Toshihiko Asano

BETA -GALACTOSIDASE-DEFICIENT MOUSE AS AN ANIMAL MODEL FOR GM1-GANGLIOSIDOSIS

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring

AbstractPurpose: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. Methods: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activate with Sr2+-containing Ca2+-free medium. Results: After thawing, 75–85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. Conclusions: Mouse round spermatids can be cryopreserved for production of normal offspring.

Characteristics of mutant mice (ICGN) with spontaneous renal lesions: a new model for human nephrotic syndrome

Spontaneous nephrotic mice (ICGN mice), a new mutant strain of mouse from outbred ICR, were clinically, macroscopically, histologically and immunohistochemically studied to establish their value as a model for human nephrotic syndrome. Most of the affected mice developed proteinuria, hypoproteinaemia and hypercholesterolaemia, and some of them developed systemic oedema. A high concentration of blood urea nitrogen (BUN) and a low haematocrit value were also observed. The kidneys of severe cases showed a decrease in size and had a yellowish granular surface. These findings indicated that the mice were terminally affected by chronic renal in-sufficiency. Histopathology demonstrated glomerular lesions consisting of thickened basement membranes of the capillary loops with irregular spike-like protrusions and enlargement of the mesangium unaccompanied by cellular proliferation. The immunofluorescence technique revealed positive granular staining for IgA, IgG and IgM and to a lesser extent for C3 along the capillary loops in affected mice. The similarity between this spontaneous disease and human nephrotic syndrome caused by idiopathic glomerular lesions is discussed. ICGN mice may be a useful animal model for this human disease.

Hereditary nephrotic syndrome with progression to renal failure in a mouse model (ICGN strain): clinical study.

The clinical course of murine hereditary nephrotic syndrome (ICGN strain) was determined by examining 201 animals under different conditions. In the early stage, significant hypoproteinemia and hypoalbuminemia developed (p < 0.001) in parallel with a progressive rise in urinary protein concentration (p < 0.001). In the middle stage, the concentrations of total cholesterol, triglyceride, and beta-lipoprotein markedly increased (p < 0.01, p < 0.001, and p < 0.001, respectively), suggesting that type IIb hyperlipoproteinemia developed as in human nephrotic patients. Systemic edema appeared in 8 of 24 animals. In the terminal stage, both BUN and creatinine values greatly increased (p < 0.001), indicating rapid deterioration of renal function. The present study suggests that ICGN mice could be a useful model to study the pathophysiology of human nephrotic syndrome and its progression to renal failure.

SCID-bg mice as xenograft recipients

SCID-bg (scid/scid, beige/beige) is a strain of double-mutant mice with impaired lymphoid development and reduced natural killer (NK) cell activity. The present study was undertaken to evaluate the usefulness of SCID-bg mice as xenograft recipients. Fetal guineapig tissues (liver, thymus, spleen) were transplanted under the kidney capsule of the mice and their serum guineapig IgG levels were measured weekly thereafter. C.B.-17-scid and anti-asialo GM1 antiserum-treated (NK-depleted) C.B.-17-scid (C.B.-17-scid-AGM1) mice that received the identical transplants were used as controls. Throughout the experimental period (1, 2, and 3 weeks after transplantation), the average serum guineapig IgG concentrations was highest in C.B.-17-scid-AGM1 mice followed by SCID-bg mice and lowest in C.B.-17-scid mice without antiserum treatment, though we could not find any statistical significance among these groups. However, SCID-bg mice always showed the smallest within-group variance (individual differencel in the serum guineapig IgG concentrations (P <0.05, versus C.B.-17-scid-AGM1 mice at 1,2, and 3 weeks and versus C.B.-17-scid mice at 2 weeks). The graft size was not significantly different among these three groups, but the spleen grafts in C.B.-17-scid mice contained fewer nucleate cells than the other two groups. These results indicate that the reduced NK cell activity by beige mutation is not crucial for the success of xenogenic transplantation, though SCID-bg mice may be useful as xenograft recipients with a consistent potential to retain the viability and function of engrafted tissues.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

NOD/LtSz‐prkdcscid/prkdcscid (non‐obese diabetic‐severe combine immunodeficiency; NOD‐scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus‐1 (HIV‐1) infection using NOD‐scid mice. Administration of interleukin‐18 (IL‐18) and IL‐12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co‐administration prevented grafting due to interferon‐γ‐dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL‐18 alone, but not in those given phosphate‐buffered saline, IL‐12 alone, or IL‐18 + IL‐12. A high rate of HIV infection was also observed in the IL‐18‐treated group. Together, these results indicate that IL‐18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class‐switching IgA. IL‐18‐administered NOD‐scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy.

