Establishment of a vascular endothelial cell-reactive type II NKT cell clone from a rat model of autoimmune vasculitis.

We previously generated a rat model that spontaneously developed small vessel vasculitis (SVV). In this study, a T cell clone reactive with rat vascular endothelial cells (REC) was established and named VASC-1. Intravenous injection of VASC-1 induced SVV in normal recipients. VASC-1 was a TCRαß/CD3-positive CD4/CD8 double-negative T cell clone with expression of NKG2D. The cytokine mRNA profile under unstimulated condition was positive for IL-4 and IFN-γ but negative for IL-2 and IL-10. After interaction with REC, the mRNA expression of IL-2, IL-5 and IL-6 was induced in VASC-1, which was inhibited by blocking of CD1d on the REC surface. Although the protein levels of these cytokines seemed to be lower than the detection limit in the culture medium, IFN-γ was detectable. The production of IFN-γ from the VASC-1 stimulated with LPS-pre-treated REC was inhibited by the CD1d blockade on the REC. These findings indicated VASC-1 as an NKT cell clone. The NKT cell pool includes two major subsets, namely types I and II. Type I NKT cells are characterized by expression of semi-invariant TCRs and the potential to bind to marine sponge-derived α-galactosylceramide (α-GalCer) loaded on CD1d; whereas, type II NKT cells do not manifest these characteristics. VASC-1 exhibited a usage of TCR other than the type I invariant TCR α chain and did not bind to α-GalCer-loaded CD1d; therefore, it was determined as a type II NKT cell clone. The collective evidence suggested that REC-reactive type II NKT cells could be involved in the pathogenesis of SVV in rats.

Prediction of response to treatment by gene expression profiling of peripheral blood in patients with microscopic polyangiitis.

The JMAAV study was an open-labeled prospective clinical trial, which proposed severity-based treatment protocols for patients with microscopic polyangiitis (MPA). The results suggest that the proposed protocols are useful (remission rate: 89.4%), but are also indicative of relapse or patient demise regardless of the treatment (recurrence rate: 19.0%; mortality rate: 10.6%). The aim of this study is to develop the method to predict response to the treatment in patients with MPA. In the present study, transcriptome analysis was performed using peripheral blood from patients enrolled in the JMAAV study before and 1-week after the beginning of treatment. The gene expression profile before treatment was not directly related to the response to the treatment. However, when the samples from 9 patients with good response (persistent remission for 18 months) were examined, the expression of 88 genes was significantly altered by the treatment. Thirty statistically reliable genes were selected, and then the alteration of expression by the treatment was examined among 22 patients, including 17 with good response, which was defined as persistent remission for 18 months and 5 with poor response, which was defined as relapse after remission or no remission. Discrimination analysis between the alteration of expression of the 30 genes by the treatment and the response identified a combination of 16 genes as the most valuable gene set to predict the response to the treatment. This preliminary study identified IRF7, IFIT1, IFIT5, OASL, CLC, GBP-1, PSMB9, HERC5, CCR1, CD36, MS4A4A, BIRC4BP, PLSCR1, DEFA1/DEFA3, DEFA4, and COL9A2 as the important genes that can predict the response to the treatment in patients with MPA at an early point during the therapy.

Pathogenesis of Vasculitis in env-pX Rats.

Transgenic rats carrying the env-pX gene of human T-cell leukemia virus type I (env-pX rats) develop necrotizing angiitis resembling human polyarteritis nodosa (PN). In the development of vasculitis in these rats, the thymus plays an important role. In this review, we provide an outline of the pathogenesis of vasculitis observed in env-pX rats, and discuss the developmental mechanism of human necrotizing angiitis such as PN with an unknown cause. (*English Translation of J Jpn Coll Angiol 2009; 49: 17-20.).

Cyclic AMP response element-binding protein is implicated in IL-6 production from arthritic synovial cells.

