Cytotoxicity Of Mononuclear Cells Research Articles

A Novel WNT5A-Mimicking Peptide Affects Leukemia Cell Survival in the Bone Marrow Microenvironment

Background: The crosstalk between hematopoietic cells and bone marrow (BM) microenvironment in hematological malignancies is related to disease initiation, maintenance and relapse. BM niche sustains a protective response against currently available treatments that have shown unwanted adverse effects and high levels of toxicity for patients. WNT5a is a glycoprotein secreted by mesenchymal stromal cells (MSC) that activates the WNT non-canonical pathway in hematopoietic cells, modulating important biological processes related to neoplasia development.Aims: To investigate WNT5a mRNA expression, protein levels and methylation pattern in Myelodysplastic Syndrome (MDS) and de novo Acute Myeloid Leukemia (AML) patients and their impact on clinical outcomes, and to analyze effects of Foxy-5 (WNTResearch), a new WNT5a-mimicking compound.Methods: WNT5a mRNA expression was analyzed in mononuclear cells (MC) from 371 AML patients (212 male, median age 61 years [range: 2-87]) (Ohsu, Nature 2018). BMMSC and BM biopsies from 5 healthy donors (HD), 6 MDS and 13 AML patients at diagnosis were submitted to analysis of WNT5a mRNA expression and methylation after Azacytidine (1μM) treatment by real-time PCR, and WNT5a protein levels by immunohistochemistry and immunofluorescence (IF). A panel of myeloid leukemia cell lines (U937, HL60, THP1, KG1a, K562) were treated with Foxy-5 (1 to 100μM) for 72h in monoculture, coculture and 3D-coculture (with MSC) and evaluated for ROS production (DCFDA dye), cell proliferation (Ki-67 stain), autophagy (acridine orange dye), chemotaxis (Transwell), actin polymerization (phalloidin stain), cell cycle (PI/RNAse stain), cell viability (MTT assay), apoptosis (Annexin-V stain) and protein expression (Western blot, WB). MC and CD34+ cells from HD were submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA or Mann-Whitney tests, as appropriate.Results: WNT5a gene expression was reduced in MC from AML patients with adverse cytogenetic risk compared to favorable and intermediate cytogenetic risk (fold-decrease [FD]: 42.9; 18.8, respectively) (P<.05) and in BMMSC from AML compared to HD (FD: 53.3) (P<.05). Accordingly, WNT5a gene expression in MDS and AML BMMSC treated with Azacytidine was restored (fold-increase [FI]: 3.99; 1.50, respectively). WNT5a protein expression were diminished in BMMSC from MDS and AML patients compared to HD onto a 3D-coculture (IF)(P<.05) and immunohistochemically detected in all BM hematopoietic lineages. Foxy-5 reduced ROS production in U937 (FD of mean fluorescence intensity [MFI]: 48.2; 46.6), HL60 (FD: 47.1; 115.0), KG1a (FD: 34.9; 20.7) and K562 (FD: 19.0; 24.3) at 100μM in monoculture and coculture, respectively (P<.05). Foxy-5 also significantly decreased proliferation in U937 (FD: 41.0), HL60 (FD: 18.0), THP1 (FD: 36.0) and K562 (FD: 68.0) at 100μM (P<.05), confirmed by a 3D-coculture containing these cell lines and MSC. Foxy-5 reduced monocyte differentiation and inhibited CD11b expression in U937 (FD: 16.4) and THP1 (FD: 14.4). Cell cycle progression was blocked in sub G0/G1 phase in all cell lines (P<.05) after Foxy-5 treatment, probably mediated by the reduction of cyclin D1 protein levels, as verified by WB. Further, Foxy-5 reduced AKT1/2/3 and ERK1/2 phosphorylation levels, possibly by beta-catenin inhibition, with disruption of actin polymerization (U937 (FD: 65.3), HL60 (FD: 35.9), THP1 (FD: 58.5), K562 (FD: 15.0)) at 100μM (P<.05) and consequent impairment of CXCL12-induced chemotaxis (U937 (FD: 27.9), HL60 (FD: 42.5), THP1 (FD: 82.4), K562 (FD: 45.1)) at 100μM (P<.05). In coculture, cell autophagy was reduced in U937 (FD: 27.8), HL60 (FD: 35.9), KG1a (FD: 16.4) and K562 (FD: 35.8) when treated with Foxy-5 at 100μM (P<.05). Finally, Foxy-5 treatment did not affect cytotoxicity in MC and CD34+ cells from HD.Conclusion: WNT5a downregulation in MDS and AML patients occurs probably by methylation and contributes to poor prognosis. Foxy-5, by restoring WNT5a levels, could represent a strategy to counterbalance several oncogenic processes present in leukemia by reducing ROS production and, consequently, inhibiting cell growth and differentiation, downregulating PI3K and MAPK pathways, disrupting actin polymerization and decreasing autophagy. Thus, Foxy-5 treatment may be an important approach to impair leukemia growth and maintenance and arises as a promising therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Mucosal-Associated Invariant T (MAIT) Cell Dysfunction and PD-1 Expression in Prostate Cancer: Implications for Immunotherapy.

