Importin ß1 regulates cell growth and survival during adult T cell leukemia/lymphoma therapy.

There is no cure for adult T cell leukemia/lymphoma (ATLL) associated with human T cell leukemia virus type 1 (HTLV-1), and novel targeted strategies are needed. NF-κB and AP-1 are crucial for ATLL, and both are transported to the nucleus by an importin (IPO)α/ß heterodimeric complex to activate target genes. In this study, we aimed to elucidate the function of IPOß1 in ATLL. The expression of IPOß1 was analyzed by western blotting and RT-PCR. Cell growth, viability, cell cycle, apoptosis and intracellular signaling cascades were examined by the water-soluble tetrazolium-8 assay, flow cytometry and western blotting. Xenograft tumors in severe combined immune deficient mice were used to evaluate the growth of ATLL cells in vivo. IPOß1 was upregulated in HTLV-1-infected T cell lines. Further, IPOß1 knockdown or the IPOß1 inhibitor importazole and the IPOα/ß1 inhibitor ivermectin reduced HTLV-1-infected T cell proliferation. However, the effect of inhibitors on uninfected T cells was less pronounced. Further, in HTLV-1-infected T cell lines, inhibitors suppressed NF-κB and AP-1 nuclear transport and DNA binding, induced apoptosis and poly (ADP-ribose) polymerase cleavage, and activated caspase-3, caspase-8 and caspase-9. Inhibitors also mediated G1 cell cycle arrest. Moreover, the expression of NF-κB- and AP-1-target proteins involved in cell cycle and apoptosis was reduced. In vivo, the IPOα/ß1 inhibitor ivermectin decreased ATLL tumor burden without side effects. IPOß1 mediated NF-κB and AP-1 translocation into ATLL cell nuclei, thereby regulating cell growth and survival, which provides new insights for targeted ATLL therapies. Thus, ivermectin, an anti-strongyloidiasis medication, could be a potent anti-ATLL agent.

Group 2 Innate Lymphoid Cells Exacerbate Amebic Liver Abscess in Mice.

Entamoeba histolytica, a protozoan parasite in the lumen of the human large intestine, occasionally spreads to the liver and induces amebic liver abscesses (ALAs). Upon infection with E. histolytica, high levels of type 2 cytokines are induced in the liver early after infection. However, the sources and functions of these initial type 2 cytokines in ALA formation remain unclear. In this study, we examined the roles of group 2 innate lymphoid cells (ILC2s) in ALA formation. Hepatic ILC2 numbers were significantly increased and they produced robust levels of IL-5. The in vivo transfer of ILC2s into Rag2-/-common γ chain (γc)-/- KO mice aggravated ALA formation accompanied by eosinophilia and neutrophilia. Furthermore, IL-33-deficient mice and IL-5-neutralized mice had less ALA formations. These results suggest that ILC2s contribute to exacerbating the pathogenesis of ALA by producing early type 2 cytokines and promoting the accumulation of eosinophils and neutrophils in the liver.

Evaluation of artesunate for the treatment of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive disease caused by infection with human T-cell leukemia virus type 1 (HTLV-1). Successful treatment is limited by resistance to chemotherapies. Therefore, there is an urgent need to develop novel effective strategies. Artesunate (ART), a widely used antimalarial compound, has been shown to exert cytotoxicity. Here, we aimed to assess the anti-ATLL activities of ART and to elucidate the possible molecular mechanisms involved in this effect. Compared with uninfected T cells, HTLV-1-infected T-cell lines were sensitive to ART-induced cytotoxicity. ART caused cell cycle arrest at G1 and/or G2/M phases, which was associated with decreased expression of cyclin dependent kinase 1/2/4/6, cyclin B1/D2/E and c-Myc, and increased expression of p21. ART-induced apoptosis corresponded to activation of caspase-8/9/3; decreased expression of Bcl-xL, Bcl-2, myeloid cell leukemia-1, survivin, X-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis 1/2; and increased expression of Bak. ART increased intracellular reactive oxygen species and activation of the DNA damage marker γ-H2AX. Moreover, ART-induced cytotoxicity was partly reversed by treatment with a reactive oxygen species scavenger, iron chelator, and necroptosis or ferroptosis inhibitor, suggesting the involvement of caspase-dependent and -independent lethal pathways. These effects were correlated with inhibition of nuclear factor-κB and activator protein-1 signaling through dephosphorylation of IκBα, IκB kinase (IKK) α and IKKß, and decreased expression of JunB and JunD. Importantly, intraperitoneal injection with ART lowered tumor burden in an ATLL murine model. These preclinical results provide a rationale for evaluating the efficacy of ART in patients with ATLL.

