Effective expansion of engrafted human hematopoietic stem cells in bone marrow of mice expressing human Jagged1.

The human immune system can be reconstituted in experimental animals by transplanting human hematopoietic stem cells (hHSCs) into immunodeficient mice. To generate such humanized mice, further improvements are required, particularly to ensure that transplanted hHSCs are maintained in mice and proliferate long enough to follow prolonged immune responses to chronic diseases or monitor therapeutic effects. To prepare the relatively human bone marrow environment in mice, we generated nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor gamma chain null (NOG) mice expressing human Jagged1 (hJ1) in an osteoblast-specific manner (hJ1-NOG mice) to examine whether Notch signaling induced by hJ1 mediates hHSC proliferation and/or maintenance in mice. The established hJ1-NOG mice possess relatively larger bone marrow space and thinner cortical bone compared with nontransgenic littermates, but the number of c-kit(+) Sca-1(+) lineage(-) cells was not significantly different between hJ1-NOG and nontransgenic littermates. In the transplantation experiments of CD34(+) cells obtained from human cord blood, CD34(+)CD38(-) cells (hHSCs) were more increased in hJ1-NOG recipient mice than in nontransgenic littermates in mouse bone marrow environment. In contrast, the transplanted mouse c-kit(+) Sca-1(+) lineage(-) cells did not show significant increase in the same hJ1-NOG mice. These results suggest that hJ1-NOG mice could contribute to the growth of transplanted human CD34(+) cells in a human-specific manner and be useful to study the in vivo behavior and/or development of human stem cells, including cancer stem cells and immune cells.

LGR5-positive colon cancer stem cells interconvert with drug-resistant LGR5-negative cells and are capable of tumor reconstitution.

The cancer stem cell (CSC) concept has been proposed as an attractive theory to explain cancer development, and CSCs themselves have been considered as targets for the development of diagnostics and therapeutics. However, many unanswered questions concerning the existence of slow cycling/quiescent, drug-resistant CSCs remain. Here we report the establishment of colon cancer CSC lines, interconversion of the CSCs between a proliferating and a drug-resistant state, and reconstitution of tumor hierarchy from the CSCs. Stable cell lines having CSC properties were established from human colon cancer after serial passages in NOD/Shi-scid, IL-2Rγ(null) (NOG) mice and subsequent adherent cell culture of these tumors. By generating specific antibodies against LGR5, we demonstrated that these cells expressed LGR5 and underwent self-renewal using symmetrical divisions. Upon exposure to irinotecan, the LGR5(+) cells transitioned into an LGR5(-) drug-resistant state. The LGR5(-) cells converted to an LGR5(+) state in the absence of the drug. DNA microarray analysis and immunohistochemistry demonstrated that HLA-DMA was specifically expressed in drug-resistant LGR5(-) cells, and epiregulin was expressed in both LGR5(+) and drug-resistant LGR5(-) cells. Both cells sustained tumor initiating activity in NOG mice, giving rise to a tumor tissue hierarchy. In addition, anti-epiregulin antibody was found to be efficacious in a metastatic model. Both LGR5(+) and LGR5(-) cells were detected in the tumor tissues of colon cancer patients. The results provide new biological insights into drug resistance of CSCs and new therapeutic options for cancer treatment.

Osteosclerosis and inhibition of human hematopoiesis in NOG mice expressing human Delta-like 1 in osteoblasts.

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34(+) hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34(+) HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34(+) HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Application of probabilistic analysis for precisely correcting the QT interval for heart rate in telemetered common marmosets.

INTRODUCTION: QT intervals are strongly influenced by preceeding heart rate history and are also characterized by rate-independent variability, leading to difficulty in precise rate-correction of the raw QT interval. The present study elucidates a novel analytical method that effectively addresses this problematic phenomenon in telemetered common marmosets. METHODS: ECGs were collected from telemetered common marmosets (male and female) and analyzed by computerized algorithms. Descriptive statistics were calculated from the mean of QT intervals for 5-ms increments of RR. The QT interval was corrected for the RR interval by applying Bazett's, Fridericia's, and individual probabilistic QT rate-correction formulae. RESULTS: The linear regression of log-transformed QT and RR intervals derived from a probabilistic approach yielded a well-correlated QT-RR fit. Assessed as the slope of the QTc-RR interval, application of individual probabilistic QT rate-corrections resulted in the most effective dissociation of the effects of rate from the raw QT interval, compared to generic rate-correction formulae. Using individual corrections, the QTc was stable while the interquartile range (IQR) of the QTc distribution was stable, spanning 5-10 ms for each subject over all physiological RR intervals. Heart rate variability distributions were centered about unity during both photoperiods and sinus arrhythmia was far less pronounced compared with measurements in dogs. DISCUSSION: Probabilistic QT rate-correction eliminated the confounding effects of heart rate and provided a stable QTc baseline. These results indicate that application of this method of analysis in telemetered common marmosets results in a high degree of sensitivity for the consistent detection of small (5-10 ms) changes in the QTc interval.

