Tatsuhiko Miyazaki

Butyltin residues in livers of humans and wild terrestrial mammals and in plastic products

Butyltin compounds (BTs) including mono-(MBT), di-(DBT) and tributyltin (TBT) were determined in livers of humans and wild terrestrial mammals, such as raccoon dogs (Nyctereutes procyonoids) and monkeys (Macaca fuscata) from Japan. In addition, 22 samples of plastic products were analyzed. BT residues were detected in all the liver samples of humans and raccoon dogs, with concentrations of <360 ng/g wet wt, whereas concentrations in the liver of monkeys were either less than the detection limit or were only in trace levels. Elevated concentrations of BTs, particularly DBT (<140,000 ng/g) and MBT (<130,000 ng/g), were found in some plastic products, such as baking parchments made from siliconized paper and gloves made up from polyurethane. The results of a cooking test using the above baking parchment indicated the transfer of BTs to foodstuffs. These observations suggest expansion of BT contamination among terrestrial mammals. BT pollution from industrial appliances, such as plastic stabilizers and catalysts other than those of marine origin as antifouling agents, are suggested as alternative sources of exposure.

Stromal cells in lymph nodes attractB-lymphoma cells via production ofstromal cell-derived factor-1

Abstract: Stromal cell‐derived factor‐1 (SDF‐1) is a chemokine produced by bone marrow stromal cells which plays an important role in B‐lymphopoiesis and the homing of hematopoietic stem cells to the bone marrow. In the present study, we investigated the role of SDF‐1 and its receptor, CXCR4, in the chemotactic interaction between non‐Hodgkin B‐lymphoma cells and lymph node stromal cells. SDF‐1 mRNA was abundantly expressed in stromal cells isolated from the lymph nodes of patients with malignant lymphoma. All B‐lymphoma cells freshly isolated from these patients and most laboratory B‐lymphoma cell lines, including follicular, diffuse large, and Burkitts lymphoma cells, expressed surface CXCR4 and migrated in the presence of recombinant human SDF‐1α. Chemotaxis assays revealed that CXCR4‐positive (but not CXCR4‐negative) B‐lymphoma cells migrated towards lymph node stromal cells, and this migration was almost completely inhibited by the addition of anti‐CXCR4 monoclonal antibody to the lymphoma cells or of anti‐SDF‐1 neutralizing antibody to the culture supernatant of the stromal cells. Down‐regulation of surface CXCR4 was detected in B‐lymphoma cells which migrated towards the stromal cells but not in those which showed no migratory response. In addition, contact between the lymphoma cells and the stromal cells resulted in down‐regulation of surface CXCR4 on the lymphoma cells. These data strongly suggest that SDF‐1/CXCR4 is the main chemokine system involved in the chemotactic interaction between B‐lymphoma cells and lymph node stromal cells.

Therapeutic effect of CXCR3-expressing regulatory T cells on liver, lung and intestinal damages in a murine acute GVHD model.

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+CD25+Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+CD25+Foxp3+ T cells (Treg cells) or naturally occurring CD4+CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.

Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice.

Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp‐Faslpr/lpr (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ‐Faslpr/lpr (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr × C3H/lpr)F2 mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method “cell‐free system” with wheat germ ribosomes, the MRL‐OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF‐α, IL‐1β and IFN‐γ in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis.

Genetic dissection of vasculitis in MRL/lpr lupus mice : A novel susceptibility locus involving the CD72c allele

An MRL/MpJ strain of mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), composed of genomes derived from LG/J, AKR/J, C3H/Di and C57BL/6J mice, develops systemic vasculitis coincidentally with other collagen diseases, but a C3H/HeJ‐lpr/lpr (C3H/lpr) strain does not. In a genome‐wide screening of the MRL background genes mediating susceptibility to collagen diseases using N2 progeny mice MRL/lpr × (MRL/lpr × C3H/lpr)F1, we previously found that each collagen disease is controlled by a different set of genes. To clarify the candidate genes for vasculitis, we extended the linkage analysis of renal vasculitis to a larger number of N2 mice and to F2 intercross mice. Two distinct recessive susceptibility loci for vasculitis were mapped on chromosome (Chr) 4 at D4Mit89 and D4Mit147 in both progenies. The former was a novel locus for lupus phenotypes, which involved the MRL allele CD72c in contrast to the C3H allele CD72b. The one on Chr 3 was a recessive locus which had an inhibitory effect on vasculitis. From their composition these loci seemed to be derived from AKR/J (for one) and LG/J (for another two) strains, and appeared to act in an additive manner on the development of vasculitis, indicating that vasculitis in MRL/lpr mice is inherited in a polygenic manner.

A novel autoimmune pancreatitis model in MRL mice treated with polyinosinic:polycytidylic acid

In this study we established a new animal model for exploring the pathogenesis of autoimmune pancreatitis. We have found previously that MRL/Mp‐+/+(MRL/+) mice develop pancreatitis spontaneously by an autoimmune mechanism but only when they are more than 34 weeks old. Because this disease might be a model of multi‐factorial diseases controlled by genetic and environmental factors, beginning at 6 weeks old, we injected polyinosinic:polycytidylic acid (poly I:C) into MRL/+ mice and in addtion, into MRL/Mp mice bearing the Fas deletion mutant gene, lpr (MRL/lpr). Poly I:C induced chronic severe pancreatitis in all the MRL/+ mice and to a lesser extent in the MRL/lpr mice by 18 weeks of age. There was no pancreatitis in control mice of both strains at the same age. Other than chronic pancreatitis, no severe autoimmune diseases were observed in MRL/+ mice. Immunohistochemical examinations revealed predominant infiltration of CD4+ T cells and Mac‐2+ activated macrophages in the pancreatic lesions. Splenic expression of the mRNAs for TNF‐α and IL‐10, which is known to suppress the development of pancreatitis, were increased in both strains of mice. These findings suggest that an MRL strain of mice treated with poly I:C might be a good model for developing new approaches to the study of the pathogenesis of autoimmune pancreatitis.

