Changes In Cytotoxic Activity Research Articles

Sequential gene expression profiling in the mouse spleen during 14 d feeding withLactobacillus brevisKB290

Some lactic acid bacteria play an important role in the immune system with potential benefits to the host. However, detailed mechanisms of immune modulation exerted by probiotics remain to be clarified. Since immune response changes in a time-related manner in some cases, we monitored changes in mRNA levels in the spleen of mice during 14 d feeding with Lactobacillus brevis KB290 (KB290). Female BALB/c mice, aged 9 weeks, commenced a diet containing KB290 (3 × 109 colony-forming units/g) or starch for a period of 1, 4, 7 or 14 d. Cytotoxic activity of the resulting splenocytes against YAC-1 cells was measured using flow cytometry. The activity was found to be significantly higher in the treated group on days 1 and 7. The highest activity appeared on day 4, but was not statistically significantly different. Gene expression profiles were analysed using DNA microarray. Gene Ontology (GO) terms related to the immune process were significantly enriched in the up-regulated gene set on days 1, 4 and 7, and GO terms related to the cellular process were enriched in the down-regulated gene set on days 4 and 7. Although the up-regulated genes involved in antigen processing and presentation for stimulation of CD8+ cytotoxic T cells were not observed on day 14, some genes involved in T-cell and natural killer cell activation remained up-regulated until day 14. For the majority of the genes tested, RT-PCR analysis was used to verify the results obtained from the DNA microarray analysis. The sequential gene expression profiling reflected changes in cytotoxic activity during KB290 feeding.

One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry

Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ( 51)Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical (51)Cr-release assay.

Biological evaluation of donor-acceptor aminonaphthoquinones as antitumor agents

Several members of the phenylamino-1,4-naphthoquinone series were prepared in order to investigate structure–activity relationships (SAR) and to explore the antitumor effects associated with this scaffold. The cytotoxic effects of the aminoquinones (EC 50) against a panel of cancer cell lines (MCF7, DU145 and T24 cells) and healthy fibroblasts (BALB/3T3) were assessed in vitro using the MTT reduction assay 48 h after drug exposure. SAR analysis of the aminonaphthoquinone series showed that insertion of a chlorine atom in the acceptor quinone nucleus and/or insertion of a methyl group at the nitrogen atom of the donor phenylamino group induced significant changes in cytotoxic activity. Quinones 7 and 9, which exhibited the highest selective indexes (5.73 and 6.29, respectively), were further characterized using the following assays: Colony formation, caspase-3 activity, and ATP content. The results showed that aminoquinone 7 strongly influenced ATP levels and impaired the proliferative capacity of T24 cells without activating caspase-3.

Abstract 4502: Synthesis and evaluation of curcumin-like compounds as inhibitors of the JAK/STAT pathway in cancer

Abstract Curcumin is an attractive lead compound for drug discovery and development. It displays a range of remarkable and diverse biological activities due to its interaction with numerous biological targets including the receptor tyrosine kinase Janus Kinase 2 (JAK2) which transduces cytokine and growth factor mediated signals by activating signal transducer and activator of transcription 3 (STAT3). The significant role of JAK-STAT pathway activation in the development, progression, and survival of cancer has been widely recognized since the 1980s. Based on our interest in developing more potent curcumin analogs which act as inhibitors of the JAK-STAT pathway we synthesized a series of monoketone curcumin analogs that bear a strong structural resemblance to AG490 and WP1066; known synthetic small molecule inhibitors of JAK2. A thorough investigation of the SAR of these compounds was pursued to elucidate which parts of the molecules are essential for the anti-proliferative activity. Thus hybrid molecules containing structural motifs found in all three classes of compounds were synthesized and screened using breast, prostate and colon cancer cell lines. Enzyme-linked immunosorbent assay (ELISA) measuring the relative amount of STAT3 phosphorylation was utilized to determine whether the anti-proliferative activity of these compounds was directly related to inhibition of the JAK-STAT pathway, and therefore whether these three classes of compounds exhibit similar cytotoxic mechanisms of action. Our results showed that although the monoketone curcumin analogs displayed potent cytotoxicity; they failed to inhibit STAT3 phosphorylation. The WP1066 analogs also showed potent cytotoxicity correlating directly to STAT3 inhibition. The pyridine and the halogen at the C3 position of the pyridine ring are essential for activity and appear to have a synergistic effect. Longer aliphatic chains at the benzylic position enhanced the cytotoxic activity and aromatic substitution usually led to a decrease in activity. Different structural modifications of the AG490 analogs by protection of the catechol moiety and/or changing the relative position of the substituents did not result in remarkable change in cytotoxic activity. We concluded that although the monoketone curcumin analogs bear remarkable structural resemblance to WP1066 and AG490, they do not inhibit the JAK-STAT pathway and may have a different anti-proliferative mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4502.

