SCID mutant mouse with functional defect of T and B cells is a good model to analyze the developmental mechanism of T and B cells because both lymphocytes do not develop for expressing their functions. It is known that antigen receptors on T and B cells require the gene rearrangement prior to their expression on protein level. Ontogenical studies of normal mouse thymocytes showed that β-chain genes of T cell receptor (TCR) initiate to rearrange in c-kit+ IL-2R+thymocytes but the germ line transcription of these genes occurs at the earlier stage. Since such transcript was not found in non-T cells such as B lymphocytes and kidney tissues. The thymocytes with germ line transcripts are thought to belong to the T cell lineage. In SCID mouse, the process of gene rearrangement is impaired : Southern blot analysis showed that TCR-β genes are not rearranged in SCID thymocytes. However, the germ line transcripts of TCR-β and -α were found in SCID thymocytes. Flowcytometric analysis showed that SCID thymocytes expressed surface markers specific for immature T cells such as Thy-1, Pgp-1 and IL-2 Ra but do not express TCR, CD2, 3, 4 and 8 which are common in mature T cells. These phenotypes were similar to c-kit +CD4-CD8- (DN) embryonic thymocytes which have not yet undergone gene rearrangement of TCR-β and α though these genes were transcribed in germ line. Therefore, it was indicated that SCID thymocytes have already been committed into T cell lineage but their development is arrested at the very early stage. Then, why is the thymocyte development arrested if TCR genes are not rearranged? Recent studies show that development of DN thymocytes into CD4⁺CD8⁺ (DP) thymocytes requires certain signaling by pre-TCR which is a heterodimer composed of pre-TCR-α (pTα) and TCR-β. We found that thymocytes of SCID mouse express pTα but not TCR-β. Thus, it is assumed that SCID thymocytes are not capable of obtaining pre-TCR mediated signal (s) for DN-DP transition. In fact, introduction of a TCR-β transgene into SCID mice induced the generation of DP thymocytes.
These observations indicate that gene rearrangement of TCR-β is essential for T cell development from DN to DP cells as well as TCR protein expression, which was evident in SCID mice.

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Recombinant IL-6 was found to function as a late-acting killer helper factor (KHF) in the differentiation of cytotoxic T lymphocytes (CTL) and to mediate an antitumor effect in our previous study. Tumor cells transfected with IL-6 cDNA using PM 5 Gneo retrovirus vector induced stronger anti-tumor effect even on non-immunogenic murine tumors. CD 4^-8⁺ CTL and CD 4⁺ T cells played a role in the in vivo anti-tumor effect of the IL-6 gene-transfectant.
We analyzed the in vivo anti-human tumor effect of IL-6 by using the SCID-PBL/hu mice construcuted with human PBL. Injection of CESS human B tumor cells transfected with the IL-6 cDNA into SCID-PBL/hu mice induced significant CD 3⁺ human CTL. When human lung cancer cells transfected with IL-6 gene were administered into SCID-PBL/hu mice constructed with syngeneic PBL, cytotoxic cells were generated and prolongation of survival was observed. Furthermore, IL-6 transgenic SCID-hu mice constructed with PBL from a lung cancer patient or a gastric cancer patient exhibited cytotoxic activity against syngenic cancer cells even when non-transfected cancer cells were injected. These IL-6 gene-transfected tumor and SCID mice might provide a useful tool for the analysis of therapeutic effect in human cancers.
Recently an anti-human tumor effect by in vivo transfer of IL-6 gene using adenovirus vector into SCID-PBL/hu mice was exhibited. When SCID-PBL/hu mice were injected with CESS human tumor cells and treated with AdexIL-6, CD 3⁺ CD 8⁺ human CTL were generated. Prolongation of survival, regression of tumor and inhibition of metastasis were also observed. Human CD 3⁺ CD 8⁺ tumor specific CTL were generated in vivo and tumor growth was inhibited in the SCID-PBL/hu mice which were constructed with PBL from patients with gastric cancer or colon cancer, injected with autologous cancer cells, and treated with AdexIL-6 i.p. Thus, the experimental model using SCID-PBL/hu and cytokine genes delivered by an adenovirus vector might provide a new strategy capable of analyzing anti-human tumor effect by cytokine gene therapy without using human body.