Overproduction of interleukin (IL)-6 from synovial cells is critically involved in the pathogenesis of rheumatoid arthritis (RA). Cyclic adenosine monophosphate (AMP) response element-binding protein (CREB), a leucine zipper transcription factor, is expressed at a high level in synovial cells of patients with RA. Although CREB transactivates IL-6 expression in vascular smooth muscle cells, the relation between CREB expression and IL-6 production from arthritic synovial cells remains unclear. In this study, to determine whether CREB is implicated in IL-6 production from arthritic synovial cells, a dominant negative molecule of activation transcription factor 1 (ATF-1) was transfected into synovial cells obtained from arthritic joints of env-pX rats. These transgenic rats carrying the env-pX gene of human T-cell leukemia virus type-1 develop destructive arthritis with high titers of serum rheumatoid factor and are thus regarded as a suitable model of RA. The dominant negative ATF-1 (ATF-1DN) constitutes a heterodimer with CREB and inhibits CREB function, as CREB/ATF-1DN heterodimers no longer bind to the target sequence of CREB. We showed that transfection of ATF-1DN significantly reduced IL-6 production from arthritic synovial cells. These findings suggest that CREB is implicated in IL-6 production from synovial cells and plays an important role in RA pathogenesis.

Human endogenous retrovirus-R Env glycoprotein as possible autoantigen in autoimmune disease.

It has long been discussed whether endogenous retroviruses (ERVs) are involved in the pathogenesis of autoimmune diseases. Among various human endogenous retroviruses (HERVs), we have focused on HERV-R. To investigate the biological roles of HERV-R, we earlier established transgenic rats carrying the full sequence of the viral genome. In these HERV-R rats, however, no disease occurred. Another trigger that induces autoimmunity may be essential for the recognition of HERV-R products by the immune system. Thus, in this study, we mated HERV-R rats with env-pX rats (transgenic rats carrying the env-pX gene of human T cell leukemia virus type I) that develop autoimmune diseases, and generated double transgenic (DTG) rats. In DTG rats, autoimmune diseases occurred similarly in env-pX rats. Interestingly, deposition of rat IgM but not IgG was observed on the glomerular endothelial cells. Such IgM deposition was never seen in the parental HERV-R or env-pX rats. We considered that in situ formation of immune complexes consisted of the HERV-R env glycoprotein and anti-HERV-R env IgM antibodies (Abs) in DTG rats, according to the following evidence: (1) No dense deposit, representing deposition of circulating immune complexes, was seen on glomerular endothelial cells. (2) IgM Abs reactive with HERV-R env glycoprotein were generated in the serum. (3) HERV-R env glycoprotein was expressed in the kidney, specifically on glomerular endothelial cells. (4) IgM deposition was partly colocalized with the HERV-R env glycoprotein on the glomeruli. These findings strongly suggest that the HERV-R env glycoprotein is recognized as an autoantigen in the host with autoimmune diseases.

Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications.

We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high- and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.

Rat CD4+CD8+ macrophages kill tumor cells through an NKG2D- and granzyme/perforin-dependent mechanism.

We previously identified a subpopulation of monocyte/macrophage lineage cells expressing both CD4 and CD8. This subpopulation was expanded in rat peripheral blood and spleen after immunization with adjuvants containing killed tuberculosis germs. CD4+CD8+ monocytes/macrophages obtained from preimmunized rats exhibited a Th1-type cytokine/chemokine profile, expressed high levels of Fas ligand, perforin, granzyme B, and NKR-P2 (rat ortholog of human NKG2D), and killed certain tumor cells. In the present study, we confirmed that CD4+CD8+ monocytes/macrophages are distinct from splenic dendritic cells (DCs) or IFN-producing killer DCs. In vitro cytotoxic assays revealed that CD4+CD8+ macrophages killed tumor cells in a cell-cell contact-dependent manner and that expression of the retinoic acid early transcript 1 (a ligand for NKG2D) made tumor cells susceptible to killing by CD4+CD8+ macrophages. Furthermore, inhibitors of granzyme and perforin significantly decreased cytotoxic activities of CD4+CD8+ macrophages. Consistent with these in vitro findings, preimmunization with adjuvants containing killed tuberculosis germs elevated the expression of granzyme B in tumor-infiltrating CD4+CD8+ macrophages and significantly inhibited the growth of inoculated tumor cells. Our current work demonstrates that CD4+CD8+ macrophages are a unique subpopulation of monocyte/macrophage lineage cells that kill tumor cells in an NKG2D- and granzyme/perforin-dependent mechanism.

Enhanced production of p24 Gag protein in HIV-1-infected rat cells fused with uninfected human cells.

