Increased NK Cell Activity Research Articles

Abstract B11: Hyperthermia and antitumor immunity

Abstract Background: A phase III randomised EORTC trial combining systemic chemotherapy and regional hyperthermia (RHT) for patients (n=341) with locally-advanced high-risk soft tissue sarcoma (STS) showed that – compared to chemotherapy alone – the addition of RHT yields a statistically significant improvement in tumor response, with doubling of disease free and local progression free survival (ASCO 2007). A phase II study in chemo-pretreated patients with STS receiving 2nd line ICE chemotherapy (ifosfamide + carboplatin + etoposide) showed clinical activity with no further toxicity if combined with RHT (ASCO 2009). The biological rationale of this approach is based on the direct cytotoxicity of heat and its chemosensitizing effect on tumor cells. Cells respond to heat by producing increasing amounts of molecular chaperones (heat shock proteins, HSPs). Some HSPs, in particular HSP70, are heat-inducible and are released by necrosis, where outside the cells they gain potent immunostimulatory functions. We provided strong evidence that heat up-regulated expression of HSP70 correlates with enhanced susceptibility to NK cell lysis of tumor cells. Besides recognizing of HSP70 on tumors as a target structure, it has been shown that NK cells can be activated ex vivo by the HSP70-derived 14-mer peptide (TKD), enhancing NK immune function. A phase I clinical study showed that reinfusion of autologous NK cells activated ex vivo by TKD in patients with advanced disease is well tolerated, feasible and initiates cytolytic activity of NK cells. In summary, this is a strong rationale that hyperthermia might have a positive impact on the immune system within the context of the heat shock response. We are implementing a prospective randomised study that investigates the impact of autologous TKD-activated NK cells adoptively transferred in patients with high-risk STS responding to chemotherapy and RHT. In a preliminary study we investigated the number and lytic activity of NK cells in patients with high-risk STS. Patients and methods: Patients at primary diagnosis were scheduled to receive an anthracyclin and ifosfamide-based first-line chemotherapy and RHT; patients with progressive disease were scheduled to receive a 2nd line regimen (ICE) and RHT. Informed consent was obtained from all patients. Blood samples taken before the start of therapy were investigated for changes in lymphocyte subpopulations and for their lytic activity cells before and after therapy. Results: Between April 2007 and December 2008 7 1st-line and 14 2nd-line patients were enrolled. 1. A progressive reduction in absolute NK cell counts but no relevant changes in total lymphocyte counts were observed over the applied cycles in second-line patients. 2. Whereas NK mediated lytic activity against K562 is detectable in healthy donors, the spontaneous NK cell activity in all but one second-line patients was completely abrogated at the start of the therapy and was not restored over 4 cycles of ICE+RHT. 3. First-line patients also showed a significant decrease in NK cell activity compared to healthy donors 4. In vitro stimulation of patients' NK cells with IL-2/TKD induced a highly significant increase in NK cell activity against K562 cells in first- and second-line patients, indicating that NK cells preserve their ability to be activated in vitro. Most interestingly, this ability persisted over the applied cycles of thermochemotherapy. Conclusions: STS patients have suppression of NK cell activity but this lytic activity can be repristinated in vivo after stimulation with TKD/IL-2. Translational studies are performed to further elucidate the potential role of hyperthermia in promoting an antitumor immune response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B11

Maitake β-glucan enhances therapeutic effect and reduces myelosupression and nephrotoxicity of cisplatin in mice

