Cytotoxicity of leukocytes from normal and Shigella-susceptible (opium-treated) guinea pigs against virulent Shigella sonnei.

Intraepithelial lymphocytes were collected from the ileum of adult Hartley strain guinea pigs and used as effector cells in a 60-min bactericidal assay with virulent Shigella sonnei as target cells. Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) were measured and correlated with the resistance of the animals to infection by S. sonnei. Normal guinea pig intraepithelial lymphocytes exhibited mean NKC and ADCC values of 22.8 +/- 5.0 and 34.1 +/- 13.6, respectively. These animals were resistant to oral challenge with virulent S. sonnei. Intraepithelial lymphocytes from guinea pigs which were fasted for 4 days demonstrated NKC and ADCC values similar to those of normal animals (31.0 +/- 8.1 and 41.7 +/- 6.7, respectively). These animals also were resistant to oral challenge. Intraepithelial lymphocytes from guinea pigs which were given 1 ml of deodorized tincture of opium 2 h before cell collection demonstrated deficient NKC (4.7 +/- 4.2) and ADCC (5.3 +/- 4.9) values but remained resistant to infection by S. sonnei. When guinea pigs were fasted for 4 days and given opium, deficient NKC (2.0 +/- 2.0) and ADCC (1.3 +/- 1.3) values were demonstrated; this group of animals was susceptible to infection by S. sonnei (P less than 0.04). These experiments demonstrated that opium treatment depresses one form of gut immunity. When combined with starvation, opium treatment may increase susceptibility to infection by shigellae by modulation of immunity in addition to the effects on gut motility and bacterial flora.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.     

Pretreatment with lipoteichoic acid sensitizes target cells to antibody-dependent cellular cytotoxicity in the presence of anti-lipoteichoic acid antibodies.

This study was performed to determine whether antibody-dependent cellular cytotoxicity (ADCC) could be directed against mammalian cells sensitized with spontaneously adhering bacterial substances. 51Cr-labeled SB leukemia cells were incubated with purified S43 group A streptococcal lipoteichoic acid (LTA; 0.001 to 100 micrograms/ml). Purified leukocyte ADCC effector cells were added to the LTA-coated target cells at various effector-to-target ratios (100:1 to 12:1), followed by the addition of rabbit anti-LTA. After incubation for 4 h, target cell lysis was calculated based on the release of label into the medium. As little as 1 ng of LTA per ml was sufficient to sensitize the target cells to ADCC lysis (12%); however, concentrations above 0.1 micrograms/ml generally resulted in 60 to 80% lysis. LTA alone was not cytotoxic to these target cells. Targeting did not occur if effector cells were sensitized or if free LTA was added to the medium. Specificity was demonstrated by cold-target inhibition, which showed that anti-LTA cytotoxicity could be inhibited only by unlabeled, LTA-treated target cells but not by cold SB cells alone. The findings indicate that certain soluble bacterial components, when bound to mammalian cells in the presence of specific antibody, can target ADCC effectors to these cells. This mechanism may be an important factor in the delayed sequelae of bacterial infections.     

血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用

目的:证实HLA-G1分子能够抑制NK细胞对同种血管内皮细胞系的杀伤作用。方法:采用脂质体介导的DNA转染技术,以构建的真核表达质粒pcDNA3-HLA-G1转染人脐静脉内皮细胞系ECV304,再用免疫荧光法检测表达的HLA-G1分子。并用MTT比色法检测HLA-G1对NK细胞杀伤活性的影响。结果:ECV304细胞上可稳定表达HLA-G1。NK细胞对空质粒pcDNA3转染的ECV304细胞的杀伤率为(50.6±18.1)%;而对pcDNA3-HLA-G1转染的ECV304细胞的杀伤率为(29.7±11.4)%,两者差异具有显著意义(P<0.01)。结论:HLA-G1分子可明显抑制NK细胞对同种血管内皮细胞的杀伤效应。     

Cell-mediated immunity to Epstein-Barr-virus-transformed lymphoblastoid cells in acute infectious mononucleosis.

Mononuclear peripheral blood leukocytes from 21 patients with infectious mononucleosis and 16 healthy controls were tested in a 51Cr-release assay for cytotoxicity against two human lymphoblastoid cell lines derived from the same donor. One line contained the Epstein-Barr virus (EBV); the other did not. Acute-phase leukocytes were significantly more cytotoxic against the EBV-infected cell line than were control leukocytes. Mean (+/- S.E.) lysis at a leukocyte-target-cell ratio of 100:1 was 10.6 +/- 1.6 per cent for patients and 3.4 +/- 0.6 per cent for controls (P less than 0.0005). Cytotoxicity correlated with the percentage of atypical lymphocytes. Cells of three patients with acute mononucleosis-like illnesses failed to show killing activity above those of normal controls. Cytotoxicity against the EBV-negative line was not significantly different for each group. The finding of cytotoxic cells in infectious-mononucleosis patients with atypical lymphocytes suggests that these cells operate in vivo to limit the proliferation of altered EBV-transformed B lymphoblasts.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

K-cell cytotoxic activity in the spleen and lymph nodes of tumour-bearing mice.