Prostate cancer is the second most common cancer in men worldwide. Despite an abundance of prostate-specific antigens, immunotherapies have yet to become a standard of care, potentially limited by T-cell dysfunction. Up to 10% of human circulating T-cells, and a significant fraction in the urogenital tract, are mucosal-associated invariant T (MAIT) cells. MAIT cells express stereotyped T-cell receptors that recognize riboflavin metabolites derived from microbes presented by MR-1. We evaluated the number, phenotype and function of circulating MAIT cells, alongside two other innate-like T (ILT) -cell subsets, in men with prostate cancer and age- and sex-matched controls. MAIT cells in men with prostate cancer circulated at similar frequencies to controls, but their cytokine production and proliferation was impaired. In contrast, the function of two other ILT-cell populations (natural killer T-cells and Vγ9Vδ2 T-cells) was not impaired. In both patients and controls, MAIT cells expressed high levels of the immune checkpoint molecule PD-1 at rest, while upregulation of PD-1 in response to the MR-1 ligand 5-amino-6D-ribitylaminouracil (5-A-RU) was greater in patients. 5-A-RU also induced upregulation of PD-L1 and -L2 RNA in primary mononuclear cells. We confirmed that circulating MAIT cell number and function were preserved before and during anti-PD1 therapy with pembrolizumab in a cohort of patients with melanoma. In vitro, 5-A-RU enhanced mononuclear cell cytotoxicity against the PD-L1 positive prostate cancer cell line PC3 in an MR-1-dependent manner. Addition of pembrolizumab enhanced this cytotoxicity, and was associated with increased MAIT cell expression of CD107a and IFN-γ. We conclude that prostate cancer is associated with MAIT-cell dysfunction, and that this might be overcome through the application of potent MR-1 ligands with PD-1 blockade. These findings may have implications for the development of cancer immunotherapies that exploit MAIT cells.

Effect of suberoylanilide hydroxamic acid on peripheral blood mononuclear cell cytotoxicity towards tumor cells in canines.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACi) that suppresses the growth of tumor cells in humans and canines. SAHA reportedly enhances the antitumor activity of human peripheral blood mononuclear cell (PBMC). However, it is unclear whether a similar effect is exerted in canines. The present study focused on the effect of SAHA on the cytotoxicity of IL-2 activated PBMC in three tumor cell lines (CTAC, CIPm, and MCM-N1). The mRNA expression of a ligand for the NKG2D receptor was upregulated in SAHA-treated cell lines. Moreover, the SAHA-treated cell lines, except MCM-N1 demonstrated a significantly higher PBMC cytotoxicity compared to the untreated cell lines. Therefore, the NKG2DL upregulation likely enhanced the interaction of NKG2D-NKG2DL, leading to enhanced cytotoxicity of PBMC. It was also revealed that activated PBMC treated with SAHA significantly attenuated their cytotoxicity toward all the cell lines. Although the NKG2D, NKp46, NKp44, and NKp30 receptors, involved in PBMC cytotoxicity, were presumed to be downregulated, there was no significant reduction in the mRNA expression of these receptors. This study revealed that SAHA not only sensitizes the canine tumor cells to cytotoxicity due to PBMC activation, but also suppresses the cytotoxicity of PBMC themselves. Therefore, our results highlight the necessity of avoiding this inhibitory action to enhance the antitumor effect of SAHA in canines.

