Terufumi Yamaguchi

All-trans retinoic acid attacks reverse transcriptase resulting in inhibition of HIV-l replication

Abstract We previously reported that all-trans retinoic acid (ATRA) inhibited growth in HTLV-l-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. Interestingly, ATRA significantly inhibited reverse transcriptase (RT) activity similar to azidothimidine (AZT) in HTLV-l-positive T-cell lines. To clarify whether ATRA has an inhibitory effect on the replication of HIV, we examined HIV proviral DNA in a HIV-l-positive cell line (8E5) using real time PCR. ATRA as well as AZT reduced the proviral DNA load of 8E5 in a dose-dependent manner. These results suggest that there is a common element of ATRA signaling in both HTLV-l and HIV. Furthermore, we examined the effects of ATRA on viral replication in primary lymphocytes of three individuals infected with HIV. ATRA reduced viral replication significantly similar to AZT. These findings suggested that ATRA acts as a RT inhibitor, reducing the HIV-l proviral DNA load. Finally, we conclude that ATRA may be a potential therapeutic agent for HIV infection.

Influence of Epstein–Barr virus infection in adult T-cell leukemia

Abstract The involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in about 50% of cases of ATL. Epstein–Barr virus (EBV) infection has often been observed in the clinical course of ATL. In this study, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both lines. As part of the characterization of these cells, an enhanced expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) or ICAM-3 (ICAM-3) (CD50), lymphocyte function-1 (LFA-1) (CD11a/CD18), and Mac-1 (CD11b/CD18) was observed. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donors lymphocytes, an EBV-infected B cell line, Raji, and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was also observed in double transfectants (EBV and HTLV-1). In the clinical course of ATL, LMP-1, EBNA-2, CD50 and CD54 were detected in lymph nodes and skin specimens by immunohistochemical staining. Furthermore, high levels of interleukin-4 (IL-4) were detected in these cell lines and transfectants. The results indicated that coinfection with HTLV-1 and EBV may induce aggressive organ involvement through the enhanced expression of adhesion molecules via IL-4 signaling. A new mechanism of ATL involvement is discussed.

Clinical importance of human herpes virus-8 and human immunodeficiency virus infection in primary effusion lymphoma

Abstract Primary effusion lymphoma (PEL) is a rare type of non-Hodgkin lymphoma that usually develops in immunosuppressed patients infected with human herpes virus-8 (HHV-8) in conjunction with human immunodeficiency virus (HIV) infection. However, there are several reports of HHV-8-related HIV-negative cases and HHV-8-unrelated HIV-negative cases, mainly in immunodeficient and elderly patients. Here, we report one case of HHV-8-related HIV-negative PEL with gastric cancer (case 1) and one case of HHV-8-unrelated HIV-negative effusion-based lymphoma (case 2), both in elderly men. A 73-year-old man and a 79-year-old man were admitted because of lymphomatous effusions, and no mass was detectable in both cases. They were diagnosed as having malignant effusion lymphoma on the basis of cytological findings indicating atypical lymphoid cells and the expression of CD20 and CD79a. To detect evidence of HHV-8 infection in neoplastic cells, immunocytochemical staining for ORF73/ latent nuclear antigen-1 (LNA-1) was performed. The results revealed that case 1 was ORF73-positive, and case 2 was ORF73-negative. Rituximab-based chemotherapy (R-THPCOP: rituximab, pirarubicin, cyclophosphamide, vincristine, prednisolone) was administered to both patients and complete remission was achieved in both. Compared to most HIV-positive PEL cases, these two cases showed a good response to chemotherapy. In cases of PEL, we should focus on HHV-8 infection and HIV status for determining prognosis.

Senescence induction therapy for the treatment of adult T-cell leukemia.

Retinoids (all-trans retinoic acid [ATRA] and tamibarotene [Am-80]) have been reported to inhibit the in vitro growth of human T-lymphotrophic virus type 1 (HTLV-I) (þ) T-cell lines and that of fresh cells obtained from patients with adult T-cell leukemia (ATL) [1,2]. The clinical efficacy of retinoid therapy in treating ATL has also been evaluated [3]. In this study, we focused on the role of retinoids in inducing cellular senescence during the treatment of ATL. Cellular senescence was detected by staining for senescence-associated bgalactosidase (SA b-Gal) [4]. SA b-Gal-positive cells were observed during spontaneous culture without retinoids (ATRA or Am-80) in HTLV-I (þ) T-cell lines (HUT102, MT-2, MT-4, ED40515, and ATL-2), but not in HTLV-I (7) T-cell lines (Jurkat and MOLT-4) [Figure 1(A)]. On treatment with ATRA or Am-80, the number of SA b-Gal-positive cells significantly increased in the HTLV-I (þ) T-cell lines, but not in the HTLV-I (7) ones [Figure 1(A)]. The same trend was observed in fresh ATL cells obtained from three patients with acute ATL [Figure 1(B)]. p16 expression was enhanced in all HTLV-I (þ) T-cell lines, but not in the HTLV-I (7) T-cell lines (Figure 2). A telomeric repeat amplification protocol (TRAP) assay (TRAPEZE Telomerase detection kit; Chemicon, Temecula, CA) revealed that telomerase activity was not inhibited in retinoid-treated HTLV-I (þ) T-cell lines; this indicated premature senescence (data not shown). We observed cellular senescence in HTLV-I (þ) T-cell lines and in fresh primary cells obtained from patients with acute ATL. The grade of cellular senescence was greater for HUT102, MT-2, MT-4, and ATL-2 cells than for ED40515 cells, which do not express Tax mRNA because of a nonsense mutation. This is an additional report pointing to Tax as an oncogene, and oncogene induced senescence (OIS) was possibly initiated. It has been reported that OIS does indeed occur in human and mouse tumor cells in vivo [5,6]. These cells cannot re-enter the cell cycle or undergo tumorigenesis once senescence is triggered [5,7]. OIS is caused by the accumulation of DNA damage. This DNA damage is, in turn, caused by oncogene-driven accumulation of reactive oxygen species (ROS) [8]. Chemotherapy using antineoplastic agents that decrease OIS and reduce cellular senescence may rejuvenate these cells and finally induce chemotherapy resistance. In conclusion, retinoids may be a reasonable agent for ATL by facilitating cellular senescence.