Sensitivity of common marmosets to detect drug-induced QT interval prolongation: moxifloxacin case study.

INTRODUCTION: Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization studies, but it has not been characterized in common marmosets which are uniquely suited to studies in early-stage development due to their small size and minimal test article requirements. The purpose of this study was to evaluate the sensitivity of the common marmoset to detect moxifloxacin-associated QT interval prolongation. METHODS: Eight telemetered common marmosets were monitored for 24 h following oral administration of moxifloxacin by gavage at 0, 10, 30, and 100 mg/kg using a Latin square design. Concurrently, a pharmacokinetic evaluation in 8 non-telemetered animals was conducted. A rate-corrected QT (QTc) interval was derived using an individual probabilistic QT rate-correction. QTc (placebo-adjusted QTc change from the individual baseline) was calculated and the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) was analyzed. RESULTS: A slight, but not significant, increase in QTc was detected with 10 mg/kg of moxifloxacin. Moxifloxacin at 30 and 100 mg/kg elicited dose-dependent increases in QTc of 14.0+/-3.6 and 35.0+/-6.2 ms, respectively, with associated total moxifloxacin C(max) values of 6.5+/-0.5 and 16.5+/-1.6 microg/mL, respectively. From the PK/PD relationship, the plasma concentration which would attain QTc of 5 to 10 ms was estimated to be 1.67-3.73 microg/mL. The results were consistent with typical clinical trial results (QTc of 6.6-14.8 ms at 2.5-3.5 microg/mL). CONCLUSIONS: The present study demonstrates that the common marmoset is highly sensitive to moxifloxacin-associated changes in cardiac repolarization, assessed as QTc. As such, this species is suitable for precise and reliable detection of small, but significant, drug-associated increases in QTc interval. Thus, the common marmoset should be regarded as a validated animal model for the detection of QT risk in early-stage drug development and represents an important addition to the current in vivo armamentarium.

Transplantation of galectin-1-expressing human neural stem cells into the injured spinal cord of adult common marmosets.

Delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord can promote functional recovery in adult rats and monkeys. To enhance the functional recovery after NS/PC transplantation, we focused on galectin-1, a carbohydrate-binding protein with pleiotropic roles in cell growth, differentiation, apoptosis, and neurite outgrowth. Here, to determine the combined therapeutic effect of NS/PC transplantation and galectin-1 on spinal cord injury (SCI), human NS/PCs were transfected by lentivirus with galectin-1 and green fluorescent protein (GFP), (Gal-NS/PCs) or GFP alone (GFP-NS/PCs), expanded in vitro, and then transplanted into the spinal cord of adult common marmosets, 9 days after contusive cervical SCI. The animals' motor function was evaluated by their spontaneous motor activity, bar grip power, and performance on a treadmill test. Histological analyses revealed that the grafted human NS/PCs survived and differentiated into neurons, astrocytes, and oligodendrocytes. There were significant differences in the myelinated area, corticospinal fibers, and serotonergic fibers among the Gal-NS/PC, GFP-NS/PC, vehicle-control, and sham-operated groups. The Gal-NS/PC-grafted animals showed a better performance on all the behavioral tests compared with the other groups. These findings suggest that Gal-NS/PCs have better therapeutic potential than NS/PCs for SCI in nonhuman primates and that human Gal-NS/PC transplantation might be a feasible treatment for human SCI.

Preimplantation development of somatic cell cloned embryos in the common marmoset (Callithrix jacchus).

The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.

Development of monoclonal antibodies for analyzing immune and hematopoietic systems of common marmoset.