Although many human molecules have been suggested to affect replication of human immunodeficiency virus type 1 (HIV-1), the distribution of such cofactors in human cell types is not well understood. Rat W31/D4R4 fibroblasts expressing human CD4 and CXCR4 receptors were infected with HIV-1. The provirus was integrated in the host genome, but only a limited amount of p24 Gag protein was produced in the cells and culture supernatants. Here we found that p24 production was significantly increased by fusing HIV-1-infected W31/D4R4 cells with uninfected human cell lines of T-cell, B-cell, or macrophage lineages. These findings suggest that human cellular factors supporting HIV-1 replication are distributed widely in cells of lymphocyte and macrophage lineages. We also examined whether the amount of p24 produced by rat-human hybrid cells was correlated with expression levels of specific human genes. The results suggested that HP68 and MHC class II transactivator (CIITA) might up- and down-regulate p24 production, respectively. It was also suggested that HIV-1 replication is affected by molecules other than those examined in this study, namely, cyclin T1, cyclin-dependent kinase 9, CRM1, HP68, and CIITA.

Resistance to experimental colitis depends on cytoprotective heat shock proteins in macrophage migration inhibitory factor null mice.

Macrophage migration inhibitory factor plays an important role in inflammatory diseases. We investigated the role of macrophage migration inhibitory factor (MIF) in the development of dextran sulfate sodium (DSS)-induced colitis using MIF null ((-/-)) mice. MIF(-/-) mice given 3% DSS showed no clinical and histological feature of colitis in contrast to wild-type (WT) mice. Lack of MIF suppressed the up-regulation of TNF-alpha and IFN-gamma as Th1-derived cytokines, and increased the level of IL-4 as Th2-derived cytokine in the colon tissues. Moreover, we found that the expressions of heat shock protein (HSP)40 and HSP70 were markedly up-regulated in the colon of MIF(-/-) mice in response to DSS compared with WT mice. Additionally, quercetin, an inhibitor of HSP synthesis, inhibited the up-regulation of HSP40 and 70 expressions and developed DSS-induced colitis in MIF(-/-) mice. Our findings in this study provide more information in the role of MIF in colitis.

Role of neuronal interferon-gamma in the development of myelopathy in rats infected with human T-cell leukemia virus type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-gamma production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-gamma in HTLV-1-infected, HAM-resistant strains. Administration of IFN-gamma suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-gamma protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-gamma after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor beta2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-gamma through the IL-12 pathway is involved in the development of HAM rat disease.

Rupture of the thyrocervical trunk branch from the subclavian artery in a patient with neurofibromatosis: a case report.

BACKGROUND: Vascular involvement in neurofibromatosis type 1 (NF1) is well recognized; however, rupture of extracranial arteries rarely occurs. We present a case of NF1 with rupture of the thyrocervical trunk, which branched from the right subclavian artery. A 76-year-old woman who has numerous café-au-lait spots and soft tumors of the skin manifested a sudden swelling of her neck accompanied with increasing pain. Radiological examinations revealed bleeding from the artery. METHODS: Histological and immunohistochemical examinations were carried out using tissues that contained the affected vessel. RESULTS: Proliferation of spindle cells positive for S-100 protein was seen in the adventitia of the ruptured vessel. Intimal thickening by proliferation of fibromuscular cells was also evident with irregularity of the media. CONCLUSIONS: These findings suggest that the artery was disrupted by NF in the vascular wall. It is considered that NF in the arterial wall causes dysplasia of the smooth muscle layer in the intima and media and leads to fragility of the vessel. Twelve cases, including the present case, with rupture of extracranial arteries in NF1 have been reported in the past 10 years; two thirds of these occurred in extravisceral sites in which there is a good deal of physical movement. This suggests that a physiological factor is one of the triggers for arterial rupture, which occurs under a background of vascular fragility in NF1.

Protective effect of geranylgeranylacetone on trinitrobenzene sulfonic acid-induced colitis in mice.

