Lactose-sensitive and -insensitive cell surface interactions of oral Streptococcus milleri strains and actinomyces

Of 158 oral Streptococcus milleri strains, 46 exhibited cellular coaggregation with the reagent strains of the actinomyces coaggregation groups A, B, and/or E. All but 1 of the 33 serotype b, e, f/F, and k/G strains belonged to streptococcus coaggregation group 2, and only 14 strains of limited seroclasses (g, i, Lancefield group F, or untypeable) appeared to be members of group 5, 3, or 4 (10, 3, and 1 strain, respectively). Thus, S. milleri infrequently exhibits lactose-inhibitable coaggregation with actinomyces.     

Representation of place by monkey hippocampal neurons in real and virtual translocation

The hippocampal formation (HF) is hypothesized as a neuronal substrate of a cognitive map, which represents environmental spatial information by an ensemble of neural activity. However, the relationships between the hippocampal place cells and the cognitive map have not been clarified in monkeys. The present study was designed to investigate how activity patterns of place-selective neurons encode spatial relationships of various environmental stimuli; to do this, we used multidimensional scaling (MDS) for hippocampal neuronal activity in the monkey during the performance of real and virtual translocation. Of 389 neurons recorded from the monkey HF and parahippocampal gyrus (PH), 166 had place fields that displayed increased activity in a specific area of an experimental field and/or on a monitor (place-selective neurons). The MDS transformed relationships among the 16 places in the experimental field and the monitor, expressed as correlation coefficients between all possible pairs of two places based on the 166 place-selective responses, into geometric relationships in a two-dimensional MDS space. In the real translocation tasks, the 16 places were distributed throughout the MDS space, and their relative positions were well correlated to real positions in the experimental laboratory. However, the correlation between the MDS space and real arrangements was significantly smaller in virtual than real translocation tasks. The present results strongly suggest that activity patterns of the HF and PH neurons represent spatial information and might provide a neurophysiological basis for a cognitive map.     

Establishment of an immortalized T-cell line from lung cancer tissue: phenotypic and functional analyses

Background. In order to investigate host defense against solid tumors, valuable information could be provided by ex-vivo analyses of functional immune cells in tumor tissues. However, available sources of fresh tumor-infiltrating T cells (TIL) are usually very limited, and it is often difficult to establish TIL lines. In this study, we analyzed the phenotypic and functional characteristics of TIL, using an immortalized cell line prepared by cotransfection of human c-myc and c-Ha-ras. Methods. A human T-cell line was established by cotransfecting c-Ha-ras and c-myc oncogenes to T lymphocytes freshly isolated from human lung large-cell carcinoma tissue. The phenotypes were assessed by flow cytometry. T-cell receptor (TCR) Vα- and β-usage was analyzed by polymerase chain reaction (PCR) and Southern blot. Cytotoxic activity against autologous and allogeneic tumor cell lines was examined by standard ⁵¹Cr-release cytotoxicity assays. Cytokine production by the established T-cell line, 904-T1, in response to stimulation by autologous tumor cells was assayed by using an enzyme-linked immunosorbent assay. Results. 904-T1 (CD3+, CD8+, CD56+, CD16-, CD161-, TCR Vα 9, 13, and Vβ 1, 5) displayed a broad range of MHC-nonrestricted tumoricidal activity against various human tumor cell lines, but did not lyse autologous B cells transformed by Epstein-Barr virus. The cytotoxicity of 904-T1 was not mediated by a T-cell antigen receptor or by Fas-ligand, but by perforin-based cytolytic pathways, and was enhanced by interleukin (IL)-12. 904-T1 cells produced large amounts of interferon (IFN)-γ, but not tumor necrosis factor (TNF)-α or IL-4 in response to autologous tumor cells, and produced high levels of IFN-γ and TNF-α, and a substantial level of IL-4 following stimulation with anti-CD3 monoclonal antibody. Conclusions. Our results suggest that 904-T1 cells were natural killer T (NKT)-like cells with regard to their nonspecific killing, cytokine repertoire, and sensitivity to IL-12, although the repertoire of the TCR variable region was not compatible with that of NKT cells. Received: January 22, 2001 / Accepted: December 19, 2001     

Successful induction of tumor-specific cytotoxic T lymphocytes from patients with non-small cell lung cancer using CD80-transfected autologous tumor cells.

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80-transfected tumor cells as stimulators of the in vitro induction of autologous tumor-specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non-small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80-transfected tumor cells (CD80-AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non-transfected tumor cells (AT). AT-stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80-AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT-stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)-class I-restricted cytokine production in response to AT, while the MHC-class I-restricted responses were found in CD80-AT-stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.     