Transportation and Routine Veterinary Interventions Alter Immune Function in the Dog

Rapid activation of the hypothalamic pituitary adrenal axis and the sympathetic nervous system are hallmarks of the acute stress response and these systems interact with the immune system by signaling though glucocorticoid and adrenergic receptors on immune cells. There is limited information about the effect of these physiologic responses on immunologic parameters of pet dogs enrolled in clinical studies. The objective of this study was to evaluate how travel, instrumentation, and hospitalization alter immunologic parameters in pet dogs. Blood was collected from healthy dogs in a home environment and from healthy dogs at the time of presentation to the hospital and after instrumentation and 24 hours of hospitalization. We found that lipopolysaccharide (LPS)-induced downregulation of toll like receptor 4 (TLR4) was blunted in dogs exposed to stress. Neutrophil and monocyte major histocompatibility complex class II (MHCII) expression increased after transportation to the veterinary hospital but then became similar to that of the control dogs at the end of hospitalization. Peripheral blood mononuclear cell cytotoxicity function was blunted in dogs exposed to the stress of transportation as well as hospitalization. Neutrophil apoptosis was greater in dogs exposed to stress compared to controls although this effect significantly decreased after hospitalization stress. Conversely, stress did not alter induced or spontaneous cytokine production from leukocytes, neutrophil or monocyte expression of TLR4, LPS-induced downregulation of monocyte TLR4, LPS-induced neutrophil and monocyte expression of MHCII or peripheral blood lymphocyte phenotype. Transportation and instrumentation/hospitalization stress should be considered when interpreting immunologic studies in pet dogs.

Loss of Heterozygosis on IFN-α Locus is a Prognostic Indicator of Bacillus Calmette-Guerin Response for Nonmuscle Invasive Bladder Cancer

We evaluated the role of loss of heterozygosity on the interferon-alpha locus to predict the response to bacillus Calmette-Guerin therapy in patients with nonmuscle invasive bladder cancer. A total of 117 consecutive patients were selected, including 77 with nonmuscle invasive bladder cancer and 40 controls. Loss of heterozygosity on the interferon-alpha locus (chromosome 9p21) was assessed in blood and urine samples before transurethral resection. All patients underwent transurethral resection and then 6 weekly bacillus Calmette-Guerin instillations. Those with nonmuscle invasive bladder cancer were assigned to groups 1 and 2 with and without loss of heterozygosity on the interferon-alpha locus, respectively. Of the 77 patients with nonmuscle invasive bladder cancer 39 (50.6%) had loss of heterozygosity on the interferon-alpha locus (group 1) and 38 (49.4%) had no alteration (group 2). Only 1 of 40 controls showed loss of heterozygosity on the interferon-alpha locus. At the end of followup 13 patients in group 1 and 27 in group 2 were alive without recurrence. We noted a significant difference between loss of heterozygosity on interferon-alpha and followup status (dF 01, LR 11.252, p = 0.003). Kaplan-Meier analysis revealed a significant difference in recurrence probability (response to bacillus Calmette-Guerin) and loss of heterozygosity on interferon-alpha (p <0.0001). On multivariate analysis loss of heterozygosity (HR 4.09, 95% CI 2.59-6.28, p = 0.002), grade (grade 3 HR 3.31, 95% CI 1.38-3.35, p = 0.03) and the number of lesions (3 or greater HR 2.31, 95% CI 1.38-3.25, p = 0.03) were independent predictors of the bacillus Calmette-Guerin response. This study highlights the predictive value of loss of heterozygosity analysis on interferon-alpha in patients with nonmuscle invasive bladder cancer treated with bacillus Calmette-Guerin.

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro.

