Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thromboxane A2 modulates interaction of dendritic cells and T cells and regulates acquired immunity

Physical interaction of T cells and dendritic cells (DCs) is essential for T cell proliferation and differentiation, but it has been unclear how this interaction is regulated physiologically. Here we show that DCs produce thromboxane A2 (TXA2), whereas naive T cells express the thromboxane receptor (TP). In vitro, a TP agonist enhances random cell movement (chemokinesis) of naive but not memory T cells, impairs DC-T cell adhesion, and inhibits DC-dependent proliferation of T cells. In vivo, immune responses to foreign antigens are enhanced in TP-deficient mice, which also develop marked lymphadenopathy with age. Similar immune responses were seen in wild-type mice treated with a TP antagonist during the sensitization period. Thus, TXA2-TP signaling modulates acquired immunity by negatively regulating DC-T cell interactions.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Rat strain differences in the early stage of porcine-serum-induced hepatic fibrosis.

Rat strain differences in the early development of porcine serum (PS)-induced hepatic fibrosis were histologically and immunohistochemically examined using Brown Norway (BN), Sprague Dawley (SD) and Wistar rats. They were injected i.p. with 0.5 ml sterile PS twice a week for 4 and 8 weeks. In addition, rats treated with physiological saline in the same way served as controls. At 4 weeks, hepatic fibrosis accompanying fibrous septa mainly composed of type III collagens developed in BN and SD rats but not in Wistar rats. In addition, the numbers of eosinophils, CD3-positive cells and ED-1-positive cells significantly increased in BN and SD rats, that of CD45RA-positive cells in BN rats, and that of alpha-smooth muscle actin (SMA)-positive cells in SD rats, respectively. Such differences in the number of inflammatory cells may be related with the absence of hepatic fibrosis in Wistar rats at 4 weeks. At 8 weeks, hepatic fibrosis with formation of many small-sized pseudolobules was observed in all strains at almost similar degree, and the numbers of infiltrating cells increased in all strains of rats with some exception. In addition, the main location of inflammatory cells was different, suggesting a different role of each inflammatory cell in the process of hepatic fibrosis.     

Microanatomical localization of PD-1 in human tonsils

PD-1 is an immunoinhibitory receptor, which belongs structurally to the CD28 family. PD-1-deficient mice show breakdown of peripheral tolerance and manifest multiple autoimmune symptoms. We previously described expression of PD-1 on activated T and B lymphocytes and myeloid cells. However, little is known about the microanatomical distribution of PD-1 in lymphoid organs. In this study, we performed immunohistochemistry using monoclonal antibodies against human PD-1. In human tonsils, PD-1 was expressed on most of T cells and a small subset of centrocytes in the light zone of germinal centers (GCs), where clonal selection of centrocytes takes place. These results suggest that PD-1 may play an important role in GC reaction.     

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats following UVA-irradiation

Ultrastructual characteristics of the dorsal skin responses to a single irradiation of UVA (1100 kJ/m2) were examined in Wistar-derived hypotichotic WBN/ILA-Ht rats (HtRs). In the epidermis, mitochondrial swelling of some keratinocytes and dissociation of keratinocytes due to intercellular edema developed at 3 hours (h) after irradiation and continued to 48 h. At 6 h, in addition to these changes, necrosis of keratinocytes accompanied with infiltration of neutrophils was also observed in some portions, and epidermal hyperplasia with many keratinocytes showing nucleolar hypertrophy and some mitotic keratinocytes was observed at 48 h. In the dermis, mitochondrial swelling and/or partial cytoplasmic destruction in capillary endothelial cells and edema with inflammatory cell infiltration were observed at and after 3 h, and extravasation of erythrocytes was found in some capillaries at 48 h. Mitochondrial swelling was also frequently found in pericytes and fibroblasts. Inflammatory cells were mainly composed of neutrophils throughout the experimental period. Mild degranulation of mast cells which also showed mitochondrial swelling was observed at and after 3 h, and a close special relationship between mast cells and fibroblasts or neutrophils was sometimes observed. In conclusion, the most prominent change in the dorsal skin of HtRs exposed to UVA was degeneration of capillary endothelial cells, resulting in edema and inflammatory cell infiltration, and the most characteristic cytopathic effect of UVA was mitochondrial swelling, and it was common to keratinocytes, capillary endothelial cells, pericytes, mast cells, and fibroblasts.     

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic ultraviolet B (UVB)-irradiation

Ultrastructural changes in the dorsal skin were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats exposed to subchronic UVB-irradiation (10 kJ/m2 per rat per day for up to 3 months). Epidermal hyperplasia developed at I month of UVB-irradiation and progressed thereafter, resulting in epidermal thickening and formation of epidermal ingrowths projecting into the dermis. In some portions of the epidermal ingrowths at 2 and 3 months, keratinocytes were somewhat pleomorphic. In addition, some of the keratinocytes showing cytoplasmic projections migrated into the dermis. The basement membrane and hemidesmosomes at the epidermal-dermal junction became to disappear along with the development of edema spreading from the upper dermis to the epidermis. However, Langerhans cells were still detected in the hyperplastic epidermis even at 3 months. In the dermis, in addition to edema, fibroblast proliferation and mast cell infiltration progressed with time, and degranulation of mast cells was obvious at 2 and 3 months. Only a few basophils as well as eosinophils were also found. In the upper dermis, especially beneath the epidermis, decrease in diameter and disintegration of collagen fibrils were observed. Ultrastructural characteristics of the dorsal skin responses to subchronic UVB-irradiation were clarified in the present study.     

Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thyroid Carcinoma in Japan and the West: Similarities and Differences

Geographic or ethnic differences in the incidence of thyroid carcinoma, as well as in the histologic distribution of thyroid carcinoma between Japan and Western countries, have been described but are still unclear. The recent establishment of histologic criteria for the diagnosis of thyroid carcinoma by the WHO committee has facilitated the comparison of clinicopathological data of patients with thyroid carcinoma all over the world. The aim of the present review article is to clarify the epidemiological and clinicopathological differences of thyroid carcinoma between Japan and Western countries. We found recently no significant differences in the incidence, mortality, and histologic distribution of thyroid carcinoma between Japan and Western countries; this was contrary to our expectation. This is likely attributable to westernization of the Japanese diet, standardized medical levels, and international standardization of histologic criteria of thyroid carcinoma.     

Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes

A mAb J43 has been produced against the product of the mousePD-1 gene, a member of the Ig gene superfamily, which was previouslyisolated from an apoptosis-induced T cell hybridoma (2B4.11)by using subtractive hybridization. Analyses by flow cytometryand immunoprecipitation using the J43 mAb revealed that thePD-1 gene product is a 50–55 kDa membrane protein expressedon the cell surface of several PD-1 cDNA transfectants and 2B4.11cells. Since the molecular weight calculated from the aminoacid sequence is 29,310, the PD-1 protein appears to be heavilyglycosylated. Normal murine lymphoid tissues such as thymus,spleen, lymph node and bone marrow contained very small numbersof PD-1⁺ cells. However, a significant PD-1⁺ population appearedin the thymocytes as well as T cells in spleen and lymph nodesby the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1antigen expression was strongly induced in distinct subsetsof thymocytes and spleen T cells by in vitro stimulation witheither anti-CD3 mAb or concanavalin A (Con A) which could leadT cells to both activation and cell death. Similarly, PD-1 expressionwas induced on spleen B cells by in vitro stimulation with anti-IgMantibody. By contrast, PD-1 was not significantly expressedon lymphocytes by treatment with growth factor deprivation,dexamethasone or lipopolysaccharide. These results suggest thatthe expression of the PD-1 antigen is tightly regulated andinduced by signal transduction through the antigen receptorand do not exclude the possibility that the PD-1 antigen mayplay a role in clonal selection of lymphocytes although PD-1expression is not required for the common pathway of apoptosis.