Incubation Of Peripheral Blood Mononuclear Cells Research Articles

The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells.

The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances.

Vitamin D interferes with glucocorticoid responsivenessin human peripheral blood mononuclear target cells.

Glucocorticoids (GCs) are widely used in the treatment of inflammatory and autoimmune diseases; however, patients are often resistant to GC effects. Current studies indicate that vitamin D reduces the risk or modifies the course of autoimmune diseases posing vitamin D supplementation as a prevention or therapeutic option. Herein, we investigated whether vitamin D can modify the response to GCs at the molecular level. To this end, peripheral blood mononuclear cells (PBMCs) were isolated from healthy vitamin D-deficient women and incubated with either the active metabolite 1,25(OH)2D3 (VitD) for 11days or dexamethasone (Dex) for the last 2days in the presence or absence of VitD. Ex vivo GC sensitivity was assessed by the expression of the glucocorticoid receptor (GR) responsive gene GILZ with RT-PCR. Long-term incubation of PBMCs with VitD significantly decreased the Dex-induced augmentation of GILZ expression. Since the intracellular concentration of GR and the GR nuclear translocation are critical determinants of GC sensitivity, we next evaluated the effect of VitD on these factors. RT-PCR and western-blot analysis revealed that VitD reduced the expression of GR. This effect was abolished by the HDAC-specific inhibitor trichostatin A, implying that HDAC was implicated in this effect. Moreover, NCoR1 mRNA was significantly decreased upon treatment with VitD either alone or as pre-treatment to Dex, suggesting that a possible increase in expression of this co-repressor was not involved. In addition, immunofluorescence analysis showed that VitD hindered the Dex-induced GRα nuclear translocation, an effect verified by subcellular fractionation and western-blot experiments. To further explore the underpinning mechanism, we examined the potential of VitD to: (1) strengthen the FK506-binding protein 5 (FKBP5) negative feedback loop and (2) modify the phosphorylation status of GR. Remarkably, VitD decreased FKBP5 expression and decreased phosphorylation at Ser211, while enhancing phosphorylation of GR at Ser203. Overall, VitD decreases the ex vivo GC sensitivity and this effect is, at least in part, attributed both to decrease of GR expression owing to a mechanism that engages HDAC and inhibition of GR translocation to nucleus via differential modulation of the phosphorylation state of GR. Our study provides, for the first time, evidence that long-term action of VitD induces GC resistance in PBMCs from healthy volunteers and offers a possible mechanistic basis for VitD-triggered attenuation of GC effects.

Small cell lung cancer: Recruitment of macrophages by circulating tumor cells

ABSTRACTTumor-associated macrophages (TAMs) play an important role in tumor progression, suppression of antitumor immunity and dissemination. Blood monocytes infiltrate the tumor region and are primed by local microenvironmental conditions to promote tumor growth and invasion. Although many of the interacting cytokines and factors are known for the tumor-macrophage interactions, the putative contribution of circulating tumor cells (CTCs) is not known so far. These specialized cells are characterized by increased mobility, ability to degrade the extracellular matrix (ECM) and to enter the blood stream and generate secondary lesions which is a leading cause of death for the majority of tumor patients. The first establishment of two permanent CTC lines, namely BHGc7 and 10, from blood samples of advanced stage small cell lung cancer (SCLC) patients allowed us to investigate the CTC-immune cell interaction. Cocultures of peripheral blood mononuclear cells (PBMNCs) with CTCs or addition of CTC-conditioned medium (CTC-CM) in vitro resulted in monocyte-macrophage differentiation and appearance of CD14+, CD163weak and CD68+ macrophages expressing markers of TAMs. Furthermore, we screened the supernatants of CTC-primed macrophages for presence of approximately 100 cytokines and compared the expression with those induced by the local metastatic SCLC26A cell line. Macrophages recruited by SCLC26A-CM showed expression of osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), IL-8, chitinase3-like 1 (CHI3L1), platelet factor (Pf4), IL-1ra and matrix metalloproteinase-9 (MMP-9) among other minor cytokines/chemokines. In contrast, BHGc7-CM induced marked overexpression of complement factor D (CFD)/adipsin and vitamin D-BP (VDBP), as well as increased secretion of OPN, lipocalin-2 (LCN2), CHI3L1, uPAR, MIP-1 and GDF-15/MIC-1. BHGc10, derived independently from relapsed SCLC, revealed an almost identical pattern with added expression of ENA-78/CXCL5. CMs of the non-tumor HEK293 cell line revealed no induction of macrophages, whereas incubation of PBMNCs with recombinant CHI3L1 gave positive results. Thus, the specific contributions of CTCs in SCLC affect CFD/adipsin, possibly involved in immunity/cachexia, VDBP which gives rise to group-specific component protein-derived macrophage-activating factor (GcMAF), GDF-15/MIC-1 which enhances the malignant phenotype of tumor cells and ENA-78/CXCL5 which attracts angiogenic neutrophils. In conclusion, CTCs are competent to specifically manipulate TAMs to increase invasiveness, angiogenesis, immunosuppression and possibly lipid catabolism.