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To develop new in vivo study system of human skin and hair, our research group has tried to produce hu-skin/SCID mice model, that is, mice xenografted with human skin or hair suitable for in vivo experiments. We found that SCID mice and nude-scid mice were good recipients of full-thickness and split-thickness skin grafts from humans. The latter mice seemed to be better, because their hairless nature was convenient for experimental procedures and shrinkage rate of the grafts was relatively small in them. The atypical keratinocytes were well survived in the grafts on the mice when lesional skin of Bowen's disease or solar keratosis was transplanted. In contrast, Paget cells of extramammary Paget's disease were almost completely eliminated from the grafts. Melanocytes of melanoma in situ were maintained only in epidermis of the grafts on nude-scid mice. Carcinogenesis experiments using various kinds of chemical carcinogens failed to produce tumors in human skin grafted on SCID mice, though many tumors developed in the control mouse skin. Hair growth was observed in the human scalp skin transplanted on SCID mice. An isolated human hair follicle obtained by dissection of scalp skin was successfully transplanted onto SCID mice and used to identify localization of stem cells of hair follicles. In conclusion, hu-skin/SCID mice system surely provides us with a very useful in vivo study system of human skin and hair.

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It has been known that human epidermal keratinocytes can be propagated in vitro and transplanted onto immunodeficient mice. However, the regenerated human epidermis like tissue is rarely retained for a long period. These tissues also lack Langerhans cells.
We show here that co-transplantation of human dermal fibroblasts facilitates the long term maintenance of mature epidermal structures reorganized after xenotransplantation of cultured human keratinocytes. These reorganized human skins on immunodeficient mice are highly reproducible. Furthermore, when human bone marrow derived hematopoietic progenitor cells are co-transplanted with skin cells, repopulation of Langerhans cells in the reorganized epidermis are observed.
We consider that the reorganized human skins on immunodeficient mice are suitable to use as human skin model in vivo.

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To establish a highly reproducible in vivo model of rhumatoid arthritis, synovial infiltrating cells obtained from patients with RA were transfered into several kinds of severe combined immunodeficient (SCID) mice and the incidence of destructive arthritis with pannus tissues was compared.
BALB/cA-bg, scid, BALB/cA-scid and CB-17-scid were used in this study. Synovial cell suspensions obtained from patients with RA, and synovial cells from patients with osteoarthritis were injected into the right knee joint and subcutanous of dorsal pedis of mice at the age of 6-7 weeks. Peripheral mononuclear cells (MNC) from healthy donors and patients with RA were injected as a control. Mice were sacrified after 5-11 weeks and histologic characteristics of their joints were examined.
Injection of synovial cell suspensions from patients with RA induced bone erosin and pannnus formation in the injected joints of three kinds of SCID mice. The highest incidence of pannus formation was 71% in male BALB/cA-bg, scid mice, compared to 44% in female BALB cA-bg, scid mice. Neither SIC from OA nor MNC from healthy donors and patients with RA induced destructive arthritis with pannus formation in any of mice. This technique represents a novel in vivo model of rheumatoid arthritis for the study of the cellular mechanisms involved in the destructive arthritis as well as for the evaluation of the effects of novel anti-rheumatic drugs.

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In vivo studies for hemoparasitic protozoan diseases of man and domestic animals will be greatly facilitated if experimental infections can be done with small laboratory animals, such as mice and rats. We have developed an innovative experimental system which makes mice susceptible to intraerythocytic protozoan parasites that are, in nature, not infectious to mice. A key of our experimental system is the use of a mutant strain of mice having severe combined immunodeficiency (SCID mice; C.B-17 scid) . Owing to the inability to generate specific immune responses, the red blood cells (RBCs) circulating in SCID mice can be substituted with RBCs of other animal species.
The RBC-substitution of SCID mice was demonstrated the best with bovine (bo) RBCs. We were able to create SCID mice having bo-RBCs by giving repetitive transfusions of bo-RBCs into SCID mice. The bo-RBC-SCID mice became susceptible to intraerythocytic parasites of cattle, such as Theileriia sergenti and Babesia bovis, and developed high levels of parasitemia as long as bo-RBC transfusions were continued. This mouse model was found to be useful to evaluate efficacy of antiprotozoal drugs and to examine ability of antibodies against parasites to inhibit parasite growth. The bo-RBC-SCID mice infected with Babesia parasites exhibited clinical symptoms and, thus, should serve as an useful model system to study mechanisms of hemoparasitic protozoan diseases, particularly in cerebral babesiosis caused by B. bovis.
Although the principle of our experimental system is applicable to human malaria, substitution of SCID mice with human (hu) RBC is found to be difficult. In SCID mice, hu-RBCs are cleared from the blood circulation much more rapidly than bo-RBCs. The rapid hu-RBC clearance in SCID mice appeared to be caused by avid phagocytosis of hu-RBCs by reticuloen-dothelial macrophages as well as deposition of complement C3 on the surface of hu-RBCs. Human serum and some carbohydrate compounds, such as yeast mannan, SCM-chitin and lactof errin, were found to decelerate the clearance of hu-RBCs in SCID mice. We were able to elaborate hu-RBC-substituted SCID mice by giving hu-RBC transfusions together with administration of human serum. Following infection with Plasmodium falciparum, parasitemia was observed in the hu-RBC-SCID mice for as long as 2 weeks, indicating a possible growth of P. falciprum in the mice.