Impairment of tissue repair in pneumonia due to ß-cell deficiency: role of endoplasmic reticulum stress in alveolar macrophages.

OBJECTIVE: Diabetes mellitus (DM) patients are susceptible to delayed resolution of pneumonia. However, the pathogenesis of the impaired tissue repair in inflamed lungs in diabetic patients is unknown. We evaluated phagocytosis of apoptotic cells (efferocytosis), hepatocyte growth factor (HGF) production in bronchoalveolar lavage fluid (BALF), and lung histology in the resolution phase following acute lung injury in streptozotocin (STZ)-induced ß-cell-depleted hyperglycemic mice. We also investigated efferocytosis and HGF production by macrophages under ß-cell depletion condition ex vivo. RESULTS: In ß-cell-depleted mice, efferocytosis was not significantly different from that in control mice; however, the concentration of HGF in BALF was decreased. In addition, diminished HGF production by alveolar macrophages and DNA synthesis in the alveolar epithelium was observed by immunohistochemistry. Ex vivo experiments confirmed that HGF production by macrophages was impaired under ß-cell depletion probably because of endoplasmic reticulum stress.

Anti-adult T­cell leukemia/lymphoma activity of cerdulatinib, a dual SYK/JAK kinase inhibitor.

Adult T­cell leukemia/lymphoma (ATLL) constitutes an aggressive malignancy caused by human T­cell leukemia virus type 1 (HTLV­1) that is resistant to available chemotherapeutics. The constitutive activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling is an important feature of ATLL, and spleen tyrosine kinase (SYK) is overexpressed in HTLV­1-transformed T­cell lines. In this study, we evaluated the effects of SYK- (PRT060318) or JAK- (JAK inhibitor 1) selective inhibitors and the dual SYK/JAK inhibitor, cerdulatinib, on the viability of HTLV­1-transformed and ATLL-derived T­cell lines. Cell proliferation, viability, cell cycle, apoptosis and intracellular signaling cascades were analyzed by the water-soluble tetrazolium-8 assay, flow cytometry and western blot analysis. HTLV­1-infected T­cell lines were sensitive to both SYK-selective and pan-JAK inhibitors, whereas cerdulatinib more potently suppressed cell proliferation and reduced cell viability than either of these agents alone. By contrast, the cytotoxic effects of cerdulatinib on uninfected T­cell lines and peripheral blood mononuclear cells from a healthy donor were less pronounced. Cerdulatinib induced cell cycle arrest in the G2/M phase, which was associated with a decreased cyclin-dependent kinase 1 and cyclin B1, and an increased p21 and p27 expression. Hoechst staining revealed chromatin condensation and nuclear fragmentation in the cells treated with cerdulatinib, and an increased fraction of apoptotic APO2.7-stained cells was detected by flow cytometry. This corresponded to the activation of caspase-8, -9 and -3, and decreased levels of the anti-apoptotic factors, Bcl-xL, survivin, X-linked inhibitor of apoptosis (XIAP) and c­FLIP. The cerdulatinib-induced decrease in cell viability was partly reversed by the caspase inhibitor, z­VAD­FMK. These anti-ATLL effects were associated with the suppression of SYK and JAK/STAT signaling, along with that of the downstream factors, AKT, ERK, activator protein­1 and nuclear factor-κB. Finally, oral dosing with cerdulatinib lowered the tumor burden in a murine model of ATLL. Thus, our findings indicate that the simultaneous inhibition of therapeutically relevant targets, such as SYK and JAK is a more effective approach than single-agent therapy for the treatment of ATLL.

Mitotic kinase PBK/TOPK as a therapeutic target for adult T­cell leukemia/lymphoma.