OBJECTIVE: Common marmosets are considered experimental animals of primates useful for medical research. We developed several monoclonal antibodies (mAbs) directed to CD molecules to gain initial insight into the immune and hematopoietic systems of this organism, and analyzed the basic cellularity and characters of marmoset lymphocytes. MATERIALS AND METHODS: Anti-marmoset CD antigen mAbs were prepared using marmoset antigen-expressing transfectants and used for flow cytometric analyses and cell fractionation. Expression of T-cell-related cytokine gene transcripts was examined in response to T-cell receptor stimulation by reverse transcription polymerase chain reaction analyses. Hematopoietic progenitor activities of marmoset bone marrow cells were examined in fractionated cells by mAbs against CD117 (c-kit) and CD34. RESULTS: CD4 and CD8 expression profiles in T-cell subsets of marmoset were essentially similar to those in mouse and human. CD4(+) and CD8(+) subsets were isolated from marmoset spleens. Detected transcripts after stimulation of T cells included Th1-, Th2-, and Th17-related cytokines in CD4(+) cells and cytotoxic proteases in CD8(+) cells, respectively. Colony-forming abilities were detected mainly in CD117 (c-kit)(+) cells, irrespective of CD34 expression. CONCLUSIONS: Marmoset immune system was basically similar to human and mouse systems.

Spontaneous thymic lymphomas in the non-obese diabetic/Shi-scid, IL-2R gamma (null) mouse.

The NOD/Shi-scid, IL-2Rgamma(null) (NOG) mouse is a severely immunodeficient mouse used for the engraftment of human tissues and cells. In this study, 2406 mice (8-62 weeks old, 503 males and 1903 females) were subcutaneously engrafted with human tissues. In 16 mice (12-26 weeks old, 1 male and 15 females), a mass was seen in the anteroventralis of the thorax on gross examination with an incidence of 0.7%. Histologically, the masses were composed of sheets of lymphoblastic cells. A 'starry sky' pattern was observed with numerous mitoses. Immunohistochemically the lymphoblastic cells were positive for Thy 1. The lymphoblastic cells were also seen in the spleen, lung, liver, kidney and heart. The gross and histopathological findings led to the diagnosis of spontaneous thymic lymphoma in NOG mice.

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.

Diffusion-tensor neuronal fiber tractography and manganese-enhanced MR imaging of primate visual pathway in the common marmoset: preliminary results.

PURPOSE: To investigate whether diffusion-tensor tractography (DTT) of neuronal fibers is useful for delineating the configuration of the neuronal fiber trajectories in the primate visual pathway, including the well-developed optic chiasm, in comparison with tract tracing at manganese-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: The handling methods used for all the animals in this study were approved by the institutional committee for animal experiments. Diffusion-tensor MR imaging was performed in four healthy common marmosets, and in two of these animals, manganese-enhanced MR imaging tract tracing was performed by using a 7.0-T MR imaging unit. The visual pathways were quantitatively investigated in terms of the manganese distribution observed on the manganese-enhanced MR images. The images obtained with DTT and manganese-enhanced MR imaging tract tracing were qualitatively compared, and the features of the visual pathway were verified through fusion of the reconstructed images obtained by using these two modalities. RESULTS: DTT provided information regarding the neuroanatomic features of the marmoset visual pathway and revealed the bilateral branching patterns of the typical primate retinogeniculate pathways, although several incorrectly tracked fibers were noted. The distribution of manganese on the manganese-enhanced MR images revealed bilateral innervation of the retinal projections and depicted the layered internal structure of the lateral geniculate nuclei bilaterally, depending on the ocularity of each layer. These morphologic findings were consistent with those of previous histopathologic studies. CONCLUSION: The findings of this preliminary study raise the possibility that DTT is useful for visualizing the neuronal fiber trajectories in primate visual pathways. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/249/3/855/DC1.

In vivo chemotherapeutic profile of human gallbladder small cell carcinoma.

Small cell carcinoma of the gallbladder is very rare, but shows high malignant potential with frequent metastasis. Chemotherapeutic regimens for the treatment of gallbladder small cell carcinoma have not yet been established. In this study, we examined in vivo chemosensitivity tests for the GB-04-JCK human gallbladder small cell carcinoma, which were previously established as a serial-transplantable xenograft in nude mice. We used four anticancer drugs: docetaxel, irinotecan, nedaplatine and gemcitabine. Docetaxel maximally suppressed xenograft tumor growth in mice (P<0.01), and showed complete tumor regression after chemotherapy day 35. Irinotecan and nedaplatine suppressed tumor growth without complete regression (P<0.01). Gemcitabine did not affect tumor growth significantly. This in vivo experimental study proposed chemotherapeutic regimens for human gallbladder small cell carcinoma.