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We established seven new mouse strains with defective T and B cells as well as innate immunological reactions, and showed that six mutant mouse strains efficiently received human PBL engraftment (hu-PBL-mouse) . Increased levels of macrophage-tropic and T-cell line-tropic HIV-1 replication were observed in the new hu-PBL-mice. In one particular strain, hu-PBL-NOD-scid, high levels of HIV-1 viremia (10⁶ infectious units per milliliter) were detected after inoculation of the virus. The viral load was about 1000 times higher than that observed in other HIV-1 infected hu-PBL-mice. Although high viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected in only hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lymph nodes, and brain. PCR in situ hybridization confirmed that HIV-1 infected cells invaded mouse brain tissue. The present study indicates that the immunological response of the NOD-scid mouse is inadequate to protect the animal against severe systemic infection. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent CNS invasion. The NOD-scid mouse represents a useful model for the study of the pathogenesis, especially brain involvement, and therapy of primary HIV-1 viremia.

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The technology to culture a cell of an organization in the living body made progress. But a cell cultured by existing culture technique has lost specialization function to appear in the living body with cultivation rapidly. It isn't enough as animal experiment estimation of the monetary value of housework in life science. In this study, we picked up bone, liver and being unspecialized cell of embryo origin as an example, and developed the technology that let specialization function appear by culture cells. And we established technique to culture the cell functioned. The technique can be utilized as toxic examination method of high sensitivity.

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We developed a new in vitro assay for chemosensitivity test using collagen gel droplet embedded culture and image analysis (CD-DST method) . In this in vitro assay, we successfully minimized the cancer cell number required for culture apporoximately 3-10×10³ cells for each 30μl collagen gel droplet, obtained the sufficient growth of cancer using serum free medium while suppressing the growth of fibroblast cells. It became possible to measure the volume of cancer cells by neglecting the contaminating fibroblast cells by the Neutral Red-stain and an image analysis in assessment. The evaluability of in vitro assay for 282 surgical specimens of several primary cancers was 85%, and the accuracy for in vitro-in vivo correlation in 11 cases was 91%. Tumor cells isolated from human tumor xenografts implanted into nude mice and cultured by CD-DST were exposed to drugs under conditions that can reproduce the plasma AUC in vitro. The in vitro sensitivity to a drug was compared with the in vitro response of the same tumor treated with the MTD of the drug. When the criterion of sensitivity was taken as 50% or less of the growth inhibition, the correlation between the in vitro and in vivo growth inhibition of MMC, CDDP and ADM tested was relatively high (86% of the true positive rate, 82% of the ture negative rate and 83% of the correlation rate) .

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We examined the effects of vitamin D metabolites on mineralization of the 10-day embryonic chick femurs in organ culture system. When 5-day cultures kept in the control medium were cultured in the several mediums containing 1α (OH) D₃, 1, 25 (OH) ₂D₃ or 24, 25 (OH) ₂D₃ at concentration of 10^-11-10^-8 M during an additional 5 days, the mineralization of cultured bones increased in the treatment with 1α (OH) D₃ at concentration of 10^-10-10^-8M, and decreased in the treatment with 1, 25 (OH) ₂D₃ at concentration of 10^-10-10^-8 M, and was no change in the treatment with 24, 25 (OH) ₂D₃. The activities of both alkaline phosphatase and acid phosphatase did not change in the cultured bones with vitamin D metabolites. These results suggested that 1α (OH) D₃ increased bone formation, 1, 25 (OH) ₂D₃ stimulated bone resorption, and 24, 25 (OH) ₂D₃ had no effect to bone-remodeling in this organ culture system.

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Osteoclasts are multinucleate giant cells playing key roles in bone resorption. These cells solubilize mineralized bone matrix by means of acid and protease action ; however, the precise mechanism of this process is not well known. Recently, we succeeded in the isolation of pure osteoclasts from rabbit bones and constructed a cDNA library. Using a differential screening procedure, two genes expressed predominantly in osteoclasts compared with spleen cells were isolated. One of them, OC-2, was found to encode a possible cysteine proteinase structurally related to cathepsins L and S.
Moreover, by use of a rabbit OC-2 fragment as a probe, its human counterpart was cloned from a cDNA library of osteoarthritic hip bone. The cloned human cDNA (hOC-2) encoded a protein of 329 amino acid residues and its deduced amino acid sequence showed 94% homology to rabbit cathepsin K. Multiple alignment of amino acid sequences of human cathepsins B, H, L, S and K showed the highest homology of cathepsin K to cathepsin S 48%. Northern blot analysis showed that cathepsin K mRNA is expressed at high levels in some osteoarthritic hip bone and at a very high level in osteoclastoma compared to very low levels in other tissues. These results suggest that cathepsin K is closely involved in human osteoclastic bone resorption.

