Takuro Watanabe

Ligand-independent signaling by overexpressed CD30 drives NF-κB activation in Hodgkin–Reed-Sternberg cells

Overexpression of CD30 and constitutive NF-κB activation characterizes tumor cells of Hodgkins disease (HD), Hodgkin and Reed-Sternberg (H–RS) cells. We report that in H–RS cells overexpression of CD30 leads to self-aggregation, recruitment of TRAF2 and TRAF5, and NF-κB activation, independent of CD30 ligand. CD30 and TRAF proteins co-localized in H–RS cell lines and in lymph nodes of HD. An adenovirus-vector carrying a decoy CD30 lacking the cytoplasmic region or a dominant negative IκBα mutant blocks NF-κB activation, down regulates IL-13 expression and induces apoptosis. Thus, in H–RS cells, ligand-independent activation of CD30 signaling drives NF-κB activation and this leads to constitutive cytokine expression, which provides a molecular basis for HD. Inhibition of NF-κB activation by adenovirus vector-mediated gene transfer may provide a novel strategy of cell- and target molecule-specific therapy for patients with HD.

Serum autotaxin measurement in haematological malignancies: a promising marker for follicular lymphoma

Autotaxin (ATX) is a tumour cell motility‐stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B‐cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients’ clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin‐2 receptor and β2‐microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.

Cytoplasmic Aggregation of TRAF2 and TRAF5 Proteins in the Hodgkin-Reed-Sternberg Cells

We previously reported that ligand-independent signaling by highly expressed CD30 in Hodgkin-Reed-Sternberg (H-RS) cells is responsible for constitutive activation of NF-kappa B. In the present study, we characterize the intracellular localization of tumor necrosis factor (TNF) receptor associated factor (TRAF) proteins in H-RS cells. Confocal immunofluorescence microscopy of cell lines derived from H-RS cells and HEK293 transformants highly expressing CD30 revealed aggregation of TRAF2 and TRAF5 in the cytoplasm as well as clustering near the cell membrane. In contrast, TRAF proteins were diffusely distributed in the cytoplasm in cell lines unrelated to Hodgkins disease (HD) and control HEK293 cells. Furthermore, the same intracellular distribution of TRAF proteins was demonstrated in H-RS cells of lymph nodes of HD, but not in lymphoma cells in lymph nodes of non-Hodgkins lymphoma. Dominant-negative TRAF2 and TRAF5 suppressed cytoplasmic aggregation along with constitutive NF-kappa B activation in H-RS cell lines. Confocal immunofluorescence microscopy also revealed co-localization of IKK alpha, NIK, and I kappa B alpha with aggregated TRAF proteins in H-RS cell lines. These results suggest involvement of TRAF protein aggregation in the signaling process of highly expressed CD30 and suggest they function as scaffolding proteins. Thus, cytoplasmic aggregation of TRAF proteins appears to reflect constitutive CD30 signaling which is characteristic of H-RS cells.

Long‐term ultra‐low‐dose acyclovir against varicella‐zoster virus reactivation after allogeneic hematopoietic stem cell transplantation

To evaluate the efficacy of long‐term prophylaxis with ultra‐low‐dose acyclovir against varicella‐zoster virus (VZV) reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2006 at our hospital. We started long‐term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least 1 year after transplantation. Sixty‐six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. Only one breakthrough reactivation occurred during long‐term acyclovir, which responded well to therapeutic dose of valacyclovir. The use of long‐term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20% vs. 50%, P < 0.0001). With this prophylaxis, visceral dissemination and serious complications other than post‐herpetic neuralgia was completely eliminated, and thereby need for hospitalization was significantly reduced (21% vs. 71%, P = 0.0034). Fifteen of the 57 patients who discontinued acyclovir developed VZV reactivation, with a cumulative incidence of 32.1%. VZV reactivation following discontinuation tended to occur in patients who were receiving immunosuppressive therapy at the cessation of acyclovir. These findings suggested that long‐term prophylaxis of ultra‐low‐dose acyclovir resulted in a successful prevention of severe VZV‐related symptoms and death, with a significantly decreased overall incidence of VZV reactivation. Prolongation of prophylactic acyclovir on profound immunosuppression might be important for thorough suppression of VZV reactivation. Am. J. Hematol., 2008.

