Osamu Shiho

AN IMPROVED COLORIMETRIC ASSAY FOR INTERLEUKIN 2

Mosmanns method for measuring the number of viable cells with a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), was modified to make it possible to measure a large number of interleukin 2 (IL-2) samples at one time with less labor and more accuracy. Each step of the method was examined in detail and modified (the modified MTT method). An IL-2-dependent mouse natural killer cell line, NKC3, was used as an indicator cell line. The incubation period before adding MTT was reduced to 24 h, A solution of 10% sodium dodecyl sulfate-0.01 N HCl was used to dissolve the MTT formazan produced. We have compared the values obtained by the modified MTT method and the conventional [3H]thymidine method (3H-TdR method), and confirmed that the estimates of IL-2 content were almost equal. The variation of IL-2 content measured by both methods was within 5% in terms of the standard error.

Comparison of the biological properties of purified natural and recombinant human interleukin-2

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.

Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicate that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Expression of neurotrophin genes in the brain of senescence-accelerated mouse (SAM) during postnatal development

We compared the expression patterns of neurotrophin genes in the brain of senescence-accelerated mouse (SAMP8) which shows age-related impairment of learning behavior, with SAMR1 control which shows normal aging. By Northern blot analysis, NT-3 mRNA levels in the cortex were higher in SAMP8 than in SAMR1 mice during development, whereas in the midbrain, hippocampus and forebrain, NT-3 expression levels in SAMP8 were lower than those in SAMR1. At early stages, although NGF mRNA levels in SAMP8 were lower than those in SAMR1, BDNF mRNA levels were almost equivalent in both strains. By in situ hybridization analysis, NT-3 mRNA signals in the CA1 and CA2 regions in SAMP8 were shown to be reduced at early stages. However, BDNF mRNA signals were almost equivalent in both SAMR1 and SAMP8.

Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity

We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination. The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry. Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites. In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons. We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ. Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3. The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization. However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice. These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.

Local immunosuppressive therapy with monoclonal anti-T cell antibody on renal allograft survival in the rat

Considerable interest in the experimental and clinical use of MoAbs as potential therapeutic agents in allograft rejection has been generated by the recent reports of striking prolongation. In this study we investigated the efficacy of the local administration of MoAb OX‐19 which is directed to the rat CD5 equivalent, through the renal artery using a rat kidney transplant model, in order to develop a potent method for modifying rejection while minimizing the systemic side effects. Untreated Lewis rats (LEW, RT‐11) rejected Brown‐Norway rat (BN, RT‐1n) kidney at 7.8±0.2 days (n=10). Mean survival time (MST) of recipients treated with OX‐19 (75 μg/kg per day) as single bolus injections via the dorsal penile vein for 7 days was 7.0 ± 0.2 days (n= 5, NS). LEW hosts receiving OX‐19 (75 μg/kg per day) continuously for 7 days via a femoral vein by using an osmotic minipump (IV‐treated group) showed a slight prolongation of graft survival (MST=8.8±0.9 days, n= 5), but this was not statistically significant. On the other hand, local continuous intrarenal arterial infusion of OX‐19 (75 μg/kg per day) for 7 days (RA‐treated group) significantly prolonged the graft survivals (MST =16.8± 1.3 days, n= 8, P<0.01). Histological examination of MoAb‐treated LEW hosts on day 6 post‐grafting revealed that kidney grafts from RA‐treated hosts showed a slight tubular necrosis, but reduced mononuclear cell infiltration, whereas kidney grafts from IV‐treated hosts displayed a severe mononuclear cell infiltration around the artery with interstitial oedema. Moreover, the local intrarenal administration of OX‐19, even when the dose is delayed until day 4 after renal grafting, has a therapeutic effect for on‐going acute allograft rejection (MST= 11.4 ± 0.8 days, n= 8) compared with administration of OX‐19 intravenously from day 4 after grafting (MST= 7.6 ±0.2 days, n= 5,P<0.01) or with no treatment (MST = 7.8±0.2 days, P<0.01). The phenotype of graft infiltrating cells (GIC) was investigated on day 6 post‐grafting. There was a significantly lower percentage of cells positive for OX‐19, OX‐8, OX‐26 (transferrin receptor), and OX‐39 (1L‐2 receptor) in the RA group than in the IV group. These results indicate that local administration of OX‐19 via the renal artery is a more efficacious treatment than systemic i.v. injection, and suggests that it may provide a localized immunosuppression of allografts, with reduction of morbidity in clinical transplant recipients.

Establishment of a novel embryonic stem cell line by a modified procedure

To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is diffucult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouseSry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice.

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analyses revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1−, Lyt 2− and Thy 1+, Lyt 1−, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1−, Lyt 2− cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2‐responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1−, Lyt 1−, and Lyt 2−. On the other hand, a predominant type of the IL 2‐responsive cells in BALB/c nu/nu mice were Thy 1−, Lyt 1−, and Lyt 2−, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.

Establishment of Human B Cell Lines Producing Antigen-specific Antibodies through SCID Mice Reconstituted with Human Peripheral Blood Lymphocytes

We have characterized the engrafted human peripheral blood lymphocytes (PBL) and the production of human immunoglobulin (Ig) in the chimeric SCID mice reconstituted with human PBL. Human cells from healthy donors were injected into the peritoneal cavity. Human lymphocytes expressing CD45 molecules migrated to lymphoid tissues of the chimeric mice and proliferated rapidly in only four days. However, B cells could not be detected in the thymus. We established several human B cell lines from human PBL in lymphoid tissues of the immunized mice that generated human IgG with specific binding activity to immunogens.