Mitsuo Honda

Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1).

Epigallocatechin gallate (EGCg), the major tea catechin, is known as a potent anti-bacterial agent. In addition, anti-tumor promoting, anti-inflammatory, anti-oxidative and antiviral activities have been reported. In the present study, we investigated possible anti-human immunodeficiency virus type-1 (HIV-1) activity of EGCg and its mechanisms of action in the viral life cycle. EGCg impinges on each step of the HIV life cycle. Thus, destruction of the viral particles, viral attachment to cells, post-adsorption entry into cells, reverse transcription (RT), viral production from chronically-infected cells, and the level of expression of viral mRNA, were analyzed using T-lymphoid (H9) and monocytoid (THP-1) cell systems, and antiviral protease activity was measured using a cell-free assay. Inhibitory effects of EGCg on specific binding of the virions to the cellular surfaces and changes in the steady state viral regulation (mRNA expression) due to EGCg were not observed. However, EGCg had a destructive effect on the viral particles, and post-adsorption entry and RT in acutely infected monocytoid cells were significantly inhibited at concentrations of EGCg greater than 1 microM, and protease kinetics were suppressed at a concentration higher than 10 microM in the cell-free study. Viral production by THP-1 cells chronically-infected with HIV-1 was also inhibited in a dose-dependent manner and the inhibitory effect was enhanced by liposome modification of EGCg. As expected, increased viral mRNA production was observed in lipopolysaccharide (LPS)-activated chronically HIV-1-infected cells. This production was significantly inhibited by EGCg treatment of THP-1 cells. In contrast, production of HIV-1 viral mRNA in unstimulated or LPS-stimulated T-lymphoid cells (H9) was not inhibited by EGCg. Anti-HIV viral activity of EGCg may thus result from an interaction with several steps in the HIV-1 life cycle.

A New Humanized Mouse Model of Epstein-Barr Virus Infection That Reproduces Persistent Infection, Lymphoproliferative Disorder, and Cell-Mediated and Humoral Immune Responses

The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (<or=1 x 10(1) TD(50)), in contrast, resulted in apparently asymptomatic persistent infection. Levels of activated CD8(+) T cells increased dramatically in the peripheral blood of infected mice, and enzyme-linked immunospot assay and flow cytometry demonstrated an EBV-specific T cell response. Immunoglobulin M antibody specific to the EBV-encoded protein BFRF3 was detected in serum from infected mice. The NOG mouse is the most comprehensive small-animal model of EBV infection described to date and should facilitate studies of the pathogenesis, prevention, and treatment of EBV infection.

Increased Expression of Cyclooxygenase-2 in Local Lesions of Endometriosis Patients

PROBLEM: Human endometrial glands contain the highest levels of cyclooxygenase (COX), although whether it is COX‐1 and/or COX‐2 has not been previously determined. Overexpression of COX‐2 may result in the pathogenesis of endometriosis.

Baicalin, an inhibitor of HIV-1 production in vitro.

The flavonoid baicalin markedly inhibits replication of human immunodeficiency virus type 1 (HIV-1) in a concentration-dependent manner in normal peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin (PHA) in vitro. The effect was more pronounced when the cells were pretreated with baicalin. Furthermore, baicalin inhibits HIV-1 replication in PHA-stimulated PBMC from asymptomatic HIV-1-seropositive carriers. The 50% inhibitory concentration for HIV-1 replication was approximately 0.5 microg/ml. At the concentration of 2 microg/ml of baicalin, copy numbers of HIV-1 proviral DNA were approximately 50 times less than in untreated controls. In a cell-free infection system, baicalin inhibited the activity of HIV-1 reverse transcriptase (RT), but not the activity of human DNA polymerases alpha and gamma (DNA polymerase beta was slightly inhibited), suggesting that the anti-HIV-1 effect of baicalin may at least partly be due to inhibition of HIV-1 RT.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes− Ccr7+ Cxcr3−, in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1− Klrg1−Ccr5− compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

HIV mucosal vaccine: nasal immunization with gp160-encapsulated hemagglutinating virus of Japan-liposome induces antigen-specific CTLs and neutralizing antibody responses.