Overexpression of hRFI inhibits 5-fluorouracil-induced apoptosis in colorectal cancer cells via activation of NF-κB and upregulation of BCL-2 and BCL-XL

Resistance to apoptosis is one of the important determinants of resistance to 5-fluorouracil (5-FU) in colorectal cancer cells. Human Ring-Finger homologous to Inhibitor of apoptosis protein type (hRFI) is a newly discovered gene that has been shown to inhibit death receptor-mediated apoptosis in colorectal cancer cells. However, the molecular mechanism of the inhibition of apoptosis is presently unknown. In order to investigate the molecular function of hRFI in the regulation of 5-FU-induced apoptosis in colorectal cancer cells, HCT116 cells were stably transfected with hRFI or LacZ as a control. hRFI overexpression resulted in cellular resistance to 5-FU through an inhibition of the mitochondrial apoptotic pathway and specific upregulation of Bcl-2 and Bcl-XL. Futhermore, hRFI overexpression resulted in the activation of nuclear factor-κB (NF-κB). Inhibition of NF-κB effectively reversed the resistance to apoptosis as well as the upregulation of Bcl-2 and Bcl-XL in the hRFI transfectant, indicating that the activation of NF-κB is the key mechanism for all these findings. Overexpression of hRFI in SW480 and COLO320 colorectal cancer cells similarly resulted in resistance to 5-FU with the activation of NF-κB and upregulation of Bcl-2 and Bcl-XL. hRFI might be a novel therapeutic target for gene therapy in colorectal cancer.

Clinical features of late cytomegalovirus infection after hematopoietic stem cell transplantation

Late cytomegalovirus (CMV) disease beyond day 100 after hematopoietic stem cell transplantation (HSCT) has become an increasing problem after the introduction of preemptive ganciclovir (GCV) administration. To clarify the risk factors and outcome for late CMV reactivation and disease, we retrospectively analyzed the records of 101 Japanese adult patients who underwent allogeneic HSCT between 1998 and 2005 at our hospital. Fifty-one developed late positive CMV antigenemia, with a cumulative incidence of 53%. Recipient CMV seropositivity, the use of alemtuzumab, chronic GVHD, and high-dose steroids were significantly associated with late positive antigenemia. Eight patients developed late CMV disease, with a cumulative incidence of 8%, including retinitis and gastrointestinal disease. None progressed to a fatal disease. The use of alemtuzumab was identified as an independent significant risk factor for late CMV disease, although it was not associated with increased non-relapse mortality. Among the 51 patients with late positive antigenemia, 28 had consistently less than three positive cells, 25 of whom showed negative conversion without antiviral agents. In conclusion, late CMV antigenemia appeared to develop frequently, especially in patients with profound immune suppression; however, a fatal outcome could be prevented by optimal preemptive therapy. Low-level antigenemia may not require antiviral treatments.

Lymphomatoid Granulomatosis Involving Central Nervous System Successfully Treated With Rituximab Alone

OBJECTIVE To report the successful treatment of a patient with lymphomatoid granulomatosis (LYG), a rare Epstein-Barr virus-positive lymphoproliferative disorder, using rituximab (anti-CD20 monoclonal antibody). The prognosis for LYG has been reported to be poor, and no satisfactory treatment has been established. Because central nervous system (CNS) involvement of LYG has been known to show poor prognosis, the establishment of an effective treatment for CNS LYG with mild adverse effects is desired. DESIGN Case report. SETTING University hospital. PATIENT A 48-year-old Japanese man presenting with slowly progressive spastic paraparesis diagnosed as LYG involving the CNS and lungs. INTERVENTIONS The patient was treated with rituximab (375 mg/m2, once weekly for 1 month) alone. Main Outcome Measure Improvement of the lesions on imaging. RESULTS The neurological signs resolved and the lesions in the CNS and lungs were mostly diminished after the rituximab monotherapy without any adverse effects. The patient stayed in remission for 18 months. CONCLUSION Rituximab monotherapy was effective in treating the patient; hence, rituximab should be considered as the initial treatment against LYG involving the CNS.