Adult T­cell leukemia/lymphoma (ATLL) is a disorder involving human T-cell leukemia virus type 1 (HTLV­1)-infected T­cells characterized by increased clonal neoplastic proliferation. PDZ-binding kinase (PBK) [also known as T­lymphokine-activated killer cell-originated protein kinase (TOPK)] is a serine/threonine kinase expressed in proliferative cells and is phosphorylated during mitosis. In this study, the expression and phosphorylation of PBK/TOPK were examined by western blot analysis and RT­PCR. We found that PBK/TOPK was upregulated and phosphorylated in HTLV­1-transformed T­cell lines and ATLL­derived T­cell lines. Notably, CDK1/cyclin B1, which phosphorylates PBK/TOPK, was overexpressed in these cells. HTLV­1 infection upregulated PBK/TOPK expression in peripheral blood mononuclear cells (PBMCs) in co-culture assays. The potent PBK/TOPK inhibitors, HI­TOPK­032, and fucoidan from brown algae, decreased the proliferation and viability of these cell lines in a dose­dependent manner. By contrast, the effect of HI­TOPK­032 on PBMCs was less pronounced. Treatment with HI­TOPK­032 resulted in G1 cell cycle arrest, and decreased CDK6 expression and pRb phosphorylation, which are critical determinants of progression through the G1 phase. In addition, HI­TOPK­032 induced apoptosis, as evidenced by morphological changes, the cleavage of poly(ADP-ribose) polymerase with the activation of caspase­3, -8 and -9, and an increase in the sub­G1 cell population and APO2.7-positive cells. Moreover, HI­TOPK­032 inhibited the expression of cellular inhibitor of apoptosis 2 (c-IAP2), X-linked inhibitor of apoptosis protein (XIAP), survivin and myeloid cell leukemia­1 (Mcl­1), and induced the expression of Bak and interferon-induced protein with tetratricopeptide repeats (IFIT)1, 2 and 3. It is noteworthy that the use of this inhibitor led to the inhibition of the phosphorylation of IκB kinase (IKK)α, IKKß, IκBα, phosphatase and tensin homolog (PTEN) and Akt, and to the decreased protein expression of JunB and JunD, suggesting that PBK/TOPK affects the nuclear factor-κB, Akt and activator protein­1 signaling pathways. In vivo, the administration of HI­TOPK­032 suppressed tumor growth in an ATLL xenograft model. Thus, on the whole, this study on the identification and functional analysis of PBK/TOPK suggests that this kinase is a promising molecular target for ATLL treatment.

Effects of NVP-BEZ235, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on HTLV-1-infected T-cell lines.

Adult T-cell leukemia (ATL) is an aggressive type of malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). In ATL, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is constitutively active, promoting cell proliferation, survival and chemoresistance. Thus, the PI3K signaling pathway is an attractive therapeutic target for ATL. In the present study, the effects of RAD001 (an mTOR inhibitor), NVP-BKM120 (a pan-PI3K inhibitor) and NVP-BEZ235 (a novel dual PI3K/mTOR inhibitor) on cultured HTLV-1-infected T-cell lines were compared. The results demonstrated that NVP-BEZ235 was more efficacious compared with RAD001 and NVP-BKM120 at inhibiting cell growth. NVP-BEZ235 exhibited cytostatic rather than cytotoxic effects on various HTLV-1-infected T-cell lines, where it induced cell cycle arrest at G1 phase. NVP-BEZ235 downregulated cyclin D1, cyclin D2, cyclin E, cyclin dependent kinase (CDK)2 and CDK4 expression, and the phosphorylation of retinoblastoma protein. In C.B-17/Icr-severe combined immune deficiency mice implanted with HTLV-1-infected HUT-102 cells, oral NVP-BEZ235 caused marked retardation of tumor growth compared with the control. The present in vitro and in vivo studies highlight the efficacious dual inhibition of PI3K, and mTOR following NVP-BEZ235 treatment. Thus, the results of the current study provide preclinical rationale for phase I clinical studies to examine the effects of NVP-BEZ235 in patients with ATL.

Schistosoma mansoni infection suppresses the growth of Plasmodium yoelii parasites in the liver and reduces gametocyte infectivity to mosquitoes.