Cisplatin is broadly used clinically as an anticancer drug. Despite its significant anticancer activity, cisplatin-induced nephrotoxicity and myelosuppression limit its use. MD-Fraction is glucan purified from maitake ( Grifola frondosa), which has β-1, 6-main chain with β-1, 3-branches, has been reported to exhibit antitumor and antimetastatic activities by enhancing the immune system. In this study, we demonstrate that MD-Fraction in combination with cisplatin significantly enhanced antitumor and antimetastatic activity compared to cisplatin alone. MD-Fraction reduced decreases in body weight, spleen weight and the number of immunocompetent cells such as macrophages, DCs and NK cells in cisplatin-treated mice. MD-Fraction also induced IL-12p70 production by splenocytes, resulting in increased NK cell activity in cisplatin-treated mice. MD-Fraction significantly increased the mRNA expression of GM-CSF, G-CSF, M-CSF, IFN-γ, IL-12 p40 in splenocytes and reduced the decrease in the number of CFU-GM colonies in cisplatin-treated bone marrow. These facts suggest that MD-Fraction can reduce cisplatin-induced myelosuppression. Moreover, treatment with MD-Fraction significantly reduced cisplatin-induced nephrotoxicity accompanied by increases in serum creatinine level, necrosis and apoptosis of renal tubular cells. These results suggest that MD-Fraction in combination with cisplatin cannot only enhance antitumor and antimentastatic acitivity, but also reduce cisplatin-induced myelotoxicity and nephrotoxicity.

Co-Transfusion of Donor NK Cells in Haploidentical Stem Cell Transplantation

39 pedatric patients with acute leukemias (ALL (n=19), AML (n=14) and MDS (n=6)) received T and B cell depleted grafts from full haplotype mismatched related donors. Depletion of the G-CSF stimulated leukapheresis products was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS device and resulted in a median number of 15.9×106 CD34 (2.5–41) stem cells, 147×106 CD56 NK-cells (9–552) and 413×106 CD14 monocytes (101–1100) per kg body weight. Median numbers of residual T and B cells were 56 000 (10 000–192 000) and 26 000 (2000–149 000) respectively. A reduced intensity regimen (melphalan (140mg/m2), thiotepa (10mg/kg), fludarabine (160mg/m2), OKT3 (0.1mg/kg)) was given in most patients. Co-transfused, HLA mismatched NK cells were traced in peripheral blood of 26 patients starting on day +1 with flow cytometry and appropriate HLA antibodies. Mean numbers of donor derived CD56+ cells/μl were: 3 (day 1), 22 (d 3), 17 (d 7), 75 (d 10), 197 (d 14). Theoretically, the mean absolute number of 4.8×106 co-transfused NK cells should have resulted in a mean number of 2000 cells/μl in peripheral blood of the patients. Comparison of this expected amount with the mean number of NK cells measured within the first week postransplant (25/μl, n=17 data points) showed, that only 1.2% of the cells remained in circulating blood. Thus, the majority of donor NKs did not circulate and probably homed to other compartments (bone marrow, lymph nodes). The number of NK cells cotransfused at day 0 partially influenced the speed of NK cell recovery: patients, who received > 100×106 donor NK cells/kg had significantly higher amounts of circulating cells at day 14 than patients, who received <100×106 donor NKs (240 vs. 140/μl, p<0.05). No significant difference was observed after d 14. Recovery of T cells was not influenced. Graft rejection occurred in 13%. This rate was similar to that of a historical control group (15% in patients who received CD34 positive selected grafts and standard conditioning regimens), although our study patients mainly received an intensity reduced regimen. We conclude, that co-transfused cells facilitated hematopoietic engraftment. Our approach resulted in low TRM (10% at d 365) and in a low relapse rate (20% at 2 years) in patients with microscopical remission (<5% blasts), but was insufficient in patients with active disease (80% relapse rate). We therefore investigated options to increase NK cell activity. Cytotoxicity against K562 cells and thymidine-uptake after PHA stimulation were measured prior and post depletion in 30 procedures. Median specific lysis at E:T ratio = 20:1 was 15% prior and 23% post depletion. Thus, NK activity was not hampered by the procedure. Specific lysis was significantly enhanced by pre-incubation with 1000 U/ml Interleukin (IL) 2 (44%, median) or 2ng/ml IL 12 (40%, median) or 1ng/ml IL 15 ( 53%, median) in vitro. In contrast, thymidine-uptake was reduced from 170 000 to 3000 counts due to profound T-cell-depletion. NK activity was weak against patient derived cryopreserved leukemic blasts without stimulation, but could be significantly increased by cytokine incubation in vitro. Therefore, a pilot study with infusions of IL 15 stimulated NK cells in vivo was started. Up to now, 6 patients received a total of 8 infusions with 12×106 - 150×106 ex vivo stimulated NK cells per kg bw without any side effects. Conclusions: co-transfusion of donor NK cells in haploidentical transplantation is feasible. Only a small portion of cells remained in circulating blood and homing to other organs is likely. NK activity could be increased by cytokines; the use of ex vivo IL 15 stimulated NK cells is currently evaluated. Clinical results suggest antileukemic and graft facilitating effects of donor NK cells.