Establishment and characterization of in vivo human tumor models in the NOD/SCID/gamma(c)(null) mouse.

Immunodeficient mice are widely used for xenografts of human cells and tissue. The purpose of the present study was to investigate the characteristics of xenograft human tumor models using engraftment of various non-hematopoietic tumors in the NOD/SCID/gamma(c) (null) mouse. For tumor models, human solid tumor tissues were serially passaged three or more times to establish tissue lines. A total of 326 fresh tumor specimens, mainly gastrointestinal and female genital tissue, were engrafted with 54 established tissue lines. The types of tissue lines varied and included tumor tissue of both epithelial and mesenchymal origin. In some cases the original surgical specimen was replaced with large mononuclear cells. In the established tumor tissue lines, differentiation and tumor structure were similar to that of the original surgical specimen. The interstitium of the xenograft tissue in the tissue lines was relatively well preserved although slightly decreased and replaced by host tissue. These results indicate that human solid tumors can be successfully engrafted into the NOD/SCID/gamma(c) (null) mouse and that tissue lines with the characteristics of the original tumors can be established. Investigators in the field of tumor research will benefit from the availability of tissue lines that allow the establishment of more relevant in vivo human tissue models.

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background.

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).

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.

Identification of a key molecular regulator of liver metastasis in human pancreatic carcinoma using a novel quantitative model of metastasis in NOD/SCID/gammacnull (NOG) mice.

We developed a reliable new model system for assaying liver metastasis using NOD/SCID/gamma(c)(null) (NOG) mice. Seven human pancreatic cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOD/SCID and NOG mice. Capan-2 and PL45 showed no metastasis when seeded at up to 10(5) cells in both strains, and no BxPC-3 metastasis was observed in NOD/SCID mice. The NOD/SCID mouse model detected liver metastasis only in the AsPC-1 cell line when inoculated with >10(3) cells. In contrast, when inoculated with only 10(2) MIA PaCa-2, AsPC-1 and PANC-1 cells, liver metastasis was evident in 71.4% (5/7), 57.1% (4/7) and 37.5% (3/8) of the NOG mice, respectively. Capan-1 and BxPC-3 cells metastasized when seeded at 10(3) cells in 50% (5/10) and in 12.5% (1/8) of the mice, respectively. Using the NOG mouse model system, we established a highly metastatic cell line, liver metastasized-BxPC-3 (LM-BxPC-3), from liver metastatic foci formed by the relatively poorly metastatic parental BxPC-3 cell line. The gene expression profiles of parental and LM-BxPC-3 cells were compared, and we identified forty-five genes that were either upregulated or downregulated >4-fold in the LM-BxPC-3 cell line. We validated 9 candidate protein-coding sequences, and examined the correlation between their expression pattern and the in vivo liver metastatic potential of all 7 pancreatic cancer cell lines. Only S100A4 expression correlated with the ability to form liver metastases, as evaluated in our quantitative model of metastasis in NOG mice. These results suggested that S100A4 is a key regulator of liver metastasis in pancreatic cancer, and demonstrated the feasibility of using the quantitative metastasis model to search for and develop new anti-cancer therapies and novel drugs against this and other key molecules.

Phenotypes of IRS-2 deficient mice produced by reproductive technology are stable.

We studied the impact of "IVF - ET" on the glucose tolerance test (GTT), insulin tolerance test (ITT) and adiponectin to investigate differences in the phenotypes of B6J- Irs2(-/-) mice. The B6J-Irs2(-/-) mice (KO-Nat group) were prepared by natural mating. Other mice were produced by IVF-ET used ICR strain recipients and surrogate mothers (KO-IVF group). Measurement of body weight, GTT, ITT and blood sampling were performed at the ages of 6, 14 and 24 weeks after birth. Body weights, impaired glucose tolerance, insulin resistance and plasma adiponectin concentrations did not differ for each gender between the KO-IVF and KO-Nat groups. Therefore, we concluded that phenotypes of Irs2(-/-) mice produced by reproductive technology are stable.

Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/gamma c(null) immunodeficient mice.