Geranylgeranylacetone (GGA) has recently been reported to have a protective effect against ischemic, injurious and apoptotic stress in several tissues. The aim of this study was to determine the effect of GGA on colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. Colitis was induced by intrarectal instillation of TNBS in 50% ethanol in BALB/c mice. Survival, change in body weight and change in wet colon weight were assessed. Histological score in the colon was evaluated 5 days after TNBS treatment. The level of myeloperoxidase (MPO) activity in the colon was also determined. Immunohistochemistry for CD4 in the colon was performed. In addition, the level of heat shock protein (HSP) 70 in the colon was determined by Western blot analysis. Mice were orally treated with GGA (300 mg/kg) 2 h before and every other day after starting TNBS administration. Treatment with GGA markedly improved the survival rate, and reduced the loss of body weight and loss of wet colon weight in mice with TNBS-induced colitis. GGA also suppressed the increase in MPO activity and the number of CD4-positive cells infiltrating the colons of mice with TNBS-induced colitis. Furthermore, treatment with GGA remarkably up-regulated the expression of HSP70 in the colons of mice with TNBS-induced colitis. Our results provide further evidence that GGA has therapeutic potential for intestinal inflammation.

Regulation of Toll-like receptor 4 expression in mouse colon by macrophage migration inhibitory factor.

Recent studies have indicated that macrophage migration inhibitory factor (MIF) and Toll-like receptor (TLR) play an important role in the regulation of innate immune responses. In this study, we investigated the effect of MIF on the expression of TLR4, a receptor that recognizes lipopolysaccharide, in colon using MIF-deficient mice. TLR4 mRNA expression in the colon tissues was determined by northern blot analysis. Western blot analysis and immunohistochemistry in the colon tissues were performed to evaluate the expression of TLR4 protein. The expressions of TLR4 mRNA and protein were remarkably down-regulated in colon tissues of MIF-deficient mice compared with wild-type mice and up-regulated by treatment with recombinant MIF. Immunohistochemical study revealed the presence of TLR4-positive staining in mononuclear cells in the lamina propria and intraepithelial mononuclear cells as well as weak staining in epithelial cells and crypts in colon tissues of wild-type mice. In contrast, MIF-deficient mice did not show TLR4-positive staining in the colonic mucosa. In MIF-deficient mice injected with recombinant mouse MIF (rMIF), TLR4-positive staining cells were observed in colon tissues similar to the findings in wild-type mice. Administration of dextran sulfate sodium (DSS) up-regulated the expression of TLR4 in the colons of WT mice but not in those of MIF-deficient mice. Furthermore, pretreatment with rMIF up-regulated the expression of TLR4 in response to DSS in MIF-deficient mice. Our results suggest that MIF affects the expression of TLR4 in mouse colon under both normal and colitic conditions.

CD4+/CD8+ macrophages infiltrating at inflammatory sites: a population of monocytes/macrophages with a cytotoxic phenotype.

We found a population of nonlymphoid cells expressing both CD4 and CD8 in peripheral blood mononuclear cells (PBMCs) of human T-cell leukemia virus type-I pX transgenic rats with autoimmune diseases. These cells, which showed a monocytic phenotype, were also found in wild-type rats, and their number increased by adjuvant-assisted immunization. GM-CSF increased the number of these double-positive (DP) monocytes in PBMCs. Consistent with the idea that DP monocytes differentiate into DP macrophages at sites of inflammation, we found infiltration of DP macrophages at the site of myosin-induced myocarditis in wild-type rats; these cells exhibited a T-helper 1 (Th1)-type cytokine/chemokine profile and expressed high levels of Fas ligand, perforin, granzyme B, and NKR-P2 (rat orthologue of human NKG2D). Adoptive transfer of GFP-positive spleen cells confirmed hematogenous origin of DP macrophages. DP monocytes had a cytotoxic phenotype similar to DP macrophages, indicating that this phenotypic specialization occurred before entry into a tissue. In line with this, DP monocytes killed tumor cells in vitro. Combined evidence indicates that certain inflammatory stimuli that induce GM-CSF trigger the expansion of a population of DP monocytes with a cytotoxic phenotype and that these cells differentiate into macrophages at inflammatory sites. Interestingly, human PBMCs also contain DP monocytes.

Altered expression of apoptosis-related genes in osteocytes exposed to high-dose steroid hormones and hypoxic stress.