Malaria and schistosomiasis are major parasitic diseases causing morbidity and mortality in the tropics. Epidemiological surveys have revealed coinfection rates of up to 30% among children in Sub-Saharan Africa. To investigate the impact of coinfection of these two parasites on disease epidemiology and pathology, we carried out coinfection studies using Plasmodium yoelii and Schistosoma mansoni in mice. Malaria parasite growth in the liver following sporozoite inoculation is significantly inhibited in mice infected with S. mansoni, so that when low numbers of sporozoites are inoculated, there is a large reduction in the percentage of mice that go on to develop blood stage malaria. Furthermore, gametocyte infectivity is much reduced in mice with S. mansoni infections. These results have profound implications for understanding the interactions between Plasmodium and Schistosoma species, and have implications for the control of malaria in schistosome endemic areas.

Intestinal Inflammation-Mediated Clearance of Amebic Parasites Is Dependent on IFN-γ.

Intestinal amebiasis is a major cause of diarrhea. However, research on host-amebae interactions has been hampered owing to a lack of appropriate animal models. Recently, a mouse model of intestinal amebiasis was established, and using it, we reported that Entamoeba moshkovskii colonized the intestine in a manner similar to that of the pathogenic Entamoeba histolytica In this study, we evaluated the protective mechanisms present against amebae using this model. CBA/J mice infected with E. histolytica had a persistent infection without apparent symptoms. In contrast, E. moshkovskii-infected mice rapidly expelled the ameba, which was associated with weight loss, diarrhea, and intestinal damage characterized by apoptosis of intestinal epithelial cells (IECs). Expression of NKG2D on intestinal intraepithelial lymphocytes (IELs) and IFN-γ-producing cells in Peyer's patches were significantly induced after infection with E. moshkovskii but not with E. histolytica IFN-γ-deficient mice infected with E. moshkovskii showed no obvious symptoms. Notably, none of these mice expelled E. moshkovskii, indicating that IFN-γ is responsible not only for intestinal symptoms but also for the expulsion of amebae. Furthermore, apoptosis of IECs and expression of NKG2D on IELs observed in E. moshkovskii-infected mice did not occur in the absence of IFN-γ. In vivo blocking of NKG2D in mice infected with E. moshkovskii enabled ameba to survive longer and remarkably reduced apoptotic IECs. Our results clearly demonstrate a novel protective mechanism exerted by IFN-γ against intestinal amebae, including induction of cytotoxicity of IELs toward IECs.

IL-17A contributes to reducing IFN-γ/IL-4 ratio and persistence of Entamoeba histolytica during intestinal amebiasis.

Amebiasis is an infectious disease caused by Entamoeba histolytica, an anaerobic protozoan parasite, and is a major public health problem worldwide, particularly in areas with inadequate sanitation and poor hygiene. Th1 responses, represented by interferon gamma (IFN-γ), play a protective role by clearing the amebae from the gut, whereas Th2 responses are responsible for chronic infection. Th17 responses preconditioned by vaccination or by modulating the intestinal microbiome protect mice from the settlement of E. histolytica. However, the role of interleukin-17A (IL-17A), which is upregulated during the natural course of intestinal amebiasis, has not been clarified. The aim of this study was to investigate the role of IL-17A during intestinal amebiasis in a mouse model. IL-17A knockout and wild-type CBA/J mice were challenged intracecally with 2×106E. histolytica trophozoites, and their infection, pathology, and immune responses were monitored. Neither the initial settlement of E. histolytica nor the inflammation of the cecum was affected by the absence of IL-17A for week 1, but the infection rate and parasite burden declined in a late stage of infection, accompanied by an increased IFN-γ/IL-4 ratio. Therefore, IL-17A contributes to the persistence of E. histolytica and modulates the immune response, including the IFN-γ/IL-4 ratio, which may be responsible for the reduction of the parasite burden in the IL-17A knockout mice during the chronic phase of intestinal amebiasis.