Improvement of a Dendritic Cell-Based Therapeutic Cancer Vaccine with Components ofToxoplasma gondii

The use of dendritic cells (DCs) as a cellular adjuvant is a promising approach to the immunotherapy of cancer. It has previously been demonstrated that DCs pulsed ex vivo with Toxoplasma gondii antigens trigger a systemic Th1-biased specific immune response and induce protective and specific antitoxoplasma immunity. In the present study, we demonstrate that tumor antigen-pulsed DCs matured in the presence of Toxoplasma gondii components induce a potent antitumor response in a mouse model of fibrosarcoma. Bone-marrow derived DCs (BMDCs) were cultured in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. After 5 days, tumor lysates with or without the T. gondii lysate were added to the culture for another 2 days. The cytokine production in the BMDC culture and the coculture supernatants of DCs and splenic cells was evaluated. For immunization, 7 days after tumor challenge, different groups of BALB/c mice received different kinds of DCs subcutaneously around the tumor site. Tumor growth was monitored, and 2 weeks after DC immunotherapy, the cytotoxic activity and the infiltration of CD8(+) T cells were monitored in different groups. According to the findings, immunotherapy with T. gondii-matured DCs led to a significant increase in the activity of cytotoxic T cells and decreased the rate of growth of the tumor in immunized animals. Immature DCs did not cause any change in cytotoxic activity or the tumor growth rate compared to that in the healthy controls. The current study suggests that a specific antitumor immune response can be induced by DCs matured with T. gondii components and provide the basis for the use of T. gondii in DC-targeted clinical therapies.

Dendritic cell maturation with CpG for tumor immunotherapy.

Bacterial DNA has immunostimulatory effects on different types of immune cells such as dendritic cells (DCs). Application of DCs as a cellular adjuvant represents a promising approach in the immunotherapy of infectious disease and cancers. To investigate the effect of tumor antigen pulsed DCs in the presence of CpG-1826 in treatment of a murine model of cancer. WEHI-164 cells (Balb/c derived fibrosarcoma cell line) were injected subcutaneously in the right flank of mice. Bone marrow cells were cultured in the presence of GM-CSF and IL-4. After 5 days, tumor lysate, CpG-1826, and oligodeoxynucleosides, as control, were added to the culture media and incubated for 2 days. Cytokine production in DCs culture media was measured by ELISA. Then DCs were injected subcutaneously around the tumor site in the right flank of mice. Tumor growth rate was monitored in case and control groups. Two weeks after DCs immunotherapy, cytotoxic assay was conducted using various amounts of effector (splenic T cells) and target cells (WEHI-164 or CT26) for 6 h. Immunotherapy with DCs treated with CpG led to a significant increase in the activity of cytotoxic T cells and decreased tumor growth in immunized mice. In the control group which received DCs without CpG treatment, no change in cytotoxic activity and tumor growth rate was detected. The current study suggests that specific anti tumor immune responses can be induced by DCs matured with CpG and proposes CpG usage in DCs targeted clinical strategies.

The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans.

The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity in peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n=6) and resting controls (CONTROL, n=4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (QRT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h. The decrease from pre- to 0 h post-exercise was seen predominately in the RUN group and was inversely correlated (r=- 0.95) to pre-exercise perforin mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry. There was no change in the proportion of NK cells expressing CD2 or CD2 density. We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.

