carrier females (G3 carriers). A total of 10 female beagles were artificially inseminated to evaluate the fertility of the frozen-thawed spermatozoa from the two affected dogs. The whelping rates of the two affected dogs were 4/5 and the litter sizes were 5.0 +/- 1.41 and 6.0 +/- 0.82, respectively. These results indicate that a canine X-linked muscular dystrophy colony has been established in Japan. We called them CXMDJ.

Grafted swine neuroepithelial stem cells can form myelinated axons and both efferent and afferent synapses with xenogeneic rat neurons.

Neuroepithelial stem cells derived from the swine mesencephalic neural tube were examined regarding their eligibility for neural xenografting as a donor material, with the aim of evaluating myelinated axon formation and both types of synaptic formation with xenogeneic host neurons as part of possible neural circuit reconstruction. The mesencephalic neural tube tissues were dissected out from swine embryos at embryonic days 17 and 18 and were implanted immediately into the striatum of the Parkinsonian model rat. The swine-derived grafts had many nestin-positive rosette-forming, neurofilament-positive, and tyrosine hydroxylase-positive cells in the rat striatum. Electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or tyrosine hydroxylase-positive donor cells in the grafts. Myelinated axons, both positive and negative for swine-specific neurofilament antibody, were mingled together in the graft. These results indicated that implanted neuroepithelial stem cells could survive well and divide asymmetrically into both nestin-expressing precursors and differentiated neurochemical marker-expressing neurons in the xenogeneic rat striatum, with the help of an immunosuppressant. Donor-derived immature neurons formed both efferent and afferent synapses with xenogeneic host neurons, and donor-derived axons were myelinated, which suggests that implanted swine neuroepithelial stem cells could possibly restore damaged neuronal circuitry in the diseased brain.

Distribution of protein gene product 9.5-immunopositive and NADPH-diaphorase-positive neurons in the common marmoset (Callithrix jacchus) accessory olfactory bulb.

The principal center of the accessory olfactory system is the accessory olfactory bulb (AOB). In primates, simians are divided into two groups, New and Old World monkeys, and the AOB is present in only New World monkeys. The common marmoset (Callithrix jacchus) is a species of New World monkey. Although the morphology of the common marmoset AOB has been demonstrated, the distribution patterns of the mitral/tufted and granule cells of the AOB remain unclear. In the present study, therefore, the distribution of the mitral/tufted and granule cells in the common marmoset AOB was examined using two histochemical markers including immuno-staining for protein gene product (PGP) 9.5 and NADPH-diaphorase staining. The vomeronasal nerves, gomeruli and mitral/tufted cells showed PGP 9.5-immunoreactivity. The mitral/tufted cells were arranged in only one or two rows along the margin of the glomerular layer to form the mitral/tufted cell layer (MTL). Since the mitral/tufted cells occurred sparsely in the common marmoset, the MTL was illegible. NADPH-diaphorase reactivity was primarily detected in the rostral and caudal areas of the AOB. In these areas, granule cells showed NADPH-diaphorase reactivity. Since the granule cells were sparse, the common marmoset AOB displayed less-developed granule cell layer. Although the functional significance of the AOB remains to be solved in the common marmoset, small-sized and less-laminated AOB may show that sexual behavior of the common marmoset has lesser dependence on the accessory olfactory system.

Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC).

Swine kidney derived CPK cells were resistant to swine complement attack in vitro while rabbit kidney derived RK13 cells were destroyed by swine complement. To rabbit complement, RK13 cells were resistant but CPK cells were sensitive. This phenomenon was known as homologous restriction (Proc. Natl. Acad. Sci. USA 78 (1981) 5118). The gC deletion mutant of pseudorabies virus (PRVdlgC) grown in CPK cells was resistant to swine complement while the same virus grown in RK13 cells was neutralized by swine complement. PRVdlgC grown in RK13 cells was more resistant to rabbit complement than the virus grown in CPK cells. Hence, the sensitivity of PRVdlgC to swine or rabbit complement was similar to that of the cells in which the virus was grown. It would appear that cell derived factors were present on the virion and they were protective against homologous complement but not against heterologous complement. The expression of gC rendered PRV more resistant to swine or rabbit complement, but the protective effect of gC was much less than that of cell derived factors. The best protection against complement was obtained when gC and cell derived factors were coexistent on the virion.

Differential alloreactivity at SLA-DR and -DQ matching in two-way mixed lymphocyte culture.

The major histocompatibility complex (MHC) class II molecules are heterodimeric cell surface glycoproteins important for antigen presentation to CD4+ T lymphocytes. Class II molecules of the pig MHC, termed SLA, identified so far include DR and DQ. Thus far, functional differences between products of different loci in SLalpha class II have not been well characterized. For detailed research on this issue, SLalpha-DRbeta1 and -DQbeta typings were newly developed by restriction fragment length polymorphism (RFLP) of the reverse transcriptase-polymerase chain reaction (RT-PCR) products. Using this method, several RFLP types were chosen from 13 CSK miniature pigs, and alloreactivities in two-way mixed lymphocyte culture (MLC) derived from these pigs were examined by cell proliferation assay using flow cytometry. The responses in MLC varied according to the degree of phenotype difference. In MLC from individuals of the same RFLP type in both SLA-DRbeta1 and -DQbeta, the proliferative responses showed slight reaction indicating that they were not so stimulated by each other. On the other hand, for the RFLP type-mismatching combination, the responses were strong indicating that they recognized each others alloantigens. The reactivity of only the DQbeta mismatching combination was as strong as those of only the DRbeta1 mismatching combination. These data indicate the important role of the DQ as well as DR molecule on alloreactivity in MLC.

The suppressive effect of dexamethasone on the proliferation of Plasmodium falciparum in squirrel monkeys.

We investigated the effects of dexamethasone (Dx) on the proliferation of Plasmodium falciparum Indochina-I/CDC in squirrel monkeys (Saimiri sciureus boliviensis). Three splenectomized squirrel monkeys each received 16 perioral Dx doses (three times per week for 36 days), at three different dosages (0.2, 0.5, and 2.0 mg/ kg). Each monkey was intravenously inoculated with 7.8 x 10(7) red blood cells infected with P. falciparum 1 week after the initiation of Dx treatment. Parasite growth was suppressed in DX-treated monkeys in a dose-dependent manner. To determine whether this suppression of parasite development was mediated by host immunity, P. falciparum was cultured in vitro in the presence of Dx. The development of P. falciparum was also markedly suppressed in vitro by Dx treatment in a dose-dependent manner. These results suggest that Dx has a direct suppressive effect on the proliferation of P. falciparum both in vivo and in vitro, via a mechanism that is not mediated by an immunological reaction of the host.