Expression and significance of Pim-3 kinase in adult T-cell leukemia.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Viral Tax protein plays a major role in ATL development. Pim family of serine/threonine kinases is composed of Pim-1, -2, and -3. The potential of Pim family as a target in ATL was analyzed. METHODS: RT-PCR and Western blotting were used to determine the expression of Pim kinases, Tax, and intracellular signal molecules. Knockdown of Pim-3 and RelA was performed using small interfering RNA. The effects on cell proliferation, viability, cell cycle, and apoptosis were analyzed by WST-8, propidium iodide, and APO2.7 assay. NF-κB DNA binding activity was investigated by electrophoretic mobility shift assay. RESULTS: Pim-3 expression was restricted to HTLV-1-infected T-cell lines. Tax induced Pim-3 expression through NF-κB. Knockdown of Pim-3 showed growth inhibition of HTLV-1-infected T cells. NJC97-NH, a novel inhibitor of the Pim-1/3 kinases, inhibited cell viability. NJC97-NH induced G2/M cell cycle arrest associated with downregulation of cyclin A and cyclin B1 expression, as well as apoptosis accompanied with downregulation of XIAP and Mcl-1 expression through inhibition of NF-κB pathway, mediated through decrease in IκBα and RelA phosphorylation. CONCLUSION: Pim-3 is a potentially suitable target for the development of novel therapeutic agents against ATL.

Butein inhibits NF-κB, AP-1 and Akt activation in adult T-cell leukemia/lymphoma.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL) but there is no effective treatment for HTLV-1-associated diseases. Herein, we determined the effect of butein, a bioactive plant polyphenol, on cell growth, apoptosis and signaling pathways in HTLV-1-infected T-cell lines and on tumor growth in SCID mice. Treatment with butein caused a decrease in viability of HTLV-1-infected T-cell lines. T cells cultured with butein showed obvious apoptosis morphology, and cleavage of poly(ADP-ribose) polymerase with activation of caspase-3, -8 and -9. Pretreatment of cells with caspase inhibitor partially blocked butein-induced inhibition of cell viability. Butein also resulted in cell cycle arrest at G1 phase. Butein markedly downregulated the protein expression levels of CDK4, CDK6, cyclin D1, cyclin D2, cyclin E, survivin, XIAP, c-IAP2 and phospho-pRb. Butein also inhibited i) total and phospho-protein levels of IκB kinase (IKK)α and IKKß, ii) degradation and phosphorylation of IκBα, iii) JunB and JunD, iv) total and phospho-protein levels of Akt, v) phosphorylation of RelA, vi) heat shock protein 90, and vii) DNA-binding activity of NF-κB and AP-1. In mice harboring ATLL xenograft tumors, butein caused a significant inhibition of tumor growth and reduced serum levels of soluble interleukin-2 receptor α chain and soluble cluster of differentiation 30. Considered together, the results indicated that butein has antiproliferative and proapoptotic properties through the suppression of NF-κB, AP-1 and Akt signaling in HTLV-1-infected T cells, both in vitro and in vivo, suggesting its therapeutic potential against HTLV-1-associated diseases including ATLL.

Peridinin, a carotenoid, inhibits proliferation and survival of HTLV-1-infected T-cell lines.

Human T-cell leukemia virus type 1 (HTLV-1) causes either adult T-cell leukemia (ATL) or chronic inflammatory disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. These diseases are not curable as yet; therefore new agents for treatment and prevention are needed. Carotenoids are natural plant compounds with anti-carcinogenic activities. Peridinin is one of the most abundant carotenoids found in nature. Based on a series of past experiments, here we investigated the effects of peridinin extracted from Okinawan coral Isis hippuris on the proliferation and survival of HTLV-1-infected T-cell lines. The results of water-soluble tetrazolium-8 assay indicated that peridinin dose-dependently inhibits cell proliferation and viability of HTLV-1-infected T-cell lines. Flow cytometry showed that low concentration of peridinin induced cell cycle arrest at G1 phase, while higher concentration induced apoptosis. Peridinin caused cleavage of caspase-3, -8 and -9. Peridinin significantly reduced the expression of G1 cell cycle regulators, including cyclin D1, cyclin D2, CDK4, CDK6 and c-Myc, and anti-apoptotic proteins, including survivin, XIAP and Bcl-2, in a dose-dependent manner. Peridinin suppressed DNA binding of NF-κB. Peridinin inhibited phosphorylation of IκBα, RelA, Akt and p70 S6 kinase, and reduced protein expression level of 3-phosphoinositide-dependent protein kinase 1. Thus, peridinin exerts its anti-proliferative and pro-apoptotic effects by suppressing NF-κB and Akt signaling in HTLV-1-infected T cells. Peridinin also reduced tumor growth in mice harboring ATL xenograft tumors. The results suggested that peridinin is a potentially suitable therapeutic agent against HTLV-1-associated diseases.