mTOR inhibition and adult T-cell leukemia.

Human T-cell leukemia virus type I (HTLV-I) is ahuman retrovirus and an aetiologic agent of adult T-cell leukemia/lymphoma (ATL/ATLL) [1,2]. ATL/ATLL have a poor prognosis with a median survivalrate of *6 months [3]. Novel therapeutic strategiesfor ATL are required. In the current study, wefocused on mammalian target of rapamycin (mTOR)[4], a serine/threonine kinase that is a downstream

Mutant Type Glutathione S-transferase Theta 1 Gene Homologue to mTOR in Myelodysplastic Syndrome: Possible Clinical Application of Rapamycin

In this study, we observed the expression of the GSTT-1 gene in patients with myelodysplastic syndrome (MDS) at the messenger RNA level. Reverse transcription-polymerase chain reaction (RT-PCR) for GSTT-1 was performed with a pair of primers complementary to the 5′ coding section and the 3′ coding section of the GSTT-1 cDNA for amplifying the 623-bp band. Among 20 patients with MDS, 8 patients showed the expected 623-bp band on RT-PCR, and 12 patients showed a 500-bp band on RT-PCR, indicating that a 123-bp sequence was deleted as a mutant of the GSTT-1 gene. Furthermore, a BLAST DNA search showed that the deletion of a 123 bp sequence creates a sequence that is 63% homologous to human FKBP-rapamycin associated protein (FRAP); this protein has been termed a mammalian target of rapamycin (mTOR). We respectively transfected the wild type and the mutant type GSTT-1 gene in an expression vector to two cell lines (K562 and HL-60). The stable transformants for the wild type and the mutant type GSTT-1 genes were made by G418 selection. Interestingly, rapamycin could induce significant growth inhibition of the stable transformants for mutant type GSTT-1, which was indicative of apoptosis, but not that of those for wild type GSTT-1. These results suggest that rapamycin could be included in the therapeutic modality for the patients with MDS who have the mTOR sequences in GSTT-1 gene.

Cytokine profiles in relapsed multiple myeloma patients undergoing febrile reactions to lenalidomide