NATURAL KILLER (NK) CELL ACTIVATION AT REST AND AFTER EXERCISE IN TRAINED AND UNTRAINED SUBJECTS

992 The aim of this study was to determine whether NK cells are activated by exercise, and if so, whether activation is related to changes in cytotoxic activity. Eight endurance trained (T) and eight sedentary (S) males, 18-29 yr, completed two sessions in balanced order: (1) 60 min cycling at 70% VO2max and (2) control (no exercise). Venous blood was obtained pre-, post- and 1.5 h post- exercise and at corresponding times in the control condition. Total (CD3-/CD 16,56+) and activated(CD3-/CD16,56+/CD69+) NK cell numbers were assessed by flow cytometry and NKCA measured by 51Cr release. All data were adjusted for diurnal variation. Resting CD16,56+/CD69+ cell numbers and percentages did not differ between T and S. CD16,56+ and CD16,56+/CD69+ cell numbers increased significantly post-exercise; CD16,56+ cell number was higher in T compared with S (P< 0.001). Total NKCA increased immediately post- but decreased 1.5 h post-exercise in both T and S (P < 0.001); suppression of NKCA 1.5 h post-exercise was greater in T than S (P < 0.05). When NKCA was calculated per CD16,56+/CD69+ (activated NK) cell, cytotoxic activity was suppressed post-exercise in both T and S (P < 0.05). These data suggest that: (1) CD69+ activated NK cells are recruited into the circulation during prolonged exercise; (2) NKCA by CD69+ NK cells is suppressed immediately post-exercise; (3) NK cell activation does not contribute to the post-exercise elevation of total NKCA; and (4) training status does not influence NK cell activation at rest and after prolonged moderate exercise.

In vitro combined effects of human interferons and interleukin-2 on natural cell-mediated cytotoxicity.

In vitro modulation of natural cell-mediated cytotoxicity (NCMC), following sequential treatment of human mononuclear cells (MNC) with cytokines was investigated. Recombinant Interleukin-2 (IL2) used in combination with interferons (IFNs) induced variable effects on the cytolytic function of different MNC preparations obtained from 16 healthy donors. When MNC were treated with IFNs on day 4, after IL2 induction of LAK cells, increase or no change in cytotoxic activity was found. On the other hand, either no change or decrease in LAK activity occurred when MNC were treated with IFNs on day 0 before exposure to IL2. In this case the effect of IFNs on NCMC did not correlate with their activity on cell proliferation or on TAC antigen expression. In conclusion the present study points out that the NCMC of MNC of healthy donors, subjected to IL2 treatment in vitro, can be significantly increased by IFNs. However this effect is largely schedule-dependent (i.e. detectable with IL2-IFNs but not with IFNs-IL2 sequence), and can be obtained in a relatively limited number of cases. Moreover it is suggested that these in vitro studies could provide preclinical bases for a rational approach to in vivo treatment with cytokine cascade in a clinical setting.

Differences in Non-MHC Restricted Cytotoxic Activities of Human Peripheral Blood Lymphocytes after Transfusion with Allogeneic Leukocytes or Platelets Possessing Class I and/or Class II MHC Molecules

MHC-unrestricted cytotoxic activity of peripheral blood lymphocytes (PBL) from 4–6 healthy donors was investigated before and after transfusion with allogeneic leukocytes or platelets. Natural killer and lectin-dependent cellular cytotoxicity (LDCC) of PBL was tested against K562 and Raji target cells in a 4-h and 16-h 51Cr-release assay, respectively. After allotransfusion with leukocytes, we found increased cytotoxic activity of each donor's PBL against all the three targets on day 3 or 7. The highest non-specific cytotoxic activity was detected against the relatively NK resistant Raji target cells. The increase of cytotoxic activity was lowest against the LDCC target (PHA-treated Raji) cells. On the contrary, no changes in cytotoxic activity against any targets were observed after allotransfusion with platelets (possessing class I HLA antigens but no HLA class 11 molecules). Our results suggest that HLA class II molecules, presumably by inducing immune responses, are essential for activation/generation of non-specific killing of tumor targets after leukocyte transfusion. Thrombocytes, known to be less immunogenic than leukocytes, are not effective in in vivo enhancing of non-specific cytotoxicity. Cellular activation of PBL following leukocyte allotransfusion was confirmed by detection of elevated serum neopterin and β-2-micro globulin levels on day 3. This was not the case after platelet allotransfusion. In addition, the expression of ICAM-1 antigen (as a molecule involved directly in MHC-unrestricted cytotoxicity) was also found to be increased in two donors' PBL on day 3 after leukocyte transfusion in contrast to transfusion with platelets.