OBJECTIVE: High-dose steroid hormones cause necrosis of the femoral head. Since steroid hormones function as blood coagulants, we hypothesized that ischemic hypoxia induced by steroid hormones is critical for apoptosis which occurs before necrosis of osteocytes. METHODS: We performed an analysis of gene expression in the process of leading osteocytes to apoptosis, using a mouse cell line. Cultured osteocytes were loaded with hypoxic stress with or without exposure to steroid hormones, and the gene expression under these conditions was investigated using a cDNA array and real-time quantitative RT-PCR. RESULTS: The proapoptotic p53 gene was downregulated under a hypoxic (1% O2) condition without exposure to steroid hormones. On the other hand, the expression of antiapoptotic Bcl-2 gene was increased by exposure to high-dose steroid hormones under a normoxic condition (20% O2). Interestingly, both proapoptotic (p53 and Bax) and antiapoptotic (Bcl-2 and MDM2)genes were downregulated in osteocytes treated with high-dose steroid hormones in the hypoxic environment. CONCLUSIONS: These findings suggest that osteocytes exposed to high-dose steroid hormones appear to be more sensitive to apoptosis in the hypoxic environment than those without exposure to steroid hormones. This concept helps to understand the pathogenesis of idiopathic necrosis of the bone.

Malignant transformation of thymoma in recipient rats by heterotopic thymus transplantation from HTLV-I transgenic rats.

Transgenic rats expressing the pX gene of human T lymphocyte virus type-I (HTLV-I) under control of the rat lymphocyte-specific protein tyrosine kinase type-I promoter (lck-pX rats) developed benign epithelial thymomas. When the thymuses of newborn lck-pX rats were transplanted into the subcapsular space of the kidney in other thymectomized lck-pX rats, similar tumors developed in the transplanted thymuses. Following the tumor growth, dissemination in the abdominal cavity and distant metastasis occurred. The tumors were histopathologically similar to the original thymomas, but prominent nuclear atypia and high mitotic activity were present. The Ki-67 index was twice as high as that in the originals. The tumors were transplantable into the subcutis of lck-pX rats, although transplantation of the originals never succeeded. All evidence indicated that malignant transformation of thymoma was induced by the heterotopic transplantation. Expression of the pX transgene in the transformed tumors were significantly reduced. Among host genes, the expression of p16ink4a/ARF, which was significantly upregulated in the originals, was never detected in the transformed tumors. Genomic Southern blots and PCR suggest that homozygous deletion of the p16ink4a/ARF gene may play important roles in malignant transformation in this model. Our model described here is a useful unique model for in vivo malignant transformation.

Evidence of dual function of macrophage migration inhibitory factor relevant to tumor progression and regression.

Macrophage migration inhibitory factor (MIF) is known to play an important role in broad-spectrum inflammation and immune responses. To evaluate the role of MIF in tumor growth, we established transgenic (Tg) mice (ICR strain) driven by cytomegalovirus (CMV) enhancer and beta-actin promoter. We inoculated Tg mice in the back with murine sarcoma cell line S-180 cells. The tumor growth rate was more enhanced in Tg mice than in littermate non-Tg mice up to day 9 after tumor inoculation. Surprisingly, most tumors embedded on the back of Tg mice regressed at day 10 after inoculation and eventually disappeared. Tumor volumes of non-Tg mice incessantly increased until death. We reinoculated the Tg mice with S-180 cells, which had been recovered from the first challenge, and found that the tumor cells were completely rejected in all cases. To identify the effector cells that eradicated the tumor cells, we prepared spleen cells from tumor-bearing Tg mice and carried out cell lysis assay. The magnitude of cytolytic activity of spleen cells obtained from Tg mice was significantly higher against S-180 cells, as well as natural killer cell-sensitive YAC-1 cells, than was the activity of cells from non-Tg mice. Furthermore, we observed that CTL activity of Tg mice against S-180 cells was significantly decreased by the deletion of CD8+ T cells or NK cells. On the other hand, the deletion of CD4+ cells minimally affected the cytolytic activity. Taken together, these results suggest that MIF has the potential to promote tumor growth and angiogenesis in the early phase and, by contrast, this protein could activate CD8+ cytotoxic T cells and NK cells, leading to tumor regression.

Chronic graft-versus-host disease-like autoimmune disorders spontaneously occurred in rats with neonatal thymus atrophy.