Efficiency of AUY922 in mice with adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATLL is associated with poor prognosis mainly due to resistance to chemotherapy, which highlights the requirement for alternative therapies. The chaperone heat shock protein (HSP) 90 assist proteins involved in the onset and progression of ATLL. In the present study, the efficacy of a second generation HSP90 inhibitor termed AUY922 was investigated in ATLL. In vitro, AUY922 induced marked inhibition of cell viability in the HTLV-1-infected T-cell lines HUT-102 and MT-4. In immunodeficient mice bearing HUT-102 xenotransplants, AUY922 markedly retarded tumor growth, compared with the control group. Apoptosis was evident in hematoxylin and eosin stained- and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-labeled tissue sections from AUY922-treated mice. In addition, AUY922 significantly reduced the serum levels of the surrogate tumor markers soluble interleukin-2 receptor and soluble cluster of differentiation 30. Overall, the present results demonstrate that AUY922 has potent anti-ATLL activity, thus providing a rationale for continuing the clinical development of HSP90 inhibitors in clinical trials for the treatment of patients with ATLL.

Induction of IκB-ζ by Epstein-Barr virus latent membrane protein-1 and CD30.

Activation of nuclear factor-κB (NF-κB) in Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HL) cells is important in the transformation and development process of these lymphomas. Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) and ligand-independent signaling by overexpressed CD30 are known to cause permanent activation of NF-κB in lymphomas. However, hyperactivation of NF-κB triggers cellular senescence and apoptosis. Here, we show that IκB-ζ, an inducible regulator of NF-κB, is constitutively expressed in BL and HL cell lines. In addition, immunohistochemical staining identified nuclear IκB-ζ­positive BL cells, and Hodgkin and Reed-Sternberg cells in lymph nodes. Expression of LMP-1 and CD30 increased IκB-ζ expression at the transcriptional level. IκB-ζ promoter was regulated by activation of the NF-κB­inducing kinase (NIK)/IκB kinase/NF-κB pathway via the carboxyl­terminal tumor necrosis factor (TNF) receptor­associated factor (TRAF)-interacting regions of LMP-1 and CD30. Interestingly, IκB-ζ inhibited NF-κB activation by LMP-1 and CD30. The results suggest that NF-κB-induced IκB-ζ negatively modulates NF-κB hyperactivation, resulting in a fine balance that ultimately endows a net evolutionary benefit to the survival of BL and HL cells.

Constitutive expression of IRF-5 in HTLV-1-infected T cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL), an aggressive and fatal leukemia of T cells. Interferon regulatory factor (IRF)-5 plays a critical role in the induction of interferon genes in viral infected cells. We examined the specific mechanisms underlying the expression and regulation of IRF-5 in HTLV-1-infected T cells. IRF-5 was constitutively transcribed into three distinct alternatively spliced isoforms (V1, V3 and V4) in HTLV-1-infected T-cell lines but not in uninfected T-cell lines. IRF-5 was also upregulated in HTLV-1-infected T-cell lines at protein level. Nuclear IRF-5 expression was noted in ATL cells present in lymph nodes and skin lesions. IRF-5 mRNA expression was induced following infection of T cells with HTLV-1, and specifically by viral oncoprotein Tax. Tax also activated V3 promoter. Microarray analysis of IRF-5-expressing uninfected T cells demonstrated that IRF-5 induced the expression of tumor necrosis factor family cytokines. The results suggest that IRF-5 is a Tax-regulated gene, and its expression may be associated with the pathogenesis of ATL.

Activation of PKC-δ in HTLV-1-infected T cells.