The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-alpha 2B secreting recombinant (r) BCG (rBCG-IFN-alpha). We demonstrated that rBCG-IFN-alpha augmented T helper type 1 (Th1) cytokine IFN-gamma production by PBMC. In this study, we further investigated whether rBCG-IFN-alpha could also enhance PBMC cytotoxicity toward bladder cancer cells. PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-alpha or control MV261 BCG, and used as effector cells in (51)Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-alpha as well as endogenously expressed IFN-gamma and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-alpha in the presence of neutralizing antibodies to IFN-alpha, IFN-gamma or IL-2. To determine the role of natural killer (NK) and CD8(+) T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in (51)Cr-release assays. Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-alpha further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-gamma and IL-2 by PBMC was observed after rBCG-IFN-alpha stimulation. Blockage of IFN-alpha, IFN-gamma or IL-2 by neutralizing antibodies during rBCG-IFN-alpha stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8(+) T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-alpha with the former cell type being more predominant. rBCG-IFN-alpha is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.

Cytotoxicity of Mononuclear Cells as Induced by Peritoneal Dialysis Fluids: Insight into Mechanisms that Regulate Osmotic Stress-Related Apoptosis

High glucose content of peritoneal dialysis fluids (PDFs) has been shown to contribute to loss of peritoneal function during long-term peritoneal dialysis. However, hyperosmolality and hypertonicity of PDF are usually seen as similar stress events inducing osmotic stress-induced programmed cell death. In this study, we examined the impact of various osmotic agents on apoptosis induced by hyperosmolar PDFs, focusing on the mechanisms underlying the lethal effects of PDFs on peripheral blood mononuclear cells (PBMCs). We assessed apoptosis and necrosis by annexin V-propidium iodide (PI) labeling, and caspase-3 activity by fluorescence assay. F-actin remodeling was measured using fluorescent phalloidin labeling. Hyperosmolality does not cause the cytotoxicity observed with PDF, but exposure to agents incapable of permeating cell membranes results in a significant increase in the percentage of apoptotic PBMCs by annexin V-PI labeling, which is confirmed by the increase in caspase-3 activity. Interestingly, inhibition of caspase-3 by Z-VAD-FMK did not suppress apoptosis. Extracellular hypertonicity produced polymerization of filamentous actin and cell shrinkage, which displayed similar time courses. Cell shrinkage was blocked by cytochalasin D, indicating an active role for actin cytoskeleton in hypertonicity-induced cell shrinkage. F-actin polymerization was related to an increase in intracellular ionic strength. Finally, we excluded a direct role for actin remodeling in osmotic stress-induced programmed cell death. Exposure to osmolytes that cannot penetrate cell membranes results in a hypertonicity-induced apoptosis that cannot be blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. In addition, extracellular hypertonicity induced by impermeant solutes produces F-actin polymerization through an increase in intracellular ionic strength. The remodeling of the cytoskeleton does not modulate apoptosis but participates in cell shrinkage.

Hepatoma cells up-regulate expression of programmed cell death-1 on T cells

To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 (PD-1), and the function of PD-1 on T cells. HepG2 or HepG2.2.1.5 cells were co-cultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL-2, INF-gamma and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Cytotoxic action of T cells was determined by MTT reduction assay-direct mononuclear cell cytotoxicity assay. The PD-1 expression on Jurkat cells increased by 16.17% +/- 2.5% and 17.43% +/- 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-gamma and IL-10 in the culture supernatant were 202.9 +/- 53.0 pg/mL, 88.6 +/- 4.6 pg/mL and 63.7 +/- 13.4 pg/mL respectively, which were significantly higher than those (102.9 +/- 53 pg/mL, 39.3 +/- 4.2 pg/mL, and 34.6 +/-13.7 pg/mL) in the control group (P < 0.05). The OD value for MTT assay in the blocking group (0.29 +/- 0.06) was significantly higher than that (0.19 +/- 0.09) in the control group (P < 0.05). PD-1 expression on Jurkat cells is up-regulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.

A Synergistic Antitumor Effect of Interleukin-2 Addition with CD80 Immunogene Therapy for Peritoneal Metastasis of Gastric Carcinoma

The co-stimulatory molecule CD80 is a ligand of CD28, which plays a key role in the induction of cell-mediated immune responses. Many tumors, including gastric cancer, decrease the expression of CD80, which results in the failure of immune recognition. We evaluated the effect of interleukin-2 addition combined with CD80 infection on the peritoneal metastasis in gastric cancer. CD80 infection combined with interleukin-2 addition significantly increased the activated cytotoxicity of mononuclear cells compared to CD80 gene infection and compared to the lacZ control group. In vivo, the survival of animals with intraperitoneal tumor was longest in those given CD80 infection with interleukin-2 addition (median survival, 46 days), followed by those given interleukin-2 (39 days), those given CD80 infection (37 days), and those given lacZ (29 days). These results suggest that interleukin-2 addition might contribute to improving the observed outcome of CD80 immunogene therapy in peritoneal metastasis of gastric carcinoma.

CD32B on Clonal Plasma Cells in Systemic Light-Chain Amyloidosis (AL) and Multiple Myeloma (MM): A Target for Immunotherapy in Both Disorders and a Prognostic Marker in MM.

Despite stem cell transplant and new therapies, nearly all patients with AL and MM die of disease or complications of treatment. Novel approaches that selectively kill clonal plasma cells are needed. CD32B, the inhibitory Fcγ receptor IIB, is a member of the Fc receptor (FcR) family on chromosome 1q. B-cells and some monocytes and dendritic cell subtypes express cell-surface CD32B, which has a cytoplasmic inhibitory motif important in regulating immune responses. Unlike the CD32B2 isoform on myeloid cells, CD32B1 on B-cells is not internalized, making it a suitable target for monoclonal antibody (MAb) therapy (Blood 2006Jun6 Epub). CD32B has not previously been found on AL and MM plasma cells. We used purified CD138+ marrow and blood cells from AL, MM and plasma cell leukemia (PCL) patients, and 5 human MM cell lines, to evaluate CD32B gene and cell-surface expression with gene expression profiles (GEP) (Affymetrix U133 PLUS 2.0), RT-PCR for CD32B1 and B2, and flow-cytometry with the 2B6 MAb for CD32B. In AL, GEP showed that CD32B expression was significantly higher than other FcR genes (p<0.01), RT-PCR showed that CD32B1 was prominently expressed on CD138+ cells from both newly diagnosed and relapsed AL patients, and flow cytometry showed intense cell-surface staining for CD32B on >98% of all AL plasma cells. In MM, public GEP data (http://lambertlab.uams) showed that CD32B expression was significantly higher than other FcR genes while RT-PCR showed CD32B1 message in CD138+ MM and PCL specimens but not in the RPMI 8226 cell line. Cytogenetic analysis then showed that 8226 cells lack t(4;14) but have 4 copies of 1q, implicating segmental uniparental tetrasomy of a mutant allele in the lack of CD32B message. Flow cytometry showed median 96% CD32B expression on CD138+ marrow cells from patients with normal or hyperdiploid cytogenetics, but significantly lower expression (median 69%, p=0.01) in patients with unfavorable cytogenetics (del 13, t(4;14)). CD32B expression was lower still on PCL specimens (median 10%) and nil on MM cell lines. We then investigated the GEP of sorted CD138+/CD32B+ and CD32B− fractions from MM patients. In CD138+/CD32B+ cells, CD45C and CXCR4 were overexpressed. In CD138+/CD32B− cells, genes on chromosome 1q were overexpressed, including those for cancer testis antigens and others possibly associated with biologic aggressivity (Cancer Cell 2006; 9:313). Notably, the CD32B-specific MAb 2B6 directs human mononuclear cell cytotoxicity against CD32B+ cell lines, reduces tumor growth and improves tumor-free survival in a mouse xenograft model. Moreover, an Fc-engineered humanized variant of 2B6 elicits ADCC-mediated specific cytotoxicity against a low-level CD32B+ MM cell line in vitro. In sum, these data show that CD32B is important in AL and MM; that CD32B on AL and the majority of MM plasma cells provides a target for MAb therapy; and that, given the results in MM, PCL and MM cell lines, CD32B expression is inversely related to biologic aggressivity. Genetic instability of chromosome 1q may provide a direct basis for this relationship thereby mechanistically linking CD32B expression in MM to prognosis. These data support further evaluation of CD32B and clinical studies of anti-CD32B MAb therapy in AL and MM.

Lipid solubility- and concentration-dependent attenuation of in vitro natural killer cell cytotoxicity by local anesthetics.

Natural killer (NK) cells constitute an essential component of the innate immune system in the defence against infected and malignant cells. In this study the in vitro effect on NK cell activity of three different local anesthetics with different lipid solubility was investigated. Venous blood from seven healthy volunteers was incubated with three amide local anesthetics with three different concentrations of lipid solubility: lidocaine 0.50, 1.00 and 2.00 mg/ml, ropivacaine 0.375, 0.75 and 1.50 mg/ml, and bupivacaine 0.25, 0.50 and 1.00 mg/ml. After 1 h of incubation, mononuclear cells were isolated and cryopreserved until tested for NK cell cytotoxicity in a 4-h 51Cr-release assay against K-562 target cells. Natural killer cell cytotoxicity of mononuclear cells incubated with isotonic saline was used as the control. A significant suppression in NK cell cytotoxicity was demonstrated for all three local anesthetic agents when the NK cell cytotoxicity was compared with the cytotoxicity estimated after incubation with the isotonic saline (P<0.004). Moreover a significant lipid solubility-dependent effect (P=0.0001) as well as an overall concentration-dependent effect (P<0.0001) on the NK cell cytotoxicity was found. The results of the present in vitro study suggest a negative association between the estimated NK cell cytotoxicity and the lipid solubility as well as the concentrations of the three local anesthetic agents tested.

Humanized anti-ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patients

Ganglioside GM2 is one of the major gangliosides expressed on the cell surface of human tumors including lung cancer. We have previously reported that a mouse–human chimeric monoclonal antibody (mAb), KM966, against GM2 promotes the lysis of lung cancer cells by human blood mononuclear cells (MNC) of healthy donors. In this study, we examined antibody-dependent cell-mediated cytotoxicity (ADCC) of MNC, using KM966 mAb and its humanized counterpart, KM8969, in 16 lung cancer patients and 18 control patients. The ADCC activity was assessed by 4-h 51Cr release from GM2 positive SBC-3 small cell lung cancer cells. MNC from lung cancer patients exhibited similar ADCC activity to those from control patients when KM966 and KM8969 were used as mAb. Moreover, effective ADCC activity was observed even in MNC from advanced lung cancer patients. These observations suggest the potential activity of humanized anti-GM2 mAb (KM8969), as well as chimeric KM966, in biological therapy for lung cancer patients.

Measurement of the activation of IL-2 on bone marrow by flow cytometry.

The changes of cell surface markers before and after activation by IL-2 were detected by flow cytometry (FCM) to establish a more convenient and precise criterion for the judgment of the activation of bone marrow. By using the measurement of the release of lactate dehydrogenase (LDH) the cytotoxicity of mononuclear cells (MNCs) from bone marrow, activated or inactivated, on tumor cell line K562 was evaluated, and at the same time the changes of surface markers on MNCs before and after activation were examined by using FCM. The results showed that the cytotoxicity of MNCs from bone marrow activated by IL-2 on tumor cell line K562 was increased obviously and the number of CD25+ and CD70+ positive cells in bone marrow MNCs was higher than before activation. The enhanced cytotoxicity of MNCs on tumor cell line K562 was synchronous with the increase of the number of CD25+ and CD70+ positive cells in 48 to 72 h. It is more direct, simple and precise to demonstrate the activation of IL-2 on bone marrow by detecting the changes of the amount of the CD25+ and CD70+ positive cells in bone marrow by FCM.

Herpes simplex virus type 1 glycoproteins gB, gC and gD are major targets for CD4 T-lymphocyte cytotoxicity in HLA-DR expressing human epidermal keratinocytes.

T lymphocytes are the main mediators of the protective immune response in recurrent herpes simplex. Early in the development of recurrent lesions, macrophages and CD4 T lymphocytes predominate in the mononuclear infiltrate surrounding infected epidermal cells. Human epidermal keratinocytes allow herpes simplex virus type 1 (HSV-1) replication and human leukocyte antigen (HLA)-DR is strongly expressed in vivo. In vitro, their pretreatment with IFN-gamma induced HLA-DR expression and partially reversed major histocompatibility complex class I down-regulation by the virus. Mononuclear cell cytotoxicity for these cells was mediated predominantly by CD4 and also by CD8 T cells. Late HSV-1 proteins were the major targets for CD4 CTL, while CD8 CTL predominantly targeted early HSV-1 proteins. Here it is shown that both mononuclear and CD4 CTL consistently recognized the major HSV-1 glycoproteins, gB, gC, gD and gH, using IFN-gamma-pretreated keratinocytes infected with vaccinia virus-HSV glycoprotein recombinants (VvgB, VvgC, VvgD or VvgH). CD4 cytotoxicity was highest for VvgD-infected keratinocytes, followed by VvgB or VvgC and then VvgH in seven patients. CD4 CTL from two of 13 patients also recognized an epitope in the HSV tegument protein VP16, demonstrated by comparing cytotoxicity for the partial deletion mutants RP3 or RP4 and the parental RP1 HSV strain. In summary, the major HSV glycoproteins gB, gC and gD were consistently the major targets for CD4 CTL in VvgB-, VvgC-, VvgD- and VvgH-infected, IFN-gamma-pretreated human epidermal keratinocytes in vitro.

