Induction of specific and flavivirus--Cross-reactive CTLs by immunization with a single dengue virus-derived CTL epitope peptide.

Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.

Enhancement of MHC class I binding and immunogenic properties of the CTL epitope peptides derived from dengue virus NS3 protein by anchor residue replacement

The immunogenecity of the defined H-2Kd-restricted, murine cytotoxic T lymphocyte (CTL) epitopesof dengue viruses were examined for CTL induction in epitope peptide / H-2Kd tetramer assays. Thepeptides used in the study included those corresponding to amino acid (a.a.) residues 298-306(GYISTRVEM) of NS3 of dengue virus types 2 and 4 (named DENV-2/4), and to a.a. residues 299-307(GYISTRVGM) of NS3 of dengue virus types 1 and 3 (named DENV-1/3), and their respective modified epitope peptides, DENV-2/4-9L (GYISTRVEL) and DENV-1/3-9L (GYISTRVGL), in which the C-terminalresidue M of the original epitope peptide was replaced by L, in order to provide the complete H-2Kd-binding motif. Immunization of BALB/c mice with the original epitope peptide, DENV-2/4 or DENV-1/3, did not induce specific CTLs, while that with the modified epitope peptide, DENV-2/4-9L or DENV-1/3-9L, induced epitope peptide/H-2Kd tetramer-binding CD8+ cells indicating specific CTLs.Competition-based binding assay with biotinylated epitope-related reference peptides (DENV-2/4-9L-Biotin and DENV-1/3-9L-Biotin) demonstrated that the modified epitope peptide, DENV-2/4-9L and DENV-1/3-9L, had higher avidity to H-2Kd than the respective original epitope peptides. These results indicate that modification of dengue virus-derived CTL epitope peptide by replacing a.a. residue at theposition of anchor residue increases the binding avidity to MHC class I, resulting in the induction ofspecific CTLs. The strategy to enhance the immunogenicity of CTL epitope peptide may contribute to investigation of CTL biology in dengue virus infection.

Diethyldithiocarbamate can induce two different type of death: apoptosis and necrosis mediating the differential MAP kinase activation and redox regulation in HL60 cells.

Although apoptosis and necrosis have been considered different pathways to cell death, only one compound induces both types of cell death. Diethyldithiocarbamate (DDC) has been shown to have antioxidant or prooxidant effects in several different systems. We observed in our present study that DDC induced not only apoptosis but also necrosis depending on its dosage in HL60 premyelocytic leukemia cells. Moreover, in hypoxia cell culture conditions, DDC-induced necrotic cells decreased but DDC-induced apoptosis continued. We investigated the DDC-induced different cell death mechanisms as they are correlated with reactive oxygen species (ROS). High-dose DDC-induced necrotic cell death is thought to depend on the increase of intracellular ROS, while low-dose DDC-induced apoptosis is thought to depend on changes of the intracellular redox state by the transporting of external metal ions. There was no sequential or quantitative change of Bcl-2 family proteins in DDC-induced apoptotic or necrotic pathways. However, the mitochondrial transmembrane potential was remarkably decreased in the DDC-induced necrosis. Finally, duration of c-Jun N-terminal kinase (JNK) activation resulted in different types of cell death.

Pretreatment with PKC inhibitor triggers TNF-alpha induced apoptosis in TNF-alpha-resistant B16 melanoma BL6 cells.

Tumor necrosis factor alpha (TNF-alpha) modulates various events through several different pathways. Many tumor cells are resistant to this cytokine. Pretreatment of these cells with actinomycin D enhances TNF-alpha-induced apoptosis. In the present study, we investigated the mechanism of this enhancement and whether or not the apoptosis of TNF-alpha-resistant cancer cells can be induced by the inhibition of Protein kinase C (PKC). When TNF-alpha was added after inhibition of PKC by H7, apoptosis was observed, and companied with the activation of nuclear factor kappa B (NF-kappaB). After the inhibition of protein kinase B (Akt) by LY294002 or p38 mitogen-activated protein kinase (p38MAPK) by SB203580, the addition of TNF-alpha did not cause apoptosis. However, after the inhibition of MAPK/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) with U0126, apoptosis was observed when TNF-alpha was added. In the Western blotting analysis, phosphorylation of MEK1/2 occurred at 60 minutes after the addition of TNF-alpha. However, it was noted that after pretreatment with H7, a significant decrease in phosphorylated MEK1/2 was observed. The present findings suggest that MEK1/2 plays an important role in TNF-alpha-resistance in TNF-alpha-resistant B16 melanoma BL6 cells. Furthermore, it was found that MEK1/2 is more important than NF-kappaB, Akt, and p38MAPK in anti-apoptotic PKC signaling and that TNF-alpha-resistance can be overcome by inhibiting MEK1/2. These results suggest the possibility of development of a new anticancer drug treatment.

