Hideki Hasegawa

Mucosal IgA responses in influenza virus infections; thoughts for vaccine design.

The current challenge in influenza vaccine design is to induce long-lasting protection not only against the vaccine strain, but also against drifted (point mutations in the surface antigens HA or NA) and even shifted (exchange of genome segments) strains. Several immune mediators that can induce cross-protection have been described, such as CD4 T-cells, CD8 T-cells and antibodies, including IgA. However, most vaccines are now administered intramuscularly or subcutaneously and subsequently relatively little is known on the role of local, mucosal responses. Since local IgA responses have been shown to play an important role in responses to natural infection, and IgA responses in mice were shown to also be involved in cross-protection, the research on mucosal influenza vaccines is currently expanding. However, the functioning of the mucosal immune system, especially in the respiratory tract, is just beginning to be revealed. Here, the current knowledge on the induction of IgA, the role of influenza specific IgA producing B-cells in anti-influenza immunity as well as the role of humoral memory responses induced upon vaccination will be reviewed.

Therapy-induced selective loss of leukemia-initiating activity in murine adult T cell leukemia

Treatment with a combination of interferon-α and arsenic trioxide ablates leukemia-initiating activity before reducing primary tumor bulk in a murine model of adult T cell leukemia.

Inhibition of the SDF-1α–CXCR4 axis by the CXCR4 antagonist AMD3100 suppresses the migration of cultured cells from ATL patients and murine lymphoblastoid cells from HTLV-I Tax transgenic mice

Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1alpha (SDF-1alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL.

Histopathological and immunohistochemical findings of 20 autopsy cases with 2009 H1N1 virus infection

Twenty autopsy cases with 2009 pandemic influenza A (2009 H1N1) virus infection, performed between August 2009 and February 2010, were histopathologically analyzed. Hematoxylin–eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalin-fixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five cases, the pathogenesis of 2009 H1N1 was confirmed to be viral infection in pneumocytes, which caused severe alveolar damage and fatal viral pneumonia. Further studies on both host and viral factors in autopsy or biopsy materials will be essential to elucidate the other pathogenic factors involved in influenza virus infection.

Identification of cancer stem cells in a Tax-transgenic (Tax-Tg) mouse model of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 10(4) SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38(-)/CD71(-)/CD117(+) cells (0.03%). Whereas lymphoma did not develop after transplantation of 10(2) SLCs, 10(2) CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117(+) CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model.

Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus.

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre‐pandemic vaccine combined with synthetic double‐stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant‐combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine‐specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA‐combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential. J. Med. Virol. 82:1754–1761, 2010.

Spi-B is critical for plasmacytoid dendritic cell function and development

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B-deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B-deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B-deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.

Pathological study of archival lung tissues from five fatal cases of avian H5N1 influenza in Vietnam

Highly pathogenic avian H5N1 influenza virus (H5N1) infection in humans causes acute respiratory distress syndrome, leading to multiple organ failure. Five fatal cases of H5N1 infection in Vietnam were analyzed pathologically to reveal virus distribution, and local proinflammatory cytokine and chemokine expression profiles in formalin-fixed, paraffin-embedded lung tissues. Our main histopathological findings showed diffuse alveolar damage in the lungs. The infiltration of myeloperoxidase-positive and/or CD68 (clone KP-1)-positive neutrophils and monocytes/macrophages was remarkable in the alveolar septa and alveolar spaces. Immunohistochemistry revealed that H5N1 mainly infected alveolar epithelial cells and monocytes/macrophages in lungs. H5N1 replication was confirmed by detecting H5N1 mRNA in epithelial cells using in situ hybridization. Quantitation of H5N1 RNA using quantitative reverse transcription PCR assays revealed that the level of H5N1 RNA was increased in cases during early phases of the disease. We quantified the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, regulated on activation normal T-cell expressed and secreted (commonly known as RANTES), and interferon-gamma-inducible protein of 10u2009kDa (IP-10) in formalin-fixed, paraffin-embedded lung sections. Their expression levels correlated with H5N1 RNA copy numbers detected in the same lung region. Double immunofluorescence staining revealed that TNF-α, IL-6, IL-8 and IP-10 were expressed in epithelial cells and/or monocytes/macrophages. In particular, IL-6 was also expressed in endothelial cells. The dissemination of H5N1 beyond respiratory organs was not confirmed in two cases examined in this study.

Intranasal vaccination with an inactivated whole influenza virus vaccine induces strong antibody responses in serum and nasal mucus of healthy adults

Haemagglutination inhibition (HI) and neutralization (NT) titers as well as haemagglutinin (HA) specific antibody responses were examined in 50 healthy adults aged between 22 and 69 y old after two intranasal administrations of an inactivated whole virus vaccine derived from A/Victoria/210/2009 virus (45 μg HA per dose) at 3 week intervals. Serum HI titers after two-doses of the nasal vaccine showed >2.5-fold rise in the ratio of geometric mean titer upon vaccination, >40% of subjects with a ≥4-fold increase in titer and >70% of subjects with a titer of ≥1:40, all parameters associated with an effective outcome of vaccination in the criteria defined by the European Medicines Agency. Serum neutralizing antibody responses correlated with HI antibody responses, although NT titers were about 2-fold higher than HI titers. These high levels of serum responses were accompanied by high levels of HI and neutralizing antibody responses in nasal mucus as measured in concentrated nasal wash samples that were about 10 times diluted compared with natural nasal mucus. Serum and nasal HI and neutralizing antibody responses consisted of HA-specific IgG and IgA antibody responses, with IgG and IgA antibodies being dominant in serum and nasal responses, respectively.

Characterization of neutralizing antibodies in adults after intranasal vaccination with an inactivated influenza vaccine

The levels and properties of neutralizing antibodies in nasal wash and serum collected from five healthy adults were examined after intranasal administration of an A/Uruguay/716/2007 (H3N2) split vaccine (45u2009µg hemagglutinin (HA) per dose; five doses, with an interval of 3 weeks between each dose). Prior to the assays, nasal wash samples were concentrated so that the total amount of antibodies was equivalent to about 1/10 of that found in the natural nasal mucus. Vaccination induced virus‐specific neutralizing antibody responses, which increased with the number of vaccine doses given. Neutralizing antibodies were produced more efficiently in the nasal passages than in the serum: A ≥4‐fold increase in nasal neutralization titres was observed after the second vaccination in four out of five subjects, whereas a rise in serum neutralization titres was observed only after the fifth vaccination. Nasal and serum neutralizing antibodies were mainly found in the polymeric IgA and monomeric IgG fractions, respectively, after gel filtration. Taken together, these results suggest that intranasal administration of an inactivated split vaccine induces high levels of nasal neutralizing antibodies (primarily polymeric IgA) and low levels of serum neutralizing antibodies (primarily monomeric IgG). J. Med. Virol. 84:336–344, 2012.