HsBAFF enhances activity of NK cells by regulation of CD4 + T lymphocyte function

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T cell activation. Recently, we have demonstrated that human soluble BAFF (hsBAFF) potently elevates NK cell activity of murine splenic cells in vivo. In this study, we further show that hsBAFF markedly enhanced NK cell activity of in vitro mouse splenic cells. Purified NK cell activities treated with hsBAFF in the presence of IL-2 or IFN-γ, but not with hsBAFF alone were obviously elevated. hsBAFF strongly increased the activity of NK cells co-incubated with mixed T and B lymphocytes or T lymphocytes alone, but not with B lymphocytes alone. We also found that culture supernatants, from mixed T and B lymphocytes or T lymphocytes alone, but not from B lymphocytes alone post-hsBAFF treatment, effectively increased NK cell activity, and noticed that IL-2 and IFN-γ levels significantly increased in the culture supernatants of hsBAFF-treated T lymphocytes. Of importance, we pinpointed that hsBAFF elicited robust population of CD4 + T lymphocytes in vitro, and further demonstrated that hsBAFF induced significantly high IL-2 and IFN-γ secretion of CD4 + T lymphocytes. These findings indicate that hsBAFF results in the elevation of NK cell activity by regulation of CD4 + T lymphocytes contributing to IL-2 and IFN-γ generation.

Immunosuppressive effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) in the rat

THI is a component of ammonia caramel, a widely used food colouring. The effect of THI on the immune system has been determined in the male F344 rat. THI was given in the drinking water at doses of 1, 10 and 50 mg/l (equivalent to 0.1, 1 and 5 mg/kg per day) to animals on a vitamin B6-deficient diet. After 1 week, the immune competence of the animals was assessed under continued THI treatment. No marked changes in thymus or spleen weight were observed after THI treatment, although there was an increased number of pyknotic cells in the thymic cortex, mainly engulfed by macrophages and there appeared to be a slight thinning of the cortex area. THI produced a significant loss in T and B lymphocytes in peripheral blood but not in the spleen. No change in natural killer (NK) cell activity against YAC-1 target cells was observed in the spleen. The observed increase in NK cell activity in peripheral blood was due to an increase in circulating large granular lymphocytes (LGL). Although the serum antibody titre against keyhole limpet haemocyanin (KLH) was not affected by THI treatment, B cells showed less proliferation when cultured with lipopolysaccharide. T cell function was impeded as measured in mitogen-induced proliferation assay, delayed-type hypersensitivity assay and host versus graft (popliteal lymph node) assay. The results indicate that THI is an immunosuppressor in the rat, in whom it can produce profound lymphopenia and suppression of cell-mediated immunity.

Mice lacking TLR4 function exhibit enhanced susceptibility to Rickettsia conorii, correlated with decreased NK cell activity (44.14)

Abstract Toll-like receptors (TLR) are critical in the initiation and polarization of protective immune responses to a wide variety of pathogens. However, the importance of TLRs in immunity to rickettsial diseases has been poorly defined. We demonstrated in a model of human rickettsiosis that mice lacking TLR4 function exhibited enhanced susceptibility to R. conorii infection compared to mice having functional TLR4 responses. Lack of TLR4 signaling resulted in overwhelming rickettsial growth in the brain and lung—the major pathologic organs. Infected mice lacking TLR4 function had decreased splenic NK cells and an increase in regulatory T-cells in peripheral lymph nodes. Further, splenocytes obtained from infected mice lacking TLR4 activity had decreased NK cell cytotoxicity activity. The lack of TLR4 signaling resulted in significantly reduced serum IL-12, IL-23, and IL-17. Thus, TLR4 is important in initiating early rickettsial immunity, possibly because of enhanced Th1 proinflammatory cytokines and increased NK cell activity.