Nasal immunization of normal mice with HIVgp160-encapsulated hemagglutinating virus of Japan (HVJ)-liposome induced high titers of gp160-specific neutralizing IgG in serum and IgA in nasal wash, saliva, fecal extract, and vaginal wash, along with both Th1- and Th2-type responses. HIVgp160-specific IgG- and IgA-producing cells were also detected in mononuclear cells isolated from spleen, nasal cavity, salivary gland, intestinal lamina propria, and vaginal tissue of nasally immunized mice. In addition, CD8+ CTLs were induced in mice nasally immunized with gp160-HVJ-liposome. These findings suggest that two layers of effective HIV-specific humoral and cellular immunity, in mucosal and systemic sites, were induced by this nasal vaccine. In immunodeficient mice, nasal immunization with gp160-HVJ-liposome induced Ag-specific immune responses for the systemic and mucosal compartments of both Th1 (IFN-γ−/−) and Th2 (IL-4−/−). In vitro Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and IgG Abs that had been induced by nasal immunization with gp160-HVJ-liposome were able to neutralize a clinically isolated strain of HIV-MN strain isolated from Japanese hemophiliac patients. Taken together, these results suggest that, for the prevention and control of AIDS, nasally administered gp160-HVJ-liposome is a powerful immunization tool that induces necessary Ag-specific immune responses at different stages of HIV infection.

Humanized NOD/SCID/IL2Rγnull Mice Transplanted with Hematopoietic Stem Cells under Nonmyeloablative Conditions Show Prolonged Life Spans and Allow Detailed Analysis of Human Immunodeficiency Virus Type 1 Pathogenesis

ABSTRACT In a previous study, we demonstrated that humanized NOD/SCID/IL2Rγnull (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.

A HIGHLY PATHOGENIC SIMIAN/HUMAN IMMUNODEFICIENCY VIRUS WITH GENETIC CHANGES IN CYNOMOLGUS MONKEY

A highly pathogenic simian/human immunodeficiency virus (SHIV), designated C2/1, was obtained by serum passages in cynomolgus monkeys of p-SHIV, an SHIV strain that contains the env gene of pathogenic human immunodeficiency virus type 1 89.6. CD4+ lymphocyte depletion was induced within 1 week of the SHIV-C2/1 infection in peripheral blood as well as in various lymphoid organs in all the animals tested, with symptoms of diarrhoea and no increase in body weight, followed by intense viraemia. Serum antibody against Env protein was detected from 4 weeks after the virus infection, while the anti-Gag antibody response was absent in the SHIV-C2/1-infected animals. In contrast, both anti-Gag and anti-Env antibody responses were present in animals infected with p-SHIV or the non-pathogenic SHIV-MN. Sequencing of the env gene of isolates of SHIV-C strains showed conserved amino acid changes in the Env C2 and V3 regions that included changes to negatively charged amino acids, in the cytoplasmic region of gp41 that included a 42 amino acid deletion, and in the Nef protein. The pathogenic SHIV-C2/1-monkey model suggests that virus-specific pathogenicity in SHIV infection may be associated with the absence of anti-Gag antibody responses in animals and may be caused by genetic changes during serum passage in vivo.

A novel NF‐κB inhibitor DHMEQ selectively targets constitutive NF‐κB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF‐κB) is a common characteristic of MM cells. In our study we successfully target NF‐κB with a novel NF‐κB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF‐κB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF‐κB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF‐κB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl‐XL and c‐FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ‐mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/γcnull (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF‐κB, DHMEQ is a unique compound that blocks the translocation of NF‐κB p65 into the nucleus and selectively targets NF‐κB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.

SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus–host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.