Therapy for pneumonitis and sialadenitis by accumulation of CCR2-expressing CD4+CD25+ regulatory T cells in MRL/lpr mice

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in animal models of autoimmune diseases. Chemokines play an important role in the development of autoimmune diseases in animal models and humans. The present study was performed to investigate whether the progression of organ-specific autoimmune diseases could be reduced more markedly by accumulating chemokine receptor-expressing CD4+CD25+ regulatory T cells efficiently in target organs in MRL/MpJ-lpr/lpr (MRL/lpr) mice. CD4+CD25+Foxp3+ T cells (Treg cells) and CD4+CD25+Foxp3+ CCR2-transfected T cells (CCR2-Treg cells) were transferred via retro-orbital injection into 12-week-old MRL/lpr mice at the early stage of pneumonitis and sialadenitis, and the pathological changes were evaluated. Expression of monocyte chemoattractant protein-1 (MCP-1)/CCL2 was observed in the lung and submandibular gland of the mice and increased age-dependently. The level of CCR2 expression and MCP-1 chemotactic activity of CCR2-Treg cells were much higher than those of Treg cells. MRL/lpr mice to which CCR2-Treg cells had been transferred showed significantly reduced progression of pneumonitis and sialadenitis in comparison with MRL/lpr mice that had received Treg cells. This was due to more pronounced migration of CCR2-Treg cells and their localization for a longer time in MCP-1-expressing lung and submandibular gland, resulting in stronger suppressive activity. We prepared chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression by accumulating in target organs. This method may provide a new therapeutic approach for organ-specific autoimmune diseases in which the target antigens remain undefined.

Mesenchymal chondrosarcoma treated with total en bloc spondylectomy for 2 consecutive lumbar vertebrae resulted in continuous disease-free survival for more than 5 years: case report.

Study Design. A case report of an extremely rare malignant spinal tumor successfully treated with total en bloc spondylectomy and chemotherapy. Objective. To describe points for consideration when an osteogenic lesion in the spine is diagnosed and treated. Summary of Background Data. Primary mesenchymal chondrosarcoma in the spine is extremely rare. There were no reports of this tumor being treated with spondylectomy to achieve total surgical resection with a wide margin followed by chemotherapy. Methods. A 44-year-old female presented with low back pain and left flank pain. Magnetic resonance imaging and computerized tomography showed an osteosclerotic tumor of the lumbar vertebrae. Tc-99m HMDP bone scintigraphy was positive, but thallium-201 scintigraphy and gallium scintigraphy were negative. The patient was diagnosed as having chondrosarcoma based on biopsy findings. Results. To resect the tumor completely, total en bloc spondylectomy for 2 consecutive lumbar vertebrae was performed. However, the postoperative pathologic diagnosis was extremely difficult because the patient was initially suspected to have osteosarcoma, but the final diagnosis was mesenchymal chondrosarcoma. Five years after surgery, there have not been any signs of local recurrence or distant metastasis, and the patient has remained continuously disease free. Conclusions. To our knowledge, we reported the first case of mesenchymal chondrosarcoma occurring from the lumbar spine treated with total en bloc spondylectomy and chemotherapy. Successful radical resection of the tumor could be accomplished. Although the effect of chemotherapy on the final results could not be clearly determined, considering that at least continuous disease-free survival was achieved, it is highly likely that chemotherapy contributed to the favorable results.

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice

An MRL strain of mice bearing a Fas‐deletion mutant gene, lpr, MRL/MpJ‐lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ‐lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr) F1 intercross, and MRL/lpr × (MRL/lpr × C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi‐squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.

Existence of Lipoprotein Lipase in Human Sarcomas and Carcinomas

Aqueous extracts of acetone/ether powders of surgically obtained specimens of human tumors hydrolyzed 3H‐labeled triolein in a dose‐dependent manner. The lipolytic activity in these extracts was inhibited by anti‐lipoprotein lipase (LPL) IgG dose‐dependently, 25 μg of anti‐LPL IgG causing 95% inhibition of the activity. Thus, LPL accounts for most of the lipolytic activity in extracts of acetone/ether powders of the tumors. All sarcomas and carcinomas examined contained LPL activity. Western blotting showed that they gave a band corresponding to that of human adipose tissue LPL (Mr=57,000). Immunocytochemical studies showed that LPL was present in cultured human osteosarcoma cells and distributed throughout the cells. We determined the proliferating cell nuclear antigen (PCNA)‐labeling index as an indicator of the proliferative activity of tumor cells and measured LPL activity in extracts of tumors in areas corresponding to those used for determining the PCNA‐labeling index. In malignant fibrous histiocytomas, the PCNA‐labeling index in area a, which corresponds to the subcapsular region, was higher than that in area b, which corresponds to the central region. The LPL activity in area a was 10 times that in area b. In rectal cancer, the index in area c, which corresponds to the subserosal region, was higher than that in area d, which corresponds to the submucosal region. The LPL activity in area c was 1.9 times that in area d. These findings indicate heterogeneity in the distributions of LPL activity within tumors and higher levels of LPL activity in tumors that are proliferating actively.