Thymic peptides improve impaired cytotoxicity of mononuclear cells from tumour patients in vitro

Thymic peptides improve impaired cytotoxicity of mononuclear cells from tumour patients <i>in vitro</i>

N-Acetylcysteine Enhances Antibody-Dependent Cellular Cytotoxicity in Neutrophils and Mononuclear Cells from Healthy Adults and Human Immunodeficiency Virus-Infected Patients

Patients with AIDS have decreased levels of the intracellular antioxidant, glutathione, in their circulating lymphocytes and plasma. N-acetylcysteine (NAC) increases intracellular stores of glutathione and has direct antioxidant properties. In this study, the effects of glutathione and NAC on the cytotoxicity of neutrophils and mononuclear cells were tested using cells from healthy controls and human immunodeficiency virus (HIV)-infected patients. NAC (1 and 5 mM) enhanced the antibody-dependent cellular cytotoxicity (ADCC) of neutrophils from healthy adult controls and HIV-infected adults and children. The antineoplastic drug, 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU), which depletes intracellular glutathione, inhibited the ADCC of neutrophils; the addition of NAC partially reversed this inhibition. Similar effects of BCNU and NAC were seen when the cytotoxicity of mononuclear cells was tested using CEM tumor cells bearing the HIV gp120 antigen as targets. Thus, NAC enhances various forms of cytotoxicity and may be beneficial to AIDS patients whose defects in leukocyte cytotoxicity may be due to glutathione depletion.

Circulating serum interferon andits effect on the activity of human natural killer cells

The interferon content in the plasma of 6 healthy donors and 10 patients with multiple sclerosis and the effect of an 1-h treatment of mononuclear cells with autologous plasma on their natural killer activity are studiedin vitro using3H-uridine-labeled (3 μCi/ml) human erythromyeloleukosis cells K-562. The serum interferon content in healthy donors is 2.3±0.82 IU/ml, whereas that in patients is higher: 5.2±0.8 IU/ml. Autologous plasma does not affect the activity of natural killer cellsin vitro, whereas it increases the cytotoxicity of mononuclear cells obtained from patients with multiple sclerosis by 35–64%.

Alcoholic liver damage–toxicity, autoimmunity and allergy

Alcoholic liver damage–toxicity, autoimmunity and allergy

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensitized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Social Drinking and the Immune Response: Impairment of Lymphokine-Activated Killer Activity

The effect of limited and intermittent alcohol ingestion on the immune response in humans has not been extensively studied. The authors, in this study, evaluate peripheral blood mononuclear cell cytotoxicity before and after alcohol ingestion in a setting designed to mimic social drinking. Eleven healthy volunteers consumed two 12 oz (355 mL) cans of beer in 30 minutes while eating pizza. Five control individuals ingested non-alcoholic beverages. Natural killer and lymphokine-activated killer activity were determined for peripheral blood mononuclear cells obtained before and 30 minutes after alcohol ingestion. Interleukin 2-induced lymphokine-activated killer activity was significantly reduced in blood samples obtained after alcohol ingestion when compared with pre-alcohol samples (p < 0.01). Natural killer activity (unstimulated) was not affected by alcohol ingestion. The authors demonstrate that ingestion of a small amount of alcohol impairs the cytotoxic capacity of peripheral blood mononuclear cells. Alcohol in the context of social drinking may have deleterious effects on the immune system's ability to clear virus-infected cells or cells that have undergone neoplastic transformation, especially for individuals with pre-existing immunosuppression.