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The Long-Evans Tokushima Lean (LETL) rat, characterized by rapid onset of insulindependent (type 1) diabetes mellitus (IDDM), no sex difference in the incidence of IDDM, autoimmune destruction of pancreatic β-cells, and no significant T-cell lymphopenia, is a desirable animal model for human IDDM. The first genetic analysis showed that an MHC-linked gene is involved in the pathogenesis of insulitis. So far, other genetic factors have not been revealed due to a low incidence (about 20%) of IDDM in this strain. We have established a diabetes-prone substrain of the LETL rat, named Komeda Diabetes-Prone (KDP) rat, showing a 100% development of insulitis and over 70% incidence of IDDM within 220 days. In this study, we performed the first genome-wide scan for non-MHC IDDM susceptibility genes in the KDP rat and identified a major IDDM susceptibility gene, termed Iddm/kdp1, on rat chromosome (Chr) 11. Homozygosity for the KDP allele at Iddm/kdp1 locus was shown to be essential for the development of severe insulitis and onset of IDDM. Comparative mapping suggests that homologs of Iddm/kdp1 are located on human Chr 3 and mouse Chr 16 and would therefore be different from previously reported IDDM susceptibility genes.

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The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is the best model for human non-insulin dependent diabetes mellitus (NIDDM) with mild obese body. OLETF rats display spontaneous diabetes with polyuria, and polydipsia at the age of approx. 20 weeks. The cross between OLETF × F344 intercrosses was conducted in a SPF barrier colony. The oral glucose tolerance test (OGTT) was performed at the age of 30 weeks old. To analyze F₂ progeny, approx. 100 polymorphic microsatellite markers were used, and then DNA fragments were amplified by the PCR techniques. Linkage analysis was performed using the MAPMAKER/ QTL computer program. Linkage studies have led to the localization of four loci, Nidd/of1, Nidd/of2, Nidde/of3, and Nidd/of4, that contribute significantly to blood glucose variation by OGTT in F₂ population (lod score >3) . Nidd/of1 affected postprandial hyperglycemia, whereas Nidd/of2, Nidd/of3, and Nidd/of4, affected fasting hyperglycemia. Also we identify one major locus bw/of1 affecting body weight on an autosomal chromosome. The localization of the loci are different from those of GK rat which is well known as animal model of non-obese type NIDDM. Our study concludes that NIDDM is devided into at least two categories with the different pathogenesis controlled by two independent gene clusters. From these results, we may propose that the distinct pathogenesis of NIDDM between OLETF and GK rats reflects the presence or absence of obesity.

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Non-insulin-dependent diabetes mellitus (NIDDM) is considered a polygenic disoder in which insulin resistance and insulin secretory defect are the major etiologic factors. Homozygous mice with insulin receptor substrate-1 (IRS-1) gene knockout showed insulin resistance, but nomal glucose tolerance due to compensatory hyperinsulinemia. Heterozygous mice with β-cell glucokinase (GK) gene knockout showed impaired glucose tolerance due to decreased insulin secretion to glucose. To elucidate the interplay between insulin resistance and insulin secretory defect for the development of NIDDM, we generated double knockout mice with disruption of IRS-1 and β-cell GK genes by crossing the mice with each of the single gene knockout. The double knockout mice developed overt diabetes and showed fasting hyperinsulinemia and selective hyperplasia of the β-cells as the IRS-1 knockout mice, but impaired insulin secretion to glucose as the GK knockout mice. In conclusion, the genetic abnormalities, each of which is non-diabetogenic by itself, can cause overt diabetes if they coexist. This study provides the first genetic reconstitution of NIDDM as a poly genic disorder in mice.

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Although glucose is the major physiologic stimulator of insulin secretion and biosynthesis in vivo, prolonged exposure of pancreatic β-cells to high glucose levels is known to cause β-cell dysfunction. Histologically, such damaged β-cells often reveal extensive degranulation, thus suggesting impaired insulin biosynthesis as a cause of the phenomenon. In this study, we established an in vitro experimental model for the evaluation of glycation effects on the β-cell functions. Glycation was introduced into HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and the effects on insulin gene transcription were examined. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control β-actin gene promoter; approximately 50% and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 days in the presence of 40 mM and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel-mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF 1/STF-1. These effect of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediator of the phenomena. These observations suggest that protein glycation in pancreatic β-cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the β-cell glucose toxicity.

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