Expression and activity of NOD1 and NOD2/RIPK2 signalling in mononuclear cells from patients with rheumatoid arthritis

Objectives: The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA).Method: mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells.Results: A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1β compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner.Conclusions: Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.

The iron chelator deferasirox affects redox signalling in haematopoietic stem/progenitor cells.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1β, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1β decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L.

Decrease of immature B cell and interleukin-10 during early-post-transplant period in renal transplant recipients under tacrolimus based immunosuppression

B lymphocytes are known to play a role in kidney transplantation (KT) outcomes. Here, we evaluated the proportion of B cell subsets before and after KT. Twenty-one patients, who showed stable allograft function without acute rejection in the month following the KT, were included in this study. Peripheral blood samples were obtained from these patients before transplantation as well as 1month after transplantation. Changes in the proportion of B cell subsets after transplantation were investigated using multi-color flow cytometry. The proportion of lymphocytes in the peripheral blood mononuclear cells (PBMCs) and of CD19+ B cells in the total leukocyte population did not change after KT. Similarly, the proportions of CD19+CD24+ lymphocytes, mature B cells (CD24IntCD38Inter/CD19+), and memory B cells (CD24+CD38−/CD19+) did not change post-KT. However, the proportion of immature B cells (CD24+CD38+/CD19+ B cells) decreased significantly after transplantation (P<0.01). The levels of IL-10, and IL-21, and expression of the B cell marker BLNK also decreased significantly after transplantation. Incubation of PBMCs with tacrolimus (0.1, 1, and 10ng/mL) and mycophenolate mofetil (200μg/mL) an immunosuppressant, resulted in significant reduction in the percentage of immature B cells. In contrast, the proportion of memory and mature B cells was not affected. Taken together, these results show that while the total B lymphocyte count and the proportion of memory/mature B cell subsets do not change after KT, the proportion of immature B cells and the associated cytokines that they secrete decrease significantly.

Do androgen deprivation drugs affect the immune cross-talk between mononuclear and prostate cancer cells?

The aim of the study was to examine the effect of androgen deprivation drugs, i.e. leuprolide and bicalutamide on the immune cross-talk between human peripheral blood mononuclear cells (PBMC) and cells from PC-3 and LNCaP human prostate cancer lines. PBMC, PC-3 and LNCaP were separately incubated without and with two androgen-deprivation drugs, i.e. leuprolide and bicalutamide, and the secretion of IL-1β, IL-6, IL-1ra and IL-10 was examined. In addition, the effect of both drugs on the production of those cytokines was carried out after 24hours incubation of PBMC with both types of cancer cells. Leuprolide or bicalutamide did not affect the production of the cytokines by PBMC or by the prostate cancer cells from the two lines. Incubation of PBMC with PC-3 or LNCaP cells caused increased production of IL-1β, IL-6 and IL-10 as compared with PBMC incubated without malignant cells. While 10−7M and 10−8M of leuprolide caused a decreased secretion of IL-1β by PBMC previously incubated with prostate cancer cells without the drug, bicalutamide did not affect this PBMC activity at any drug concentration. This observation suggests the existence of an additional mechanism explaining the effect of androgen deprivation therapy in prostate cancer patients.

CD137 stimulation and p38 MAPK inhibition improve reactivity in an in vitro model of glioblastoma immunotherapy.

Dendritic cell vaccination has become an interesting option for cancer immunotherapy. Tumor-lysate-pulsed dendritic cells (DC) can prime naïve T cells and induce the regression of established tumors including gliomas as shown in various animal models. Despite hopeful results even in clinical studies, the outcome for many patients is still unsatisfying. In the present study, we tested the combination of tumor-lysate-pulsed dendritic cells (TPDC) with a monoclonal antibody against CD137, a monoclonal antibody against CD25 (daclizumab) and a specific p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for improving immunostimulation in an in vitro model of immunotherapy for human gliomas. We observed a higher secretion of interferon gamma by TPDC-primed peripheral blood mononuclear cells (PBMC) that were incubated with an antibody against CD137 or the p38 MAPK inhibitor. In addition, we observed higher specific lysis of tumor cells after incubation of PBMC with the p38 MAPK inhibitor or the anti-CD137 antibody. In contrast, incubation of TPDC-primed PBMC with the anti-CD25 antibody did enhance neither interferon gamma secretion nor cellular cytotoxicity. Cell depletion experiments demonstrated that the immune reaction induced by TPDC is strongly dependent on CD4-positive and CD8-positive cells. Incubation of DC during maturation and antigen loading with the anti-CD137 antibody did not enhance cytotoxicity and interferon gamma secretion in comparison with application of the anti-CD137 antibody during priming. In conclusion, our data suggest that p38 MAPK inhibition and anti-CD137 antibodies can enhance the immune response against glioblastoma cells.