An electron microscopic study of glomerular lesions in hereditary nephrotic mice (ICGN strain)

Glomerular lesions in hereditary nephrotic mice (ICGN strain) were investigated by electron microscopy. The glomeruli of unaffected animals, which appeared normal by light microscopy, had developed an ultrastructural change in the glomerular capillary basement membrane (GCBM). There was a partial thickening of the GCBM with bilaminar splitting of the lamina densa and an electron-dense fibrillar material exhibiting cross-striations. In affected animals, light microscopy revealed a marked thickening of GCBM and an increase of mesangial matrix without cellular proliferaton. By electron microscopy, multilaminar splitting of the lamina densa in the thickened GCBMs and fusion of the epithelial foot processes were observed. In some severely affected animals, immune complex deposition was found in GCBM, but little if any was observed in other animals. In the end, the glomeruli were globally sclerosed. Our findings suggest that initial structural abnormalities in GCBM may play an important role in the onset and development of the disease, though subsequent events such as immune complex deposition would modify the disease.

Localization of HIV-1 in human thymic implant in SCID-hu mice after intravenous inoculation

Human immunodeficiency virus type 1 (HIV‐1) was immunohistochemically and ultrastructurally localized in human thymus implants in SCID‐hu mice 3 weeks after intravenous (i.v.) inoculation of the virus. A viral antigen (gp120) was predominantly distributed in and around the epithelial cells in Hassall’s corpuscles as demonstrated by fluorescence immunohistochemistry. Occasional solitary round cells positive for the viral antigen but negative for cytokeratin were detected in the perivascular areas. Ultrastructural examinations clearly revealed a number of mature viral particles in the intercellular spaces of the Hassalls corpuscles. Thus the present study indicates the possibility that thymic epithelial cells in Hassalls corpuscles act as a target and/or reservoir in an early stage of HIV infection.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte

Oral–genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2mnull mice grafted with human peripheral blood leucocytes (hu‐PBL) and stimulated with interleukin (IL)‐4, and second to investigate whether oral exposure to cell‐free R5 and X4 HIV‐1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell‐free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu‐PBL‐NOD/SCID and NOD/SCID Β2mnull mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL‐4‐administrated NOD/SCID B2mnull mice are useful as a small‐humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments.

TCRβ-independent development of CD4 + CD8 + thymocytes observed in a strain of scid mice

Mice homozygous for severe combined immunodeficiency (scid) mutation usually halt their thymocyte development at the CD4−CD8− double negative (DN) stage due to their inability of TCR gene rearrangement. In this study, we report that SCID‐bg mice, which were originally generated by mating CB‐17‐scid mice with KSN‐bg mice. spontaneously develop dominant CD4+CD8+ double positive (DP) thymocytes. Their thymi were mainly composed of DN, CD4−CD8+ and DP cells, and the majority of them did not present CD3. Similarly, they lacked TCRβ expression both on cell surface and in cytoplasm, which suggests that the thymocyte development to the DP stage observed m SCID‐bg mice was independent of CD3 and TCRβ expression. In spite of significant DP thymocytes in SCID‐bg mice, the histology of their thymi was not so different from those of CB‐17‐scid mice. Analysis of bone marrow cells in SCID‐bg mice showed that the development of B lineage cells was not altered when compared with CB‐17‐scid mice. These findings point out the fact that thymocytes in SCID‐bg mice have a peculiar characteristic compared to CB‐17‐scid mice, and provide evidence of TCRβ‐independent development of thymocytes to the DP stage.