Substance P modulation of anti-tumor responses: enhanced tumor resistance by adoptive transfer of SP-treated T cells and increased NK cell activity (50.5)

Abstract Substance P (SP) is a neuropeptide that has been shown to have immunoregulatory properties including effects on mediators involved in anti-tumor immunity. In previous studies we have shown that treatment of mice with SP provides protection against tumor growth. This protection requires both T cells and NK cells, and adoptive transfer of cells from SP-treated animals can transfer tumor protection. These studies suggest a model in which SP treatment prior to tumor challenge primes immune mediators to prevent or delay tumor establishment. In this study we examined the role of NK cells and T cells in tumor protection. The cytotoxic activity of NK cells against Yac-1 targets was examined by labeling cells with annexin-V and measuring apoptotic cell death by flow cytometry. NK cells from mice treated with SP had significantly higher cytotoxic activity compared to NK cells from controls. To examine whether SP-primed T cells could increase tumor resistance, purified T cells from SP-treated donor mice were adoptively transferred into animals prior to tumor challenge. Mice adoptively transferred with T cells from SP-treated donors developed significantly fewer tumors than did controls. These results suggest that SP mediates anti-tumor immunity in part by increasing NK cytotoxic activity. Since SP-primed T cells can transfer tumor protection, these cells may play a role in this increase in NK activity.

Direct vaccination with pseudotype baculovirus expressing murine telomerase induces anti-tumor immunity comparable with RNA-electroporated dendritic cells in a murine glioma model

Baculovirus pseudotyped with vesicular stomatitis virus G protein (Bac-VSV-G) was found to efficiently transduce and express transgenes on mammalian cells. In this study, this recombinant virus was used for induction of anti-tumor immunity against murine telomerase reverse transcriptase (mTERT) and was compared with RNA-electroporated dendritic cells (DCs) in a murine glioma model. Splenocytes from the mice vaccinated with Bac-VSV-G expressing mTERT (Bac-VSVG-mTERT) showed significantly increased numbers of mTERT-specific IFN-γ-secreting T cells using an ELISPOT technique, and also showed increased NK cell activity. In addition, the TERT-specific T cells activated by Bac-VSVG-mTERT and mTERT RNA-electroporated DCs were predominantly CD4 + T cells and CD8 + T cells, respectively. The protective anti-tumor effect of Bac-VSVG-mTERT was similar to that of mTERT RNA-electroporated DCs. These results suggest that the pseudotype baculovirus expressing TERT may be a good candidate for a genetic vaccine for use in the treatment of malignant gliomas.

Pretherapy predictive immunomodulation of NK cell activity and expression of activating and inhibitory receptors in stage IV melanoma patients