Modulation of Cytokine Release and Gene Expression by the Immunosuppressive Domain of gp41 of HIV-1

The transmembrane envelope protein gp41 of the human immunodeficiency virus HIV-1 plays an important role during infection allowing fusion of the viral and cellular membrane. In addition, there is increasing evidence that gp41 may contribute to the immunodeficiency induced by HIV-1. Recombinant gp41 and a synthetic peptide corresponding to a highly conserved domain in gp41, the immunosuppressive (isu) domain, have been shown to inhibit mitogen-induced activation of human peripheral blood mononuclear cells (PBMCs) and to increase release of IL-6 and IL-10 from these cells. We recently reported that a single mutation in the isu domain of gp41 abrogated the immunosuppressive properties and that HIV-1 sequences containing such abrogating mutations had never been isolated from infected individuals. Here, we studied the influence of the isu peptide on the release of 66 cytokines and the expression of 27,000 genes in PBMCs. Incubation of PBMCs with isu peptide homopolymers increased the expression of 16 cytokines among them IL-6 and IL-10, and decreased that of IL-2 and CXCL9. Interestingly, the extend of cytokine modulation was donor-dependent. Among the genes up-regulated were IL-6, IL-8, IL-10 but also MMP-1, TREM-1 and IL-1beta. Most importantly, genes involved in innate immunity such as FCN1 and SEPP1 were found down-regulated. Many changes in cytokine expression demonstrated in our experiments were also found in HIV-1 infected individuals. These data indicate that the isu domain of gp41 has a broad impact on gene expression and cytokine release and therefore may be involved in HIV-1 induced immunopathogenesis.

ESAT-6-ELISpot and interferon γ in the diagnosis of pleural tuberculosis

BackgroundAppropriate diagnostic methods for tuberculous pleural effusion are vital. The IFN-γ tests using specific Mycobacterium Tuberculos is antigens in samples from the site of infection may be promising in diagnosis of tuberculosis. Objective we examined the ability of ELISpot test using circulating peripheral blood mononuclear cells (PBMC) and compartmentalized pleural fluid mononuclear cells (PFMC) for diagnosis of active TB infection in patients with tuberculous pleural effusion. Methods PBMC and PFMC-based ELISpot test for IFN-γ test using specific M. tuberculosis antigen: Early Secretory Antigen Target-6 protein (ESAT-6) was used for diagnosis of active TB infection. Thirty-five patients with clinically suspected tuberculous pleural effusion were enrolled over a 12-month period. Results 11 patients out of 35 were positive by culture and PCR (31.4%). Incubation of PBMC with ESAT-6 for 8h showed sensitivity and specificity of 82% and 92%, respectively, for the PBMC–ELISpot as compared to PFMC–ELISpot that was 54% and 96% respectively. With 24h incubation of ESAT-6 there was around 2.5 fold increase in the median number of spot forming cells (SFCs) in PFMC from 30 to 74, whereas there was minimal increase of median number of SFCs in PBMC from 55 to 60. Conclusion ESAT-6 – ELISpot using PBMC and PFMC is useful as a tool for diagnosis of TB effusion. PFMC needs longer period of incubation for processing of ESAT-6 than PBMC. Moreover, IFN-γ in pleural effusion (PE) is another useful way for diagnosis of TB pleurisy which is sensitive, simple and cheap.

1α,25-dihydroxyvitamin D3 modulates the interaction between immune and colon cancer cells

Following observations indicating the existence of a relationship between immune and cancer cells in the course of tumor development, it was the aim of the study to establish the role of 1α,25-dihydroxyvitamin D3 (vit. D) in the functional equilibrium between cells from two human colon carcinoma lines-induced cytokine production by peripheral blood mononuclear cells (PBMC). PBMC were incubated with HT-29 and RKO human colon cancer cells with and without vit. D at final concentrations of 10−8, 10−7 or 10−6M. The production of the cytokines TNF-α, IL-6 and IL-10, as well as the effect of the vitamin on tumor cell proliferation were evaluated. Incubation of PBMC with either HT-29 or RKO cells caused a significant stimulation of both pro- and anti-inflammatory cytokine generation. Addition of vit. D to the incubation mixture containing PBMC and cells of both colon carcinoma lines caused a marked inhibition of the generation of the pro-inflammatory cytokines TNF-α and IL-6 and to a lesser extent–of the IL-10. Vit. D did not affect the proliferation of the cancer cells. The results indicate the existence of a functional “dialog” between immune and cancer cells expressed by an alteration of their capacity for cytokine production and support the role of inflammation in carcinogenesis. The ability of vit. D to attenuate production of the pro-inflammatory cytokines when added to the incubation mixture containing PBMC and cancer cells endorses observations as for the beneficial role of the vitamin in suppressing inflammation with subsequent colon cancer prevention.

Human prostate cancer cells induce inflammatory cytokine secretion by peripheral blood mononuclear cells

A substantial number of studies provide evidence that inflammation may play a significant role in the pathogenesis of prostate cancer via increased activity of inflammatory cytokines, particularly IL-6. We have previously shown that peripheral blood mononuclear cells (PBMC) are capable of carrying out an in vitro “immunomodulