Takushi Tadakuma

Adenylyl Cyclase α and cAMP Signaling Mediate Plasmodium Sporozoite Apical Regulated Exocytosis and Hepatocyte Infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase α (ACα), a gene containing regions with high homology to adenylyl cyclases. PbACα-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACα, as re-introduction of ACα in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACα and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

Nuclear translocation of extracellular superoxide dismutase.

Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression.

Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma.

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5‐fluorouracil (5‐FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5‐FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5‐FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC. (Cancer Sci 2006; 97: 1115–1124)

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.

Regulation of the expression of caspase-9 by the transcription factor activator protein-4 in glucocorticoid-induced apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5′-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at –1.55 and –1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases –1140 and –944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Mouse CD8+ CD122+ T Cells with Intermediate TCR Increasing with Age Provide a Source of Early IFN-γ Production

Although CD8+ IL-2Rβ (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rβ-negative or -low (CD122−) T cells conversely decreased. The IFN-γ production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-γ than did CD8+ CD122− T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-γ production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-γ production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-γ. The CD3-stimulated IFN-γ production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-γ than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-γ production and are suggested to be involved in the immunological changes with aging.

Selective Inhibition of Hepatoma Cells Using Diphtheria Toxin A under the Control of the Promoter/Enhancer Region of the Human α‐Fetoprotein Gene

We constructed a plasmid containing human α‐fetoprotein (AFP) promoter/enhancer to direct the cell type‐specific expression of diphtheria toxin fragment A (DTA), designated as pAF‐DTA, to AFP‐producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE‐C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF‐DTA was transfected, the growth of AFP‐positive HuH‐7 cells was inhibited, whereas growth inhibition was not observed in AFP‐negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF‐DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF‐DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH‐7 and splenic stromal cells were co‐cultured revealed the growth inhibition by pAF‐DTA to be selective in HuH‐7 cells. Finally, the growth of HuH‐7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF‐DTA/liposome complex into a tumor mass. These results suggest that use of pAF‐DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.

Functional and Vβ repertoire characterization of human CD8+ T‐cell subsets with natural killer cell markers, CD56+ CD57− T cells, CD56+ CD57+ T cells and CD56− CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK‐type T cell); CD56 single positive (CD56)‐T cells, CD56/CD57 double positive (DP)‐T cells and CD57 single positive (CD57)‐T cells in the peripheral blood. All NK‐type T‐cell populations expressed CD122 and intermediate levels of T‐cell receptor (TCR; regular CD8+ T cells are CD122− and express high levels of TCR). The number of both DP‐T cells and CD57‐T cells, but not CD56‐T cells, gradually increased with age. All NK‐type T‐cell populations produced larger amounts of interferon‐γ than did regular CD8+ T cells after stimulation with interleukin (IL)‐2, IL‐12 and IL‐15. However, CD56‐T cells and CD57‐T cells but not DP‐T cells showed a potent antitumour cytotoxity to NK‐sensitive K562 cells, whereas only CD56‐T cells showed a potent cytotoxity to NK‐resistant Raji cells. Furthermore, although NK‐type T cells produced large amounts of soluble Fas‐ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VβT cells were found in each NK‐type T‐cell population. The non‐variant CDR3 region(s) for the TCRβ chain(s) showed CD57‐T cells and CD56‐T cells to be derived from distinct origins, while the DP‐T cell population consisted of a mixture of the clones seen in both CD56‐T cells and CD57‐T cells. Our results suggest that CD57‐T cells and CD56‐T cells are functionally and ontogenically different populations while DP‐T cells appear to originate from both CD56‐T cells and CD57‐T cells.

Adenovirus mediated prostate specific enzyme prodrug gene therapy using prostate specific antigen promoter enhanced by the Cre-loxP system

PURPOSE Tissue or tumor specific gene delivery is crucial for achieving successful results in suicide gene therapy. Prostate specific antigen (PSA) promoter is known to be highly specific in prostate tissue but its promoter activity is much weaker than that of constitutive viral promoters. In the current study we enhanced PSA promoter activity by combining it with the Cre-loxP system. We also applied this system to adenovirus mediated suicide gene therapy with the cytosine deaminase (CD) gene. MATERIALS AND METHODS The Cre-loxP DNA recombination system was used to enhance PSA promoter. A plasmid with the PSA promoter-enhancer combination was constructed to drive Cre recombinase. Another plasmid contained the cytomegalovirus promoter-loxP-flanked stop signal-luciferase gene. LNCaP human prostate cancer cells were co-transfected with these 2 plasmids and luciferase activity was measured to assess promoter activities. Adenoviral vectors with the CD suicide gene were constructed in similar fashion and tested in LNCaP cells in in vitro/in vivo prostate cancer models. RESULTS Promoter activity of the combined PSA promoter/enhancer and Cre-loxP system was 3 times stronger than that of PSA promoter/enhancer alone. It was further enhanced 7-fold in the presence of testosterone. Application of this system to CD suicide gene therapy by adenoviral vectors inhibited subcutaneous LNCaP tumor growth in nude mice. CONCLUSIONS Combining the Cre-loxP system with PSA promoter/enhancer amplified promoter activity and was found to inhibit the growth of PSA producing prostate cancer cells in vivo.

Administration of plasmids expressing interleukin‐4 and interleukin‐10 causes BALB/c mice to induce a T helper 2‐type response despite the expected T helper 1‐type response with a low‐dose infection ofLeishmania major

BALB/c mice are susceptible to developing an infection with Leishmania major as a result of a fatal T helper 2 (Th2)‐type response. However, mice infected with a low dose of parasites are reported to be able to overcome the lesions associated with a T helper 1 (Th1)‐type response. To clarify why a difference in the dose of parasites induces a difference in the polarization of the Th phenotype, we first attempted to measure cytokine production. Soon after infection, the mice given high doses of parasites produced elevated levels of both Th1 [interferon‐γ (IFN‐γ)] and Th2 [interleukin (IL)‐4 and IL‐10] cytokines. However, when assessed at 1 and 2 weeks after infection, no significant difference in the balance of Th1 and Th2 cytokines could be detected between mice infected with low or high doses of L. major. These results support the notion that the Th2 cytokine levels at an early phase of infection could be a key factor for the induction of a Th2 response. In order to assess the efficacy of Th2 cytokines, the mice infected with low doses of L. major were co‐administered IL‐4 plasmid and IL‐10 plasmid. Consequently, the mice (which originally exhibited a Th1 response) showed progressive disease and developed a Th2 response. However, administration of these plasmids at 7 days postinfection could not alter the Th polarization. Furthermore, production of IL‐12 from the spleen cells stimulated by L. major was suppressed in the presence of IL‐4 and IL‐10. These results strongly suggest that the susceptibility to L. major in BALB/c mice depends on the persistence of Th2 cytokine levels at an early phase of infection.