The cytotoxic activity of spleen and lymph node cells from female CBA mice bearing a methylcholanthrene-induced fibrosarcoma on target cells coated with anti-target cell antibody has been investigated. Spleen cells from tumour-bearing mice caused a significantly greater degree of target cell lysis than did spleen cells from control mice. This increase in cytotoxicity was most apparent when a ratio of ten lymphoid cells to one target cell was used. With control mice, the mean cytotoxic index was 13.54 +/- 1.05 (s.e.m.) as compared to 55.00 +/- 6.11 with tumour-bearing mice. An increase in the cytotoxic activity of lymph node cells was also noted. In control mice, the cytotoxic index, using a ratio of 100:1 was 8.55 +/- 2.27. In tumour-bearing mice, the mean cytotoxic activity of the draining lymph node was 21.66 +/- 4.96, and of the contralateral lymph node, 13.00 +/- 3.46.     

Immune modulation of natural killer cell cytotoxicity against herpes infected target cells in pregnancy

Natural killer cell cytotoxicity (NKC) is a nonspecific, primary immunodefense system active against a variety of pathogens, including herpes simplex virus (HSV). Evidence suggests that during pregnancy, NKC is attenuated. The regulatory mechanisms for this immune attenuation have yet to be defined. We examined two cytokines (interleukin-2 [IL-2] and alpha interferon [IFN]) for their ability to alter NKC responsiveness during pregnancy, utilizing an HSV-infected target cell model. Peripheral mononuclear effector cells were isolated from 19 pregnant and 19 nonpregnant subjects by Ficoll-Paque separation. These cells were incubated with IFN, IL-2, or media alone, and analyzed for %NKC by an 18 h chromium release assay. The percentage of NKC was lower using the effector cells from the pregnant subjects as compared to nonpregnant controls. Incubation with either IFN or IL-2 resulted in a significant augmentation of NKC in both the pregnant and nonpregnant derived cells. There were no differences in IL-2 dose requirements or levels of cytotoxicity achieved (43.1 +/- 6.8% vs. 44.4 +/- 6.8%, respectively) between pregnant and nonpregnant derived cells. The IFN-mediated augmentation of NKC was somewhat blunted in pregnancy both in terms of absolute levels of cytotoxicity achieved (26.1 +/- 3.9% vs. 37.2 +/- 4.9%, respectively) and dose response curves generated. These results demonstrate that NKC against HSV infected cells is attenuated during pregnancy and can be immunoregulated with the use of either IFN and IL-2. The restoration of NKC responsiveness with IFN, however, remains incomplete during pregnancy, suggesting that this cytokine's mechanism of action differs from that of IL-2.     

Assessment of physiologic natural killer cell cytotoxicity in vitro

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.     

Production of TNF-alpha and bone resorbing activity by macrophages in response to different types of bone cement particles

We have compared the capacity of clinically relevant wear debris from seven different cement types to activate macrophages to produce TNF-alpha, IL-1beta, IL-6 and bone resorbing activity in vitro. The bone cements were: CMW 1 original (PMMA only); CMW 1RO (1 microm BaSO4; 9.2%); CMW copolymer bone cement 1 (10 microm BaSO4; 10%); CMW copolymer bone cement 2 (1 microm BaSO4; 10%); Palacos R (10 microm ZrO2; 15.6%); CMW Calcium phosphate cement 20% (10 microm tri-calcium phosphate; 20%) and CMW calcium phosphate cement 30% (10 microm tri-calcium phosphate; 30%). Cement debris was produced aseptically using a simple configuration wear test. The majority of particles were in the size range 0.1-0.5 microm for each cement type. The cement particles were co-cultured with the U937 macrophage cell line at ratios of 10 and 100 microm3 particle volumes to macrophage cell numbers for 24 h. At the 10:1 ratio the particles had no effect on the cells. At the 100:1 ratio, the major cytokine produced was TNF-alpha and there were no statistical differences between the different types of cement debris. The bone resorption activity of the co-culture supernatants was significantly greater than the control (U937 cells without particles) for particles of CMW 1RO, CMW copolymer bone cement 1, CMW copolymer bone cement 2 and Palacos R (P < 0.05, ANOVA). However there were no statistical differences between the levels of bone resoprtion evoked by these four cement types. The CMW1 original and CMW calcium phosphate containing cements failed to induce the macrophages to elaborate bone resorption activity at the 100:1 ratio. These data suggest that the addition of radio-opaque additives to bone cement may increase the capacity of the debris to induce osteolysis.     