12514 Background: As melanoma (MM) is an immunogenic tumor with poor response to chemotherapy immunomodulating agents are applied in order to potentiate cytotoxic effect of chemotherapy and enhance antitumor immune response. Beside therapeutic benefit of IFN-α and IL-2, 13-cis retinoic acid (RA), as an antiproliferative, differentiating and immunomodulating agent is also investigated. The effect of these agents on NK cells, as main innate immune system effectors, is being investigated. Methods: 35 patients with MM in stage IV prior to therapy and 20 controls were investigated. We evaluated NK activity and the expression of activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptors on freshly isolated PBL. Predicitive immunomodulation was performed in 18 h in vitro treated PBL with rh IL-2 (200U/ml), IFN (250U/ml), RA (10−6M), and their combination. Results: Native NK cell cytotoxic activity and expression of NKG2D and CD161 activating receptors on fresh NK cells in MM patients is significantly decreased compared to controls. Predictive treatments with IL-2, IFN, IFN and RA, unlike RA alone, gave a significant increase in NK cell activity of MM patients. Singly, IFN also induced a significant increase in CD161 expression on NK cells in patients. The treatments gave no change in the expression of CD158b, while RA, alone, induced significant decrease in the expression of the inhibitory CD158a antigen. Evaluation of mRNA of transcription molecule IRF-1 shows that it is promptly up-regulated by IFNα, more by IFNα and RA, while single RA has no effect on mRNA induction. Conclusions: Considering that the mechanism of applied immunomodulating agents is continually investigated in order to optimize the dose and schedule of their administration and, also, considering controversial clinical results of RA application, alone, or with IFN, in this study we give novel results that show no significant effect of RA, whereas, IFN-induced increase in NK cell activity of MM patients is for the first time associated with the increase in the expression of NKG2D and CD161 receptors on CD16+NK cells, and not with the decrease in some inhibitory receptors, as the activity of NK cells is regulated by the balance of these two types of signals. No significant financial relationships to disclose.

Effects of Methamphetamine on Cortisone Concentration, NK Cell Activity and Mitogen Response of T-lymphocytes in Female Cynomolgus Monkeys

As a model for studying methamphetamine (MAP) abuse, which has become a social problem in Japan, we investigated the changes in serum cortisone, NK cell activity and mitogenic response of T-lymphocytes after a single injection of MAP (3.0 mg/kg) in female cynomolgus monkeys. Serum cortisol concentration was significantly elevated to 2.66 times pre-injection levels at 6 h post-injection, and the effect was still observed 24 h later. NK cell activity was significantly elevated at 6 h after MAP injection, but at 24 h after injection had dropped markedly to 49.5% of baseline. Mitogen (PHA) response of lymphocytes was elevated when MAP was injected, and this increased level continued up to 24 h. We speculate that the transient increase in NK cell activity followed by a distinct drop, as well as the changes in T-lymphocytes, may be strongly related to the cortisone concentration.

Anti-cancer Effect of Hematopoietic Stem Cell-derived Allogeneic-DC Vaccine in Melanoma Metastasis Model

Background: Dendritic cell (DC)-based cancer immunotherapy is studied for several years. However, it is mainly derived from autologous PBMC or leukapheresis from patient, which has limitations about yield and ability of DC production according to individual status. In order to solve these problems, inquiries about allogeneic DCs are performed but there are no preclinical trial answers for effect or toxicity of allogeneic DC to use for clinical trial. In this study, we compared the anti-tumor effect of allogeneic and autologous DCs from mouse bone marrow stem cells in mouse metastatic melanoma model. Methods: B16F10 melanoma cells (/mouse) were injected intravenously into the C57BL/6 mouse. Therapeutic DCs were differentiated from autologous (C57BL/6: CDC) or allogeneic (B6C3F1: BDC) bone marrow stem cells with GM-CSF, SCF and IL-4 for 13days and pulsed with B16F10 tumor cell lysate (Blys) for 18hrs. DC intra-peritoneal injections began on the 8th day after the tumor cell injection by twice with one week interval. Results: Anti-tumor response was observed by DC treatment without any toxicity especially in allogeneic DC treated mice (tumor burden score: for saline, CDC/unpulsed-DC: U-DC, CDC/Blys-DC, BDC/U-DC and BDC/Blys-DC, respectively). IFN- secretion was significantly increased in allogeneic DC group stimulated with B16F10 cell lysate ( cells for saline, BDC/U-DC and BDC/Blys-DC, respectively) with increased NK cell activity. Conclusion: Conclusively, promising data was obtained that allogeneic DC can be used for DC-based cancer immunotherapy.