Protein kinase C (PKC)-δ is a member of the PKC family. It has been implicated in tumor suppression as well as survival of various cancers. The aggressive malignancy of T lymphocytes known as adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we show that HTLV-1-infected T cells are characterized by phosphorylation and nuclear translocation of PKC-δ. Expression of HTLV-1 regulatory protein Tax increased PKC-δ phosphorylation. Blockade of PKC-δ by rottlerin suppressed PKC-δ phosphorylation and inhibited cell viability in HTLV-1-infected T-cell lines and primary ATL cells. Rottlerin induced cell cycle arrest at the G1 phase and caspase-mediated apoptosis of HTLV-1-infected T cells. Rottlerin downregulated the expression of proteins involved in G1/S cell cycle transition, cyclin D2, CDK4 and 6, and c-Myc, resulting in dephosphorylation of retinoblastoma protein (pRb). Furthermore, rottlerin reduced the expression of important anti-apoptotic proteins (e.g., survivin, XIAP, Bcl-xL and c-FLIP) and Bcl-2 phosphorylation, and activated the pro-apoptotic protein Bax. Our results showed that permanent activation of nuclear factor-κB (NF-κB) by HTLV-1 Tax allows infected cells to escape cell cycle arrest and apoptosis and that PKC-δ mediates Tax-induced activation of NF-κB. Based on these findings, new therapies designed to target PKC-δ could be potentially useful in the treatment of ATL.

Protective role of TNF-α, IL-10 and IL-2 in mice infected with the Oshima strain of Tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) causes acute central nervous system disease. Here, we investigated the roles of the TNF-α, IL-10 and other cytokines in appropriate KO mice following infection with Oshima and Sofjin strains of TBEV. Following infection with the Oshima strain, mortality rates were significantly increased in TNF-α KO and IL-10 KO mice compared with wild type (WT) mice. These results suggested that TNF-α and IL-10 play protective roles against fatal infection due to Oshima strain infection. However, viral loads and proinflammatory cytokine levels in the brain of TNF-α KO andIL-10 KO mice were not significantly different compared with those of WT mice. On the other hand, all WT, TNF-α KO and IL-10 KO mice died following infection with Sofjin strain. Interestingly, Sofjin-infected mice did not exhibit an up-regulated mRNA level of IL-2 in the spleen in all groups of mice, whereas Oshima-infected mice showed significantly increased level of IL-2 compared with mock-infected mice. From these results, we suggest that TNF-α, IL-10 and IL-2 are key factors for disease remission from fatal encephalitis due to infection with Oshima strain of TBEV.

Involvement of IL-18 in the expansion of unique hepatic T cells with unconventional cytokine profiles during Schistosoma mansoni infection.

Infection with schistosomes invokes severe fibrotic granulomatous responses in the liver of the host. Schistosoma mansoni infection induces dramatic fluctuations in Th1 or Th2 cytokine responses systemically; Th1 reactions are provoked in the early phase, whilst Th2 responses become dominant after oviposition begins. In the liver, various unique immune cells distinct from those of conventional immune competent organs or tissues exist, resulting in a unique immunological environment. Recently, we demonstrated that S. mansoni infection induces unique CD4+ T cell populations exhibiting unconventional cytokine profiles in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. They produce both IFN-γ and IL-4 or both IFN-γ and IL-13 simultaneously. Moreover, T cells secreting triple cytokines IFN-γ, IL-13 and IL-4 were also induced. We term these cells Multiple Cytokine Producing Hepatic T cells (MCPHT cells). During the transition phase, when MCPHT cells increase, IL-18 secretion was up-regulated in the liver and sera. In S. mansoni-infected IL-18-deficient mice, expansion of MCPHT cells was curtailed. Thus our data suggest that IL-18 produced during S. mansoni infection play a role in the expansion of MCPHT cells.

Cytotoxic effects of fucoidan nanoparticles against osteosarcoma.

In this study, we analyzed the size-dependent bioactivities of fucoidan by comparing the cytotoxic effects of native fucoidan and fucoidan lipid nanoparticles on osteosarcoma in vitro and in vivo. In vitro experiments indicated that nanoparticle fucoidan induced apoptosis of an osteosarcoma cell line more efficiently than native fucoidan. The more potent effects of nanoparticle fucoidan, relative to native fucoidan, were confirmed in vivo using a xenograft osteosarcoma model. Caco-2 cell transport studies showed that permeation of nanoparticle fucoidan was higher than native fucoidan. The higher bioactivity and superior bioavailability of nanoparticle fucoidan could potentially be utilized to develop novel therapies for osteosarcoma.