We earlier reported that the human T cell leukemia virus type-1 pX gene transduced into rat thymic epithelial cells had an impact on biology of the cells. We report here that FW-pX rats born by mating of F344 transgenic rats expressing the pX gene without tissue specificity with nontransgenic Wistar rats developed disorders, including atrophy of the thymus, lymphocytopenia, and inflammatory cell infiltration into multiple organs, similar to events in chronic graft-vs.-host disease (GVHD). Vanishment of thymic epithelial cells especially in the cortex and marked depletion of CD4 CD8 double-positive thymocytes were evident in the neonatal thymus in these rats. The relative abundance of CD8 compared to CD4 T cells may be related to dominant infiltration of CD8 T cells into the affected organs. Additionally, adoptive transfer of FW-pX splenocytes could induce lymphocytic infiltration into sublethally irradiated wild-type syngeneic recipients. Analysis of the expression level of the Foxp3 gene in peripheral blood mononuclear cells revealed that the numbers of immunoregulatory T cells were less in FW-pX rats than in wild-type rats. The collective evidence suggested that the FW-pX rats spontaneously developed chronic GVHD-like autoimmune diseases, following abortive differentiation of T cells in the thymus in early days of the newborn. This rat model may shed light on the pathogenesis of chronic GVHD and also other systemic autoimmune diseases, the etiology of which is unknown.

Aberrant gene expression by CD25+CD4+ immunoregulatory T cells in autoimmune-prone rats carrying the human T cell leukemia virus type-I gene.

Transgenic rats expressing the env-pX gene of human T cell leukemia virus type-I under the control of the viral long terminal repeat promoter (env-pX rats) developed systemic autoimmune diseases. Prior to disease manifestation, the immunosuppressive function of CD25(+)CD4(+) T (T-reg) cells was impaired in these rats. Since T cell differentiation appeared to be disordered in env-pX rats, we assumed that the impairment of T-reg cells might be caused by an abortive differentiation in the thymus. However, reciprocal bone marrow transfers between env-pX and wild-type rats revealed that direct effects of the transgene unrelated to the thymus framework induced the abnormality of T-reg cells. To identify molecular changes, comparative analyses were done between env-pX and wild-type T-reg cells. Expression of the Foxp3 gene and cell-surface markers supported a naive phenotype for env-pX T-reg cells. Array analyses of gene expression showed some interesting profiles, e.g. up-regulation of genes associated with the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in env-pX T-reg cells. Additionally, expression of the suppressor of cytokine signaling (SOCS) family genes, which inhibit the JAK/STAT signals, was extremely low in env-pX T-reg cells. These findings suggest that the transgene may mediate the down-regulation of the SOCS family genes and that subsequent excess signals through the JAK/STAT pathways may result in the loss of function of env-pX T-reg cells. We suggest that investigation of the pathology of T-reg cells in our autoimmune-prone rat model may aid in understanding the roles of T-reg cells in human autoimmune diseases.

Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination: morphologic alteration and reduced expression of beta1 integrin subsets.

Peritoneal dissemination is one of the main causes of death in cancer patients. Pathophysiology of metastasis has been well investigated, but the mechanism of diffuse spread of tumor colonies in the peritoneal cavity is not fully understood. CD9 is a member of tetraspanin and its down-regulation is known to be involved in poor prognosis. To investigate the significance of the down-regulation of CD9, HTOA, an ovarian carcinoma cell line that highly expressed CD9, was transiently transfected with small interfering RNA (siRNA) against CD9, and CD9-negative cells (HTOA(CD9-)) were purified. HTOA(CD9-) showed altered adhesion patterns on Matrigel, collagen, fibronectin, and laminin compared with those of control siRNA-transfected HTOA (control-HTOA). Flow cytometry and fluorescence cytostainings revealed that the expression levels of integrins beta1, alpha2, alpha3beta1, alpha5, and alpha6 were lower in HTOA(CD9-) than those of control-HTOA. HTOA(CD9-) showed altered expression of junctional and cytoskeletal molecules. By time-lapse video microscopy, control-HTOA showed solid adhesion to extracellular matrix and formed cobblestone pattern, whereas HTOA(CD9-) showed weaker adhesion and were distributed as diffuse spots. To examine whether the expression level of CD9 change during tumor dissemination, HTOA-P, a highly disseminative subclone of HTOA, was established. HTOA-P showed distinctive down-regulation of CD9 at mRNA and protein levels, and showed similar morphologic alteration as HTOA(CD9-) did. These findings indicate that the down-regulation of CD9 may be an acquired event in the process of tumor dissemination. Down-regulated CD9 may attenuate the expression of several integrins and rearrange junctional and cytoskeletal molecules that might contribute to dissemination of ovarian carcinomas.