Case report: persistent cytomegalovirus (CMV) infection after haploidentical hematopoietic stem cell transplantation using in vivo alemtuzumab: emergence of resistant CMV due to mutations in the UL97 and UL54 genes.

Addition of in vivo alemtuzumab to the conditioning regimen enabled 2‐ or 3‐locus‐mismatched hematopoietic stem cell transplantation with an acceptable incidence of graft‐versus‐host‐disease. However, the procedure was associated with a high incidence of cytomegalovirus (CMV) reactivation. Although preemptive therapy with ganciclovir prevented successfully severe CMV diseases and CMV‐related mortality, a patient developed persistent positive CMV antigenemia for more than 1 year after transplantation and CMV disease, despite the use of ganciclovir and foscarnet. The in vitro susceptibility assay showed that the clinical isolate was resistant to foscarnet, moderately resistant to ganciclovir, but sensitive to cidofovir. Therefore, cidofovir was administered. CMV antigenemia became negative within 2 weeks and never developed again. Nucleotide sequence of the UL54 and UL97 of the clinical isolate showed 4 amino acid substitutions (V11L, Q578H, S655L, and G874R) in UL54 and 2 mutations (A140V and A594V) in UL97 compared with the Towne and AD169 strains. Ganciclovir resistance was suspected to be caused by both A594V of UL97 and Q578H of UL54, whereas foscarnet resistance was due mainly to Q578H of UL54. In conclusion, the in vitro susceptibility assay as well as nucleotide sequence of clinical isolate is important to choose appropriate antiviral agents for patients who have persistent CMV reactivation after stem cell transplantation. J. Med. Virol. 80:1769–1775, 2008.

Acute eosinophilic pneumonia is a non-infectious lung complication after allogeneic hematopoietic stem cell transplantation

Acute eosinophilic pneumonia (AEP) is an acute febrile illness with respiratory impairment, diffuse pulmonary infiltrates, and eosinophilia in bronchoalveolar lavage (BAL) fluid. We report an adult male who developed severe cough and dyspnea with slight fever on day 78 after allogeneic hematopoietic stem transplantation. The symptoms coexisted with skin and gut GVHD. The imaging test demonstrated interstitial infiltrates and BAL analysis revealed marked increase of eosinophils and no sign of infection. We made a diagnosis of AEP and steroid was started. AEP remitted with other GVHD symptoms but exacerbated partially when steroid was decreased. This case suggests a potential link between AEP and GVHD.

A tele-micro-surgery system across the Internet with a fixed viewpoint/operation-point

This paper describes the necessary functions and the construction of a micro-surgery system which can be operated from a separate place. In particular, a vision system which has a fixed visual point and a slave manipulator, which has the same fixed point at the end of the tool, are proposed. In the vision system, the axes of all rotational degrees of freedom intersect at the focal point of the microscope. In the slave manipulator, the axes of all rotational degrees of freedom also intersect at this point. These mechanisms provide high accessibility to small objects independent of the approach angle from the microscope and the manipulator to the object. Another feature of the system is the transformation of multi-axis force to auditory information to enhance the operability of the system by indicating the contact state of the end-effector and object. Furthermore, the visual information acquisition and display systems are controlled in accordance with the inferred intention of the operator. Finally, a tele-micro-handling experiment was performed using the Internet and two artificial satellites.