Autologous Tumor-specific Cytotoxic T Lymphocytes in a Patient with Lung Adenocarcinoma: Implications of the Shared Antigens Expressed in HLA-A24 Lung Cancer Cells

Human lung adenocarcinoma-specific cytotoxic T lymphocytes (CTL) were generated by multiple stimulations with autologous tumor cells (named A110L) from regional lymph node lymphocytes and tumor-infiltrating lymphocytes expanded by solid-phase anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2. The CTL lysed A110L but failed to kill either autologous B lymphocytes immortalized by the Epstein-Barr virus or K562. The killing activity of the CTL against autologous A110L was inhibited by anti-MHC class I mAb (W6/32), but not by anti-MHC class II mAb. The CTL produced interferon-γ and GM-CSF in response to A110L and the production was completely blocked by the addition of anti-MHC class I mAb. The HLA type of the CTL was HLA-A2/A24, B52/B54, Cw1/-. Allele-specific deletion of HLA-A2 molecules was observed in A110L by staining with anti-HLA-A2 mAb. A partial blocking effect on the cytokine production from the CTL was also obtained with anti-CD8, and anti-HLA-A24 mAbs, but not with anti-MHC class II, anti-CD4 and anti-HLA-A2 mAbs. To analyze further the mechanism of antigen recognition by the CTL, the cross reactivity of the CTL against several HLA-A locus-matched (HLA-A24+) and mismatched allogeneic tumor cells (HLA-A24-) was investigated. The A110L-specific CTL showed a weak but significant cytotoxicity against some HLA-A24 positive lung cancer cell lines, such as Sq-1 (HLA-A11/A24, squamous cell carcinoma) and PC-9 (HLA-A2/A24, adenocarcinoma), but failed to kill HLA-A locus-mismatched allogeneic tumors. This cross reactivity of the CTL against Sq-1 and PC-9 was blocked by anti-MHC class I mAb. These results thus demonstrate that shared common tumor antigens might exist among lung cancer cells in the context of HLA-A24.     

Cellular coaggregation of oral Streptococcus milleri with actinomyces.

Oral isolates of Streptococcus milleri were examined for their ability to coaggregate with actinomyces. Of the 68 S. milleri strains tested, including 3 reference strains, 40 strains coaggregated with Actinomyces naeslundii WVU45 (actinomyces coaggregation group B) and 36 strains coaggregated with Actinomyces viscosus T14V (actinomyces coaggregation group A). All S. milleri strains of serotypes b (4 strains), e (2 strains), and f (24 strains) coaggregated with both of the actinomyces. The coaggregation reactions between the S. milleri cells and A. naeslundii WVU45 cells were optimal at about pH 7.0 and were Ca2+ or Mg2+ dependent, but they were not inhibited by the presence of simple sugars or amino sugars, including lactose (up to 0.5 M). Treatment of the S. milleri cells with heat (100 degrees C, 3 min) or proteases (trypsin, 1.0 mg/ml; pronase, 0.25 mg/ml; 37 degrees C; 3 h) and of the actinomyces cells with periodate (0.01 M, 4 degrees C, 16 h) destroyed their coaggregating abilities. The coaggregations between cells of the S. milleri strains, we well as cells of the Streptococcus sanguis H1 (reference strain for streptococcus coaggregation group 2) and the actinomyces strains (WVU45 and T14V), were inhibited by AFH1 (a carbohydrate receptor on T14V cells for a lectin on H1 cells). These interactions were also inhibited by anti-AFH1 immunoglobulin G (IgG) and by anti-b, anti-e, and anti-f S. milleri IgG or anti-f IgG Fab fragments. These results suggest that S. milleri, at least strains of serotypes b, e, and f, belongs to streptococcus coaggregation group 2.     

Successful Induction of Tumor-specific Cytotoxic T Lymphocytes from Patients with Non-small Cell Lung Cancer Using CD80-transfected Autologous Tumor Cells

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80-transfected tumor cells as stimulators of the in vitro induction of autologous tumor-specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non-small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80-transfected tumor cells (CD80-AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non-transfected tumor cells (AT). AT-stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80-AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT-stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)-class I-restricted cytokine production in response to AT, while the MHC-class I-restricted responses were found in CD80-AT-stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.     

Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes

BACKGROUND: The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). METHODS: MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. RESULTS: Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. CONCLUSION: These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.     

Effects of T-588, a cognitive enhancer compound, on synaptic plasticity in the dentate gyrus of freely moving rats.

(1R)-1-benzo [b] thiophen-5-yl-2-[2-(diethylamino) ethoxy] ethan-1-ol hydrochloride (T-588) is a compound for the treatment of neurodegenerative disorders, including Alzheimer's disease and cerebrovascular diseases. T-588 reportedly alleviates learning and memory deficits in animal models of dementia. In the present study, we investigated the effects of T-588 on the induction and decay of long-term potentiation (LTP) and on the responses to paired-pulse (pp) stimulation in freely moving rats. Perforant path-evoked field potentials were recorded in the dentate gyrus by chronically implanted electrodes. LTP was induced by high-frequency stimulation 30 min after oral administration of T-588 (0.3 or 3 mg/kg). T-588 significantly augmented the increase in population spike amplitude and field excitatory postsynaptic potential slope after LTP induction. T-588 also prolonged the decay of augmented population spike amplitude, but had no significant effect on the response to pp stimulation. These results suggest that T-588 facilitates long-term synaptic plasticity, but not short-term synaptic plasticity in the dentate gyrus of freely moving rats. The effect of T-588 on long-term synaptic plasticity may contribute to the alleviation of learning and memory dysfunction seen in animal models.