Type IV collagen, type IV collagenase activity and ability of cell proliferation in human thyroid tumours.

BACKGROUND: The processes of malignant tumour invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase degrades type IV collagen, a main component of the basement membrane. METHODS: In our study, type IV collagenase activity in human thyroid tumours was measured by the Liotta method. The degree of destruction of diseased regions of thyroid tumours was immunohistochemically determined by anti-type IV collagen antibody staining. Cell proliferation in the tumours was estimated using anti-proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR). RESULTS: T4 thyroid carcinomas with higher type IV collagenase activity and very weak type IV discontinuous immunostaining for type IV collagen of follicular basement membranes, exhibited many PCNA or EGFR positive cells. In benign tumours, normofollicular- or macrofollicular-type tumours with low type IV collagenase activity showed few PCNA and EGFR positive cells and intact type IV collagen of basement membranes, as seen in normal thyroids. Conversely, an atypical adenoma with higher type IV collagenase activity showed many PCNA and EGFR positive cells and weak type IV discontinuous immunostaining for type IV collagen, as in thyroid carcinomas. CONCLUSION: These findings suggest that staining for type IV collagen and type IV collagenase activity reflect the ability of cell proliferation, and help predict the aggressiveness of invasion and metastasis in human thyroid tumours.

Histopathological findings of human thyroid tumors and dynamic MRI.

OBJECTIVE: Time intensity curves for gadolinium-diethylenetriaminepentaceticacid (Gd-DTPA) enhanced magnetic resonance imaging (MRI), namely dynamic MRI, were determined for thyroid diseases, and compared with findings of histopathologic examination. METHODS: Time intensity curves for the thyroid tumors were determined. Three different patterns of time intensity curves were observed: a rapid washout pattern, a delayed washout pattern and no change. Cell proliferating activities of thyroid tumors were estimated immunohistochemically by proliferating cell nuclear antigen (PCNA). RESULTS: In most of malignant diseases and a few benign diseases that had marked cell proliferative activity with showing more than 20% on labeling indexes of PCNA, the time intensity curve displayed the delayed washout pattern, in which intensity was above half-maximal value within 10 min after injection Gd-DTPA. Almost all benign diseases and a few well differentiated carcinomas displayed the rapid washout pattern, in which intensity was decreased to lower than half of peak grade within 10 min following injection and showed from 10 to 20% on labeling indexes of PCNA. Benign diseases that displayed no change of time intensity curve did not show PCNA positive cells. CONCLUSION: These findings suggested that the time intensity curve obtained from dynamic MRI might indicate cell proliferating activity of thyroid tumors.

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma.

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.

Correlation between morphology of colonies formed by human thyroid tumor cells in collagen gel culture and invasive ability.

Two different types of colonies from human thyroid papillary carcinomas were observed in collagen gel culture. One was a branching type, with many three-dimensional outgrowths. The other was a spherical type, with no outgrowths. As viewed by electron microscopy, the tumor cells of spherical-type colonies had more microvilli on their surfaces than did cells in the branching-type colonies. Histochemically, the tumor cells of spherical-type colonies had stronger positive reactions than did tumor cells of branching-type colonies. This suggests that tumor cells of spherical-type colonies have a higher potential for dissemination and invasion than do tumor cells of branching-type colonies. In fact, cases with higher percentages of spherical-type colonies could show extracapsular invasion histopathologically. Our results indicate that collagen gel culture is a useful method for studying the heterogeneity of tumor cells as well as their invasive ability in vitro.