Lenalidomide plays a central role in the treatment of multiple myeloma (MM). Ozaki et al. [1] very recently reported two cases that had developed inflammatory reactions to lenalidomide. We also experienced three similar cases among eight cases treated in our hospital from August 2010 to July 2011 (Table 1). Case 1 received lenalidomide (10 mg/day on days 1–21) plus dexamethasone (20 mg on days 1–4) on a 28-day cycle. On day 7 at cycle 1, the patient developed high fever without apparent infection. After tapering the dose of lenalidomide to 5 mg/day without administration of antibiotics, his fever slowly receded. Case 2 received lenalidomide monotherapy (15 mg/day on days 1–21) on a 28-day cycle. On day 14 at cycle 1, the patient developed mild fever without focus of infection. We reduced the dose to 10 mg/day. The fever subsequently receded. Case 3 received lenalidomide (15 mg/day on days 1–21) plus dexamethasone (8 mg on days 1–4) on a 28-day cycle. On day 6 at cycle 1, the patient developed high fever without focus of infection. Five days after stopping lenalidomide, the fever receded. Cases 4–8 received lenalidomide (15 mg on days 1–21) plus dexamethasone therapy (20 mg/day on days 1, 8, 15, 22) on a 28-day cycle. The patient in Case 4 developed high fever due to the oral cavity infection on day 15, while febrile events were not observed in Cases 5–8. To clarify the mechanism underlying the febrile reactions to lenalidomide, we measured serum cytokine levels in these cases by enzyme-linked immunosorbent assay (ELISA) in SRL, Inc. (Tokyo, Japan) using residual serum samples, after obtaining informed consent. As for Cases 1–3, lenalidomide alone or in combination with dexamethasone drastically reduced serum tumor necrosis factor (TNF)-a levels in spite of the febrile reactions (12.2–1.5 pg/ml in Case 1; 2.9–1.8 pg/ml in Case 2; 71.0 to 4.5 pg/ml in Case 3) (Table 1). Lenalidomide also dramatically decreased serum interleukin (IL)-6 levels in Cases 1 and 2 (200.0–8.0 pg/ml in Case 1; 9.0–4.2 pg/ml in Case 2), but not in Case 3 (2.4–54.7 pg/ml). Levels of other cytokines, including pro-inflammatory cytokine IL-1b and anti-inflammatory cytokine IL-10, were scarcely influenced by lenalidomide in these three cases. These results indicate that, although the cytokine(s) involved in the febrile reactions to lenalidomide remains unknown, it appears that, except for the possible involvement of IL-6 in Case 3, these reactions were caused independently of IL-6 and TNF-a. Similarly, lenalidomide reduced serum levels of TNF-a and IL-6 in Cases 4–8, whereas their degrees were considerably different among the cases. Although we cannot deny the possibility that dexamethasone influences the serum cytokine levels, it is believed that serum IL-6 and TNF-a levels are reduced by the standard combination therapy with lenalidomide and dexamethasone in most MM patients. We also examined whether anti-MM effects of lenalidomide might not be affected by the febrile reactions. However, after one cycle of lenalidomide treatment, the % M-protein Y. Morita (&) T. Shimada T. Yamaguchi S. Rai C. Hirase M. Emoto K. Serizawa Y. Taniguchi M. Ojima Y. Tatsumi T. Ashida I. Matsumura Division of Hematology, Department of Internal Medicine, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589-8511, Japan e-mail: moriyasu@med.kindai.ac.jp

Induction of molecular remission by using anti-CC-chemokine receptor 4 (anti-CCR4) antibodies for adult T-cell leukemia: a risk of opportunistic infection after treatment with anti-CCR4 antibodies

The CC-chemokine receptor 4 (CCR4) is expressed in almost ATLL cells. Thus, anti-CCR4 antibodies can be used as a treatment strategy for ATLL. Mogamulizumab (MOG), which is a defucosylated anti-CCR4 monoclonal antibody, showed good results even in patients with recurrent ATLL in phase I or II studies. We treated 8 elderly patients with ATL who were resistant to chemotherapy using MOG monotherapy. All patients received 1.0 mg/kg of mogamulizumab (MOG) once per week for 8 weeks by intravenous infusion. In the present study, we observed CCR4-specific ADCC against CCR4-positive ATL cells. All patients showed CR with a marked decrease in the number of ATL cells. However, 2 patients contracted viral infection because of severe lymphopenia. One patient died because of severe cytomegalovirus infection despite adequate treatment. One patient had Stevens’ Johnson syndrome. These results suggested that MOG was effective in chemotherapy-resistant ATL patients. However, CCR4 is not only on ATL cells but also on endogenous Treg. The decrease in the number of Treg after MOG monotherapy has been expected to boost the antitumor activity and to be involved in the development of immune disorders, including autoimmune diseases. Furthermore, a decrease in CD4+ T cells led to viral infection. In conclusion, several treatments for prophylaxis of opportunistic infection, including CMV infection, should be recommended.

A Case of Secondary Leukemia Subsequent to Myelodysplastic Syndromes Successfully Treated with Azacitidine

Elderly patients with secondary acute myeloid leukemia (AML) following myelodysplastic syndrome (MDS) are often medically unfit for or resistant to chemotherapy, and their prognosis is dismal. In the present paper, we reported a case of secondary leukemia following MDS in an 80-year-old male patient who was deemed unfit for chemotherapy owing to his old age and poor physical condition. Despite a high tumor burden, treatment with AZA exerted a remarkable response, leading to an immediate cytoreduction in our case. Our results suggest that AZA can be an attractive therapeutic option for elderly MDS or AML patients, offering adequate efficacy and high tolerability.

Interleukin-8 in the pathogenesis of primary central nervous system lymphoma in association with HIV infection

Abstract The pathogenesis of acquired immunodeficiency syndrome-associated primary central nervous system lymphoma (AIDS-associated PCNSL) remains unclear. However, cell adhesion molecules have been reported to be strongly associated with PCNSL. In this study, we established Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) from HIV-positive patients (LCLHIV) and normal individuals (LCLN). The expression of CD18 antigen by LCLHIV was stronger than that by LCLN. We performed a cell adhesion assay using ISO-HAS, which is the human hemangiosarcoma cell line and expresses intercellular adhesion molecule 1 (CD54). The binding rates of LCLHIV and ISO-HAS without stimulation were higher than those of LCLN. Further, we demonstrated that azidothymidine or simvastatin inhibited the binding rates of LCLHIV and ISO-HAS more significantly than those of LCLN. Further, the levels of interleukin (IL)-8, a CD18 inducer, were higher in LCLHIV than in LCLN. We conclude that interaction between IL-8 and CD18 may be critical to AIDS-related PCNSL.