An autologous tumor-derived heat shock protein vaccine for high risk ovarian cancer

9592 Background: Five-year survival for stage III and IV ovarian cancer of the epithelial cell origin is less than 30%, despite surgery and chemotherapy, due largely to disease relapse. Tumor-derived heat shock protein (HSP) gp96-peptide complexes can induce tumor-protective T cell immunity in multiple preclinical models. We have initiated the first study of consolidation therapy with autologous gp96 vaccine for stage III/IV ovarian cancer. Methods: All patients have the diagnosis of stage III or IV ovarian cancers. Gp96-peptide complexes are purified from autologous tumors procured at the initial surgery. Patients who have completed standard therapy (Carboplatin/Taxane) and have no evidence of progressive disease are eligible to receive the vaccine. The first 11 patients receive gp96, 25 μg i.d., weekly for 8 weeks. The second group of 11 patients will receive the same dose and schedule of gp96 vaccine, mixed with 100 μg GM-CSF. Patients are followed clinically for toxicity and relapse-free survival, and immunologically for T cell and NK cell responses. Results: Twenty patients signed consent and their tumors were procured. So far, gp96 was purified successfully for all 8 patients. Five have completed the vaccination with gp96. Three are being immunized. No patients received gp96 and GM-CSF just yet. Grade II or higher toxicity was not observed. All 5 patients demonstrated increased NK cell activity measured by ELISPOT against K562 cells. Three of 5 patients showed increased IFNγ-producing cells in the peripheral blood against autologous gp96-peptide complexes that are MHC class I-dependent. Conclusions: This is the first trial to test an HSP-based vaccine against ovarian cancer in a consolidation setting. Our results demonstrated that it is feasible and well-tolerated. In addition, measurable immunological responses were observed. Future studies will determine the clinical efficacy of gp96 vaccine for ovarian cancer and the potential benefits of adding GM-CSF in HSP vaccines. Acknowledgement: This work is supported in part by the Pamela Weinstein Ovarian Cancer Endowment Fund. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antigenics Antigenics

Cutting edge: increased NK cell activity in HIV-1-exposed but uninfected Vietnamese intravascular drug users.

We addressed the role of innate immunity in the protection against HIV-1 infection by studying NK cell function in 37 Vietnamese intravascular drug users (IDUs), who appeared to remain HIV-1 uninfected despite many years of high-risk exposure (exposed uninfected, EU), 10 IDUs who underwent seroconversion and 28 unexposed blood donors. Main results were: NK cell lytic activities against both the NK-susceptible K562 cell line and the NK-resistant Daudi cell line were significantly augmented in EU IDUs compared with either controls or seroconverters before or after seroconversion; NK cells producing the cytokines IFN-gamma and TNF-alpha and the beta chemokines CCL3, CCL4, and CCL5 were also increased in the EU IDUs, either after in vitro activation or without stimulation. The finding of an enhanced NK cell function in EU IDUs, especially compared with IDUs who became HIV-1 infected, supports the hypothesis that NK cells contribute to the protection against HIV-1 infection.

Inhibition of NF-κB Activity in T and NK Cells Results in Defective Effector Cell Expansion and Production of IFN-γ Required for Resistance to Toxoplasma gondii

To define the role of NF-kappa B in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (Delta N) form of I kappa B alpha, an inhibitor of NF-kappa B, were challenged with T. gondii and their response to infection compared with control mice. I kappa B alpha(Delta N)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-gamma, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4(+) T cells was similar in Wt and Tg mice, but expansion of activated CD4(+)T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4(+) T cells from infected mice to produce IFN-gamma. Together, these studies reveal that inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to T. gondii.

Effects of Coronary Artery Bypass Grafting on Cellular Immunity with or without Cardiopulmonary Bypass: Changes in Lymphocytes Subsets