Indomethacin augments lymphokine-activated killer cell generation by patients with malignant mesothelioma

Human malignant mesothelioma (MM) cells are resistant to natural killer (NK) cell lysis but susceptible to lysis by lymphokine-activated killer (LAK) cells from control individuals. The present study was performed to determine the capacity of patients with MM (n = 22) and individuals occupationally exposed to asbestos (the major population at risk of developing this disease, n = 52) to generate LAK cells capable of effectively lysing human mesothelioma cells. Compared to controls (n = 20), both patient groups demonstrated significantly depressed LAK cell activity against mesothelioma tumor cell targets (55 +/- 3% lysis by controls vs 34 +/- 3% lysis by patients with MM, P less than 0.005; and 45 +/- 3% lysis by asbestos-exposed individuals, P less than 0.025). Addition of 10 micrograms/ml indomethacin during LAK cell generation restored normal LAK cell activity for patients with MM (52 +/- 6% lysis of cultured human MM cells, P = NS compared to controls), suggesting that the defective cytolytic cell function observed in some patients with MM is a result of prostaglandin-induced immunosuppression. The ability of indomethacin to restore suppressed LAK cell activity in patients with MM suggests that the concomitant use of this agent in ex vivo LAK cell generation and in patients undergoing interleukin/LAK cell therapy may be beneficial.     

Natural Killer Cytotoxicity and Antibody-Dependent Cell Cytotoxicity to Herpes Simplex Virus-Infected Target Cells in Murine Pregnancy

ABSTRACT: In vitro natural killer cytotoxicity (NKC) and antibody-dependent cell cytotoxicity (ADCC) activity against herpes simplex virus (HSV)-infected cells were evaluated in a pregnant murine model (C₅₇B1₆inbred strain). Virgin (n = 16) and pregnant (late gestation) mice (n = 15) were infected intraperitoneally with HSV, type 1. After 18 hr, a 0.5-ml aliquot of the peritoneal wash was frozen for virus plaque assay, and the cells were cultured in the ⁵¹chromium release assay for NKC and ADCC. %NKC (mean ± S.E.) to HSV-infected targets was significantly suppressed (P < 0.05) in pregnant mice, 10.3% ± 1.9, compared to that of virgin mice, 32.5% ± 2.5. This suppression was abrogated with HSV-specific antisera (%ADCC); 53.9% ± 4.4 (pregnant) compared to 49.1% ± 3.6 (virgin). The diminished NKC activity in pregnant mice was reflected in an increased mean number of virus particles in the peritoneal wash, 266 + 66 PFU/ml, compared to 38 ± 11 PFU/ml in virgin mice (P < 0.05). We concluded that NKC, but not ADCC, to HSV-infected targets was suppressed and that HSV elimination was impaired in pregnant mice.     

T-cell cytokine pattern at three time points during specific immunotherapy for mite-sensitive asthma

BACKGROUNDS: Several lines of evidence indicate that specific immunotherapy may act by modifying the patterns of cytokines produced by helper T cells. However, different protocols have been used and different results obtained. OBJECTIVES: To quantify the effect of specific immunotherapy on the TH1/TH2 T-cell cytokine pattern at the single cell level. METHODS: We examined the interferon-gamma/interleukin-4 ratio in peripheral blood CD4+ and CD8+ T cells from 12 subjects with house dust mite-sensitive asthma using a flow cytometric method of intracellular cytokine detection. Cytokine production was determined following stimulation with phorbol myristate acetate/ionomycin, a policlonal activator. Subjects were examined at three occasions: before specific immunotherapy, after the 3-months dose increase phase and after 1 year of treatment. During the treatment year patients kept a diary in which they recorded: (a) symptoms of asthma according to a 0-3 grading (0 = absent, 1 = mild, 2 = moderate, 3 = severe); (b) number of puffs (100 microg) per day of salbutamol required to control symptoms; and (c) peak expiratory flow. RESULTS: Specific immunotherapy improved clinical indices of disease activity including symptom scores and medication use during the treatment year, and had a marked effect in increasing the interferon-gamma/interleukin-4 ratio in peripheral blood CD4+ T cells already after the dose increase phase (5.47 +/- 1.5 vs 4.07 +/- 1.49%, P = 0.03) with and a further rise after 1 year's treatment (16.12 +/- 2.8 vs 4.07 +/- 1.49 and 16.12 +/- 2.8 vs 5.47 +/- 1.5%, P = 0.001 and P = 0.002, respectively). There were no significant changes in the interferon-gamma/interleukin-4 ratio in peripheral blood CD8+ T cells at the three times of the study. CONCLUSIONS: These data add to view that the efficacy of specific immunotherapy may be attributed to a modified cytokine secretion of CD4+ T cells.     

Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation.

Immunotherapy may potentially improve the outcome of autologous hematopoietic cell transplantation (HCT). Poor effector cell proliferation and marginal antitumor activity limit attempts to use immunotherapy. We have characterized the ex vivo expansion, up to 1000-fold, of CD3+ CD56+ lymphocytes from the peripheral blood lymphocytes (PBL) of healthy donors. Expanded cells termed cytokine-induced killer (CIK) cells induce non-major histocompatibility complex-restricted lysis of tumor cells and demonstrate cytolytic activity superior to lymphokine-activated killer cells without the requirement of interleukin (IL)-2 treatment in vivo. To determine whether cytolytic cells could be expanded from patient material, we evaluated samples of peripheral blood progenitor cells (PBPCs) from 25 patients undergoing autologous HCT. The PBPCs were expanded by priming with interferon-gamma followed by anti-CD3 monoclonal antibody and IL-2 the next day. Fluorescence-activated cell sorting analysis was performed on days 0, 15, 21, and 28 of cell culture. The median T-cell content rose from 15.3% (range, 1.1% to 89.7%) on day 0 to 97.2% (range, 83.6% to 99.5%) by day 15. By day 21, T cells expanded 21.8-fold (range, 1.7- to 420.0-fold) and CD3+ CD56+ cells expanded 44.8-fold (range, 5.1- to 747.0-fold). CIK cells were used as effector cells against B-cell lymphoma targets (OCI-Ly8) with a median of 24% (range, 3% to 67%) and 42% (range, 6% to 96%) specific lysis of target cells on days 21 and 28, respectively. CIK cells derived from PBL of 2 additional patients with acute myelogenous leukemia demonstrated 39% and 78% specific lysis of OCI-Ly8 and 26% and 58% specific lysis of autologous leukemic blasts at an effector:target ratio of 40:1. CIK cells may be expanded from granulocyte colony-stimulating factor-mobilized PBPCs of patients undergoing autologous HCT. CIK cells may provide a potent tool for use in posttransplantation adoptive immunotherapy.     

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.     

Identification of lymphocyte subsets in peripheral blood and thyroid gland in Graves' disease by alpha-naphthyl-acetate-esterase reaction

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.     

The comparison of cytotoxic T-lymphocyte effects of dendritic cells stimulated by the folate binding protein peptide cultured with IL-15 and IL-2 in solid tumor

The current modalities for treating cancer employ not only single but multiple approaches involving surgery, radiotherapy and chemotherapy. Unfortunately, the survival outcome is not promising even with these approaches. Alternative approaches for cancer therapy are now emerging. Immunotherapy is aiming at both increasing the power, and in redirecting the specificity of the patients' immune system to attack the tumor cells. Recently, many studies using tumor associated lymphocytes (TAL) isolated from malignant ascites cultured in a media containing interleukin-2 exhibit antitumor responses. IL-2 is a lymphokine produced by T-cells. It facilitates activation, sustained growth and rescue from apoptosis. Lately, newly developed IL-15 has also exhibited antitumor activity similar to IL-2. IL-15 is a newly described cytokine produced from monocytes-marcrophages and T-cells. It has a different molecular structure but it functions like IL-2 by binding to the IL-2R beta and gammac chain. These antitumor responses are mediated by the cytotoxic T lymphocytes (CTL) that recognize the antigen in the context of the MHC molecules using the T cell receptors. CD8+-CTL recognize the peptide epitopes that are processed from the cellular proteins in the context of the MHC class I molecules. These peptides have a restricted length of 8-11 amino acids. The folate binding protein (FBP) is overexpressed in over 90% of ovarian and 20-50% in breast cancers. The FBP is the source of the antigenic peptides that are recognized by a number of these CTL-TAL, and is antigenic to both ovarian and breast cancer in vivo. To define the antitumor response of IL-15 and its' FBP immunogenicity, a peptide defining epitope E39 and E75 were presented by the PMBC derived dendritic cells (DC) from healthy donors isolated by the CD14 method to ovarian and breast CTL-TAL. Stimulating both ovarian and breast CTL- TAL by E39 or E75 pulsed DC (DC-E39, DC-E75), in the presence of IL-15 and IL-2 can rapidly enhance or induce the E39 or E75 specific CTL activity. The antitumor activities were measured by a chromium release assay for the tumor specific lysis activity using the ovarian and breast cancer cell lines. The tumor specific lysis activity for the ovarian TALs for IL-15 vs IL-2 were 28.6 +/- 3.9% and 30.3 +/- 3.2%, respectively and in the breast TALs, they were 14.8 +/- 3.1% vs 13.5 +/- 2.9%, respectively. Using autologous tumor cells, a slightly higher tumor specific lysis activity was obtained for the ovarian TALs cultured in IL-15 compared to IL-2 (72.0 +/- 8.2% vs 68.5 +/- 3.6%). However, for the breast TALs, they were 39.5 +/- 4.2% vs 41.5 +/- 3.3%, respectively. IL-15 is a newly developed cytokine that shows promising antitumor activity similar to IL-2. However, it requires lower dosage and is less toxic. Therefore, IL-15 might be a potential anticancer immunotherapeutic agent.     