Human uterine endometrium exhibits unique properties of cyclical regeneration and remodeling throughout reproductive life and also is subject to endometriosis through ectopic implantation of retrogradely shed endometrial fragments during menstruation. Here we show that functional endometrium can be regenerated from singly dispersed human endometrial cells transplanted beneath the kidney capsule of NOD/SCID/gamma(c)(null) immunodeficient mice. In addition to the endometrium-like structure, hormone-dependent changes, including proliferation, differentiation, and tissue breakdown and shedding (menstruation), can be reproduced in the reconstructed endometrium, the blood to which is supplied predominantly by human vessels invading into the mouse kidney parenchyma. Furthermore, the hormone-dependent behavior of the endometrium regenerated from lentivirally engineered endometrial cells expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescence imaging. These results indicate that singly dispersed endometrial cells have potential applications for tissue reconstitution, angiogenesis, and human-mouse chimeric vessel formation, providing implications for mechanisms underlying the physiological endometrial regeneration during the menstrual cycle and the establishment of endometriotic lesions. This animal system can be applied as the unique model of endometriosis or for other various types of neoplastic diseases with the capacity of noninvasive and real-time evaluation of the effect of therapeutic agents and gene targeting when the relevant cells are transplanted beneath the kidney capsule.

Development of human-human hybridoma from anti-Her-2 peptide-producing B cells in immunized NOG mouse.

OBJECTIVE: Numerous monoclonal antibodies have been developed for the purpose of medical treatments, including cancer treatment. For clinical application, the most useful are human-derived antibodies. In this study, we tried to prepare designed antigen-specific antibodies of completely human origin using immunodeficient mouse. METHODS: Nonobese diabetic/severe combined immunodeficient/IL-2 receptor gamma null mouse (NOG) mouse was used to reconstitute the human immune system with umbilical cord blood hematopoietic stem cells (CB-NOG mouse) and to prepare human-derived Her-2-epitope-specific antibodies. Hybridoma lines were prepared by fusing the human myeloma cell line Karpas707H. RESULTS: Serum of immunized NOG mouse contained human-derived immunoglobulin M (IgM) antibodies specific for a short peptide sequence of 20 amino acids, including the epitope peptide of apoptotic Her-2 antibody CH401. Hybridoma lines were successfully prepared with spleen B cells obtained from the immunized CB-NOG mouse. One of these cell lines produced human IgM against the epitope peptide that can recognize surface Her-2 molecule. CONCLUSION: We could produce human-derived IgM antibody against Her-2 epitope peptide in CB-NOG mouse, succeeding in generation of human hybridoma-secreting IgM against a given peptide.

Ontogenetic characteristics of enzyme activities and plasma metabolites in C57BL/6J:Jcl mice deficient in insulin receptor substrate 2.

We have established an inbred line of mice deficient in insulin receptor substrate 2 (IRS2) on a C57BL/6J Jcl genetic background (B6J-IRS2(-/-) mice) as an animal model for typical type 2 diabetes mellitus (DM). We investigated the effect of age and sex on glucose tolerance and insulin resistance and on the activities of enzymes related to lipid metabolism in the liver and skeletal muscle of B6J-IRS2( -/-) mice. Glucose tolerance tests (GTT), insulin tolerance tests (ITT), and sampling for chemical analysis were performed at ages of 6,14, and 24 wk. GTT showed that both genders of B6J-IRs2(-/-) mice had impaired glucose tolerance at the ages of 6 and 14 wk, whereas 24-wk-old female B6J-IRs2(-/-) mice showed glucose tolerance almost comparable to that of wild-type mice; 24-wk-old male B6J-IRs2(-/-) mice still showed impaired glucose tolerance. ITT revealed that both male and female B6J-IRS2(-/-) mice remained insulin-resistant at all time points. Hepatic lipogenetic enzyme activities were higher in B6J-IRS2(-/-) mice than in wild-type mice at 6, 14 and 24 wk of age. In addition, plasma glucose, triglyceride, free fatty acid, total cholesterol, and insulin concentrations in B6J-IRS2(-/-) mice were significantly higher than those in wild-type mice at most time points; plasma triglycerides in 14-wk-old B6J-IRS2(-/-) mice were lower than those of wild-type mice. These findings suggest that young B6J-IRS2(-/-) mice are useful as type 2 DM models.