Cell-mediated immunity responses decrease after all kinds of surgical procedures. Either anesthesia or surgical trauma plays an important role in this effect. Identification of functional lymphocyte subsets, by using appropriate monoclonal antibodies and analysis of flow cytometry data, appears to provide an accurate measurement of cellular immune competence. We found a significant decrease in the total number of T helper/inducer cells ( p&lt;0.035), B cells ( p&lt;0.043) and natural killer cells (NK) ( p&lt;0.018) but in contrast, increase in NK cell activity ( p&lt;0.012) in the peripheral arterial blood of ten patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (group 1) immediately after surgery and postoperative day 1 (POD1). On the other hand, there was no significant change of these parameters occurred in the peripheral arterial blood of ten patients (group 2) who were undergoing coronary artery bypass grafting without cardiopulmonary bypass. Therefore, we conclude that coronary artery bypass grafting (CABG) with cardiopulmonary bypass induce a greater decrease in immunologic response than CABG without cardiopulmonary bypass (off pump) operations. Nevertheless, off pump CABG operations do not induce a greater decrease in immunologic response than other surgical operations.

Effects of coronary artery bypass grafting on cellular immunity with or without cardiopulmonary bypass: changes in lymphocytes subsets

Cell-mediated immunity responses decrease after all kinds of surgical procedures. Either anesthesia or surgical trauma plays an important role in this effect. Identification of functional lymphocyte subsets, by using appropriate monoclonal antibodies and analysis of flow cytometry data, appears to provide an accurate measurement of cellular immune competence. We found a significant decrease in the total number of T helper/inducer cells ( p<0.035), B cells ( p<0.043) and natural killer cells (NK) ( p<0.018) but in contrast, increase in NK cell activity ( p<0.012) in the peripheral arterial blood of ten patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (group 1) immediately after surgery and postoperative day 1 (POD1). On the other hand, there was no significant change of these parameters occurred in the peripheral arterial blood of ten patients (group 2) who were undergoing coronary artery bypass grafting without cardiopulmonary bypass. Therefore, we conclude that coronary artery bypass grafting (CABG) with cardiopulmonary bypass induce a greater decrease in immunologic response than CABG without cardiopulmonary bypass (off pump) operations. Nevertheless, off pump CABG operations do not induce a greater decrease in immunologic response than other surgical operations.

Genistein Modulates Splenic Natural Killer Cell Activity, Antibody-Forming Cell Response, and Phenotypic Marker Expression in F0 and F1 Generations of Sprague-Dawley Rats

The potential effects of the phytoestrogen genistein (GEN) on the immune system were evaluated in both F0 (dams) and F1 generations of Sprague-Dawley rats exposed to a soy-free diet containing low (L: 25 ppm), middle (M: 250 ppm), and high (H: 1250 ppm) levels of GEN. In dams, exposure to GEN from Gestation Day 7 to Postpartum Day 51 (totally 65 days) produced a significant increase in NK cell activity (M and H), while a decrease in the percentage of helper T cells (H). In F1 males, exposure to GEN gestationally, lactationally, and through feed from Postnatal Days 22 to 64 (total 78 days) produced an increase in the relative weights (% body) of spleen (L and H) and thymus (L). Furthermore, exposure to GEN increased the number of splenic B cells (H), T cells (L, M, and H), and T-cell subsets (L, M, and H). Although GEN decreased the percentages of splenic NK cells (L, M, and H), no effect on the activity of NK cells was observed. In F1 females, exposure to GEN produced a decrease in terminal body weight (H), with an increase in the relative weight of spleen (L, M, and H). Exposure to GEN also increased the number of splenic B cells (L), macrophages (L and M), T cells (H), helper T cells (L and H), and cytotoxic T cells (M and H). Additionally, exposure to GEN increased the percentages of T cells (M and H), helper T cells (H), and cytotoxic T cells (M and H). Moreover, the spleen IgM antibody-forming cell response to sheep red blood cells was enhanced (H), although the percentages of B cells were decreased (M and H). No effect on the activity of NK cells was observed; however, the percentages of splenic NK cells were decreased by GEN (L and H). In conclusion, these results demonstrate that exposure to GEN can modulate the immune responses in Sprague-Dawley rats. Furthermore, the sexual dimorphic effects of GEN in F1 male and female rats suggest that there may be interactions between GEN and the responses modulated by sex hormones.

Chronic ethanol inhibits NK cell cytolytic activity: role of opioid peptide beta-endorphin.

The role of beta-endorphin (beta-EP) in ethanol-altered NK cell cytolytic activity is studied using male Fischer-344 rats as an animal model. Ethanol was administered for 1, 2, 3, or 4 wk in a liquid diet containing 8.7% ethanol (v/v), which means that 37% of the total calories were derived from ethanol. Rats treated with ethanol for 1 wk showed an increase in hypothalamic and plasma levels of immunoreactive (IR)-beta-EP, but displayed no significant effect on NK cell activity determined by (51)Cr release assay, as compared with those in pair-fed and ad libitum-fed animals. However, animals treated with ethanol for 2, 3, or 4 wk showed decreased hypothalamic and plasma levels of IR-beta-EP and decreased splenic NK cell activity. No significant decrease in the number of splenocytes and NK cells or in the percentage of NK cells was seen until after 3 and 4 wk of ethanol treatment. Exposure in vitro of splenic lymphocytes obtained from control animals to various concentrations of beta-EP increased NK cell activity. The opiate antagonist naltrexone blocked the beta-EP-stimulated effect. The in vitro NK cell response to beta-EP was reduced in the splenocytes obtained from animals treated with ethanol for 2 wk, but not in those obtained from animals treated with ethanol for 1 wk as compared with those in control animals. Additionally, beta-EP administration into the paraventricular nucleus of the hypothalamus stimulated NK cell cytolytic activity, whereas the opiate blocker administration reduced NK cell activity. The NK cell responses to paraventricular nucleus beta-EP were reduced in the animals treated with ethanol for 2 wk. These data provide evidence for the first time that ethanol inhibits NK cell cytolytic activity, possibly by reducing beta-EP-regulated splenic NK cell function.

Increased natural killer-cell activity is associated with infertile women.

An increase in natural killer (NK)-cell activity has been observed in women with unexplained recurrent miscarriages. Because of the many similarities between infertility and early pregnancy loss patients, we investigated whether infertile women had raised NK-cell activity. We tested 94 infertile women who, in spite of treatment, were unable to conceive for 6 or more months. NK-cell activity was measured by using a chromium-51 release cytotoxicity assay. with K562 human myeloid leukemia cells as targets. NK-cell activity of the infertile group (mean +/- SD; 40.2%+/-14.7) was significantly higher than the control group (31.5%+/-11.9, P < 0.0001). The increased NK-cell activity was not associated with age, infertile duration, depression scores, treated hyperprolactinemia, or treated endometriosis. In certain patients, elevated NK-cell activity may be considered an independent risk factor for infertility.

Total Sleep Deprivation Induces an Acute and Transient Increase in NK Cell Activity in Healthy Young Volunteers

To investigate the effects of one night's total sleep deprivation (TSD) on NK cell activity, with rigorous control of circadian phase of sampling points as well as physical exercise level in association with sleep deprivation. The mean sleep onset time of each subject before starting the study was defined as his 0000 h. This study was composed of a Sleep-Sleep session (sleep times, 00:00 h - 08:00 h and 24:00 h - 32:00 h) and a Sleep-Wake session (sleep time, 00:00 h - 08:00 h) with TSD (24:00 h - 32:00 h) placed in a cross-over design with 2-week interval between each session. In each session, the subjects were rested in the supine position under dim light from - 06:00 h to 36:00 h (for 42 hours). University-based sleep and chronobiology laboratory 10 healthy adult men (mean age, 20.9 y; age range, 19-23 y) NA. NK cell activity was measured every 4 hours from 12:00 h. NK cell activity during TSD (at 28:00 h) has been revealed to significantly increase (p=0.01) compared with the corresponding value in the Sleep-Sleep session. This effect was weaker at their usual waking time 32:00 h (p=0.07), and disappeared until 36:00 h (4 hours after awakening). The circadian rhythm phases (dim light melatonin onset time) were coincident between the 2 sessions. The present findings suggest that one night TSD induces an acute and transient increase in NK cell activity that is not influenced by the effects of circadian rhythm or the amount of physical exercise undertaken during TSD.