Hajime Nomura

Coexistent anaplastic and differentiated thyroid carcinoma : An immunohistochemical study

The aim of the present study was to clarify the underlying molecules that might contribute to the highly aggressive behavior of anaplastic thyroid carcinoma. We selected 5 cases of anaplastic thyroid carcinoma that had a differentiated area to determine differences in the molecules of undifferentiated and differentiated cancer cells. We immunohistochemically examined the localization of nuclear antigen (Ki-67), proliferating cell nuclear antigen (PCNA), p53, apoptotic protease-activating factor-1 (Apaf-1), CD26, galectin-3, E-cadherin, and CD147. We found increased Ki-67, PCNA, and p53 labeling indices; decreased levels of Apaf-1, CD26, galectin-3, and E-cadherin; and overexpression of CD147 in the undifferentiated area compared with the differentiated area. These findings indicate high proliferative properties, suppression of apoptosis, disruption of cell-cell interaction, and induction of matrix metalloproteinases in the undifferentiated areas. Thus the molecules examined might be useful for evaluating the aggressive nature of this tumor and the prognosis.

Coexistent Anaplastic and Differentiated Thyroid Carcinoma.

The aim of the present study was to clarify the underlying molecules that might contribute to the highly aggressive behavior of anaplastic thyroid carcinoma. We selected 5 cases of anaplastic thyroid carcinoma that had a differentiated area to determine differences in the molecules of undifferentiated and differentiated cancer cells. We immunohistochemically examined the localization of nuclear antigen (Ki-67), proliferating cell nuclear antigen (PCNA), p53, apoptotic protease-activating factor-1 (Apaf-1), CD26, galectin-3, E-cadherin, and CD147. We found an increased Ki-67, PCNA, and p53 labeling indices; decreased levels of Apaf-1, CD26, galectin-3, and E-cadherin; and overexpression of CD147 in the undifferentiated area compared with the differentiated area. These findings indicate high proliferative properties, suppression of apoptosis, disruption of cell-cell interaction, and induction of matrix metalloproteinases in the undifferentiated areas. Thus the molecules examined might be useful for evaluating the aggressive nature of this tumor and the prognosis.

Cloning and characterization of the rat p130, a member of the retinoblastoma gene family.

A cDNA clone encoding rat p130, a member of the retinoblastoma (Rb) gene family, was isolated based on the sequence homology of the E1A-binding domain. The 4.87 kb cDNA contained an 1135-amino acid open reading frame with high homologies to the human and mouse p130 and a partial homology to the pRb protein. p130 showed difference in distribution of potential phosphorylation sites from pRb in the N-terminal and the B pocket regions. p130 mRNA was detected in most rat tissues. The p130 gene was mapped to rat chromosome 19p11-13 by fluorescence in situ hybridization.

Defective human T‐lymphotropic virus type 1 provirus in asymptomatic carriers

Few studies have specifically examined defective provirus in asymptomatic human T‐lymphotropic virus Type 1 (HTLV‐1) carriers and its relation to proviral DNA loads (PVLs). To assess the significance of defective provirus in asymptomatic carriers, we examined PVLs in peripheral blood mononuclear cells of 208 asymptomatic HTLV‐1 carriers. The mean PVLs determined using primers for the pol region were less than that for the pX region in these carriers. Analysis of seven carriers with high PVLs for the pX region but lower PVLs for the pol region showed that four had single nucleotide polymorphisms of proviral genomes for the pol region and three had HTLV‐1‐infected cells with defective provirus. Three carriers with defective provirus showed high PVLs at their initial screens, and PVLs increased after a 10‐ to 12‐year interval in two carriers. Southern blot assay showed clonal expansion of HTLV‐1‐infected cells, and the predominant clones changed during the observation period. These data suggest that although HTLV‐1‐infected cells with defective provirus may have a growth advantage, the predominant clones of HTLV‐1‐infected cells do not always survive for many years in asymptomatic carriers.

Multiple integrations of human T-lymphotropic virus type 1 proviruses in the engrafted cells from the asymptomatic carriers in NOD/SCID/gammacnull mice.

Objectives: Successful engraftment of human T-lymphotropic virus type 1 (HTLV-1)-infected cells and a marked increase of proviral DNA loads (PVLs) in non-obese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull (NOG) mice have been reported. Whether the increased PVL in transplanted mice is due to the new infection of HTLV-1 was examined. Methods: Mononuclear cells from 3 NOG mice with primary engraftment from asymptomatic HTLV-1 carriers were transplanted into a second group of NOG mice. HTLV-1 PVL, proviral integration by fluorescence in situ hybridization assay, expression of viral antigen, and T-cell clonality were analyzed. Results: The PVLs in the secondarily transplanted NOG mice were significantly higher than those of primarily transplanted NOG mice. Multiple signals of HTLV-1 proviruses in the nucleus of the infected cells were revealed by fluorescence in situ hybridization analysis. Expression of HTLV-1 tax/rex mRNA and antigen was observed. The variety of T-cell clones was limited in the transplanted NOG mice. Conclusions: Multiple proviral integrations were considered to be due to the new infection from HTLV-1-infected cells to the other cells. Only a certain fraction of T cells seemed to have selectively survived in NOG mice after engraftment.

Infection of defective human T-lymphotropic virus type 1.

In a previous study, we reported that an identical defective provirus had integrated into multiple sites of the genome of a representative human T-lymphotropic virus type 1 (HTLV-1) cell line, MT-2. A possible explanation for this may be the repeated infection of this defective provirus to a cell. Therefore, we attempted to determine whether a defective provirus could transmit during the co-culture of HTLV-1 uninfected human T-cell line, Jurkat, with MT-2 cells treated with mitomycin C. As a result, we established not only a cell line with the integration of one complete provirus, but also a cell line with the integration of one defective provirus. The rearrangement of the T-cell receptor -γ gene of these cell lines showed them to be derived from Jurkat cells. Both HTLV-1 Tax/Rex and HBZ RNA were detected in the cell line, which harbors a complete provirus. On the other hand, HBZ RNA and transcriptional product specific for the defective provirus were detected in the cell line, which harbors a defective HTLV-1 provirus only. These results suggested that a defective HTLV-1 provirus with large depletion of internal sequence could transmit to other cells. Moreover, the defective provirus can be transcriptionally active. This suggested the possibility that the defective HTLV-1 provirus found in the lymphocytes of HTLV-1 carriers and patients with adult T-cell leukemia may transmit to other T-cells in vivo. The results also suggested that defective provirus in HTLV-1 carriers could be functional and may play a role in leukemogenesis.

The time-sequential changes of risk factors for adult T-cell leukemia development in human T-cell leukemia virus-positive patients with rheumatoid arthritis: a retrospective cohort study

Abstract Objective: This study aimed to investigate the time-sequential changes of risk factors for adult T-cell leukemia (ATL) development in human T-cell leukemia virus type 1 (HTLV-1)-positive rheumatoid arthritis (RA) patients. Methods: HTLV-1 infection was screened using particle agglutination assay and confirmed via western blotting in 365 RA patients. Twenty-three HTLV-1-positive RA patients were included in the study cohort. Blood samples were obtained from these patients at each observation time point. The values of HTLV-1 proviral load (PVL) and serum soluble IL-2 receptor (sIL2-R), which are risk factors for ATL development, were measured using real-time PCR and enzyme immunoassay, respectively. Results: The study cohort comprised 79 person-years. The median HTLV-1 PVL and sIL2-R values of the HTLV-1-positive RA patients were 0.44 copies per 100 white blood cells (WBCs) and 406 U/mL, respectively. Three HTLV-1-positive RA patients showed a high PVL value. No remarkable changes were observed in the PVL and sIL2-R values during the observation period. However, one elderly HTLV-1-positive RA patient who had a high PVL value developed ATL during treatment with methotrexate and infliximab. Conclusion: A thorough clinical assessment of the risk factors for ATL development may be necessary in daily clinical practice for RA patients in HTLV-1-endemic areas in Japan.

AB0021 Human T cell leukemia virus type 1 (HTLV-1) exacerbates rheumatoid arthritis; exosomes and IFN-GAMMA derived from HTLV-1 infected cells enhance the inflammatory response of rheumatoid arthritis synovial fibroblasts via pattern recognition receptor, RIG-I

Background Human T cell leukemia type 1 (HTLV-1) positive rheumatoid arthritis (RA) patients show severe inflammatory state and resistance to anti-rheumatic therapy, including biologic agents (1). HTLV-1 infected T cells was increased in the synovial fluid and tissue from an HTLV-1 positive RA patients (2). However the mechanism of worsening RA by HTLV-1 infection remains unclear. We focused on the role of HTLV-1 infected T cells as a key player in the exacerbation of RA. Objectives To clarify the role of HTLV-1 infected T cells in the pathogenesis of RA. We investigate inflammatory mediators derived from HTLV-1 infected cells. Methods Peripheral blood mononuclear cells (PBMCs) were collected from asymptomatic HTLV-1 carriers (AC) (n=5) and healthy subjects (HS) (n=5). Rheumatoid arthritis synovial fibroblasts (RASFs) were co-cultured with PBMCs for 5 days. Cytokine profiles of supernatants were analyzed by multiplex. Exosomes were isolated and purified from cultured medium of HTLV-1 infected cell line (MT2). RASF was cultured with MT2 derived exosomes with and without IFN-gamma for 24hours. Total RNA was extracted using TRIZOL method. The expression of RIG-I, IL-6, CXCL10, and CCL5 mRNA in RASF was measured using real-time quantitative PCR. The expression of pattern recognition receptor, RIG-I was determined by immune blotting. Silencing of RIG-I in RASF was performed by transfection of siRNA against RIG-I. Results The levels of cytokine, including IFN-gamma, IL-2, IL-9, IL-13, IL-6, and CCL20, were higher in supernatants co-cultured with HTLV-1 positive PBMCs than in those of negative PBMC (p<0.05). The expression of CXCL10 and IL-6 mRNA was increased in RASF co-cultured with HTLV-1 positive PBMCs compared to those of negative PBMCs. IFN-gamma is well known to be an important cytokine in the pathogenesis of HTLV-1 associated inflammatory diseases. IFN-gamma induced the expression of IL-6, CCL5, and CXCL10 mRNA in RASF. HTLV-1 infected cell line, MT2, autonomously released a large amount of exosomes which contain nucleic acids such as RNA and DNA. MT2 derived exosomes significantly enhanced the expression of CXCL10 mRNA, but not IL-6 and CCL5, in RASF activated by IFN-gamma. Therefore, we hypothesized that exosomes play the role of ligand for pattern recognition receptors. IFN-gamma increased the expression of RIG-I protein in RASF in a dose-dependent manner. The expression of RIG-I protein also increased in RASF co-cultured with HTLV-1 positive PBMCs compared to those of negative PBMCs. Finally, the silencing of RIG-I suppressed the expression of CXCL10 in RASF induced by co-stimulation of both exosomes and IFN-gamma. Conclusions It is possible that HTLV-1 infected T cells exacerbate the inflammatory responses of RASFs. Exosomes derived from HTLV-1 infected cells enhance the expression of CXCL10 in RASF induced by IFN-gamma via pattern recognition receptor, RIG-I. References Umekita K, et al. Arthritis Care Res (Hoboken). 2014 May;66(5):788–92. Yakova M, et al. Retrovirology. 2005 Feb 1;2:4. Disclosure of Interest None declared

AB0027 A Novel Transcription Factor NFAT5 Plays An Important Role as Critical Regulator in The Inflammatory Response of Rheumatoid Arthritis Fibroblasts Mediated via Toll-Like Receptor 4 Signaling Pathways

Background Damage-associated molecular patterns (DAMPs) are proposed to drive aberrant stimulation of Toll-like receptors (TLRs) in the rheumatoid arthritis (RA) joints resulting in increased expression of proinflammatory cytokines and chemokines. In the recent study we demonstrated that the neutrophil-derived lactoferrin (LTF) induces inflammatory response in RA synovial fibroblasts (RASF) via TLR-4 (Ref.). However, the molecular mechanisms of TLR-4 signaling in activated RASF are still unclear. Objectives To clarify the molecular mechanisms of TLR-4 signaling pathways in activated RASF. Methods Recombinant human neutrophil-derived LTF was used as one of the TLR-4 ligands. RASF were treated with LTF and/or TNF-α, and the expression of proinflammatory cytokines and chemokines, such as IL-6, IL-8 and CCL20 in RASF was measured by RT-qPCR and ELISA. To repress the TLR-4 signaling pathways, a small molecular inhibitor of TLR-4 (TAK-242), TAK1 inhibitor (5Z-7-Oxozeaenol), nuclear factor kappa B (NF-kB) inhibitor (BMS345541), and p38 mitogen activated protein kinase (MAPK) inhibitor (SB202190) were used. The role of nuclear factor of activated T cells 5 (NFAT5) in the TLR-4 signaling in RASF was investigated using a small interfering RNA targeting NFAT5. Results Stimulation of RASF with LTF significantly increased the expression of IL-6, IL-8 and CCL20 mRNA and proteins (p=0.01). LTF enhanced the expression of these cytokine and chemokine mRNA in RASF stimulated by TNFα. TAK-242 completely repressed the expression of these cytokines and chemokines in RASF stimulated by LTF, while the TAK-1 inhibitor did not suppress the expression of these cytokines and chemokines in RASF. The NF-kB inhibitor, but not the p38MAPK inhibitor, partially repressed the expression of IL-6 and IL-8 mRNA induced by LTF. However, neither the NF-kB inhibitor nor p38MAPK inhibitor repressed the expression of CCL20 mRNA. Interestingly, silencing of NFAT5 significantly decreased the basal expression of IL-6, IL-8 and CCL20 mRNA in RASF. Additionally, silencing of NFAT5 significantly repressed the expression of not only IL-6, IL-8, but also of CCL20 mRNA in RASF treated by LTF. Conclusions These findings suggest that NFAT5 plays an important role as a critical regulator in the proinflammatory response of RASF mediated by the TLR-4 signaling pathway. References Umekita K, et al. Neutrophil-Derived Lactoferrin Regulates the Activity of NFAT5 in Rheumatoid Arthritis Synovial Fibroblasts Via Toll-like Receptor 4. 2015 ACR/ARHP Annual Meeting (San Francisco) Acknowledgement We thank Ms. Yuki Kaseda, Ms. Ayaka Miyamoto and Dr. Yatsuki Aratake for their excellent technical assistance. Disclosure of Interest None declared