Prednisolone suppresses NK cell cytotoxicity in vitro in women with a history of infertility and elevated NK cell cytotoxicity

PROBLEM: To evaluate the effect of prednisolone on NK cell cytotoxicity in vitro environment and also to compare the effect of prednisolone versus immunoglobulin-G (IVIG) on NK cell cytotoxicity using in vitro co-culture with K562 cells. METHOD OF STUDY: The following is a prospective observational study, between August 2006 and February 2007, was carried out on blood samples from 110 patients with a history of recurrent miscarriage or recurrent failed implantation. Peripheral blood mononuclear cells containing NK cells were isolated and co-cultured with target cell K562 in three different effector-to-target (E:T) ratios of 50:1, 25:1 and 12.5:1. Prednisolone or IVIG was then added to the tube with E:T ratio of 50:1 to assess suppressive effect. The percentage killing was recorded and statistical analysis performed using Student's t-test. RESULTS: In the experiments with an E:T ratio of 50:1 without prednisolone or IVIG in the co-culture, the mean target cell killing percentage was 26.4%. In cultures using the same E:T ratio, this killing percentage was significantly reduced in the presence of IVIG (9.9%) or prednisolone (13.6%), (P<0.001 in both analyses). On comparing the reduction in killing percentage of target cells by prednisolone versus IVIG, a slightly lower reduction in the prednisolone co-culture was noted but this was not statistically significant (P>0.05). CONCLUSION: The results of this study show that prednisolone is able to suppress the cytolytic activity of the NK cell. Prednisolone and IVIG are almost equally effective in suppressing in vitro NK cell cytolytic activity.     

Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry.

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.     

Influence of plaque biofilm removal on reestablishment of the biocompatibility of contaminated titanium surfaces

The aim of the present study was to evaluate the influence of plaque biofilm removal on the mitochondrial activity of human SaOs-2 osteoblasts grown on titanium surfaces. Volunteers wore acrylic splints with structured titanium discs for 72 h to build up plaque biofilms (n = 30). Specimens were randomly instrumented using either (1) an ultrasonic system at two power settings (EMS1, EMS2) + chlorhexidine (CHX), or (2) plastic curettes + CHX. Untreated (NC, n = 10) and sterile (C, n = 10) titanium discs served as controls. Specimens were incubated with SaOs-2 cells for 6 days. Treatment time (T), residual plaque biofilm (RPB)/clean implant surface areas (%), mitochondrial cell activity (MA) (counts/second), and cell morphology (SEM) were assessed. Statistical analysis revealed the following mean scores (+/-SD): RPB areas: P (58.5 +/- 4.9) > EMS1 (38.4 +/- 4.1) > EMS2 (28.3 +/- 2.0); T: PC (292 +/- 30) = EMS1 (244 +/- 24) > EMS2 (199 +/- 25); MA: C (1.544.661 +/- 203.442) > PC (597.559 +/- 566.984) = EMS2 (389.875 +/- 409.300) = EMS1 (356.653 +/- 293.863; n.s.) > NC (138.676 +/- 86.666). In NC and PC groups, cells were predominantly rounded in shape. However, in the EMS groups, some cells had started to spread, showing complete cytoplasmatic extensions of the cell body on the titanium surface. A monolayer of flattened cells was generally observed in the C group. Within the limits of the present study, it was concluded that MA seemed to be impaired by the presence of RPB areas. However, its removal alone might not be the crucial step in the reestablishment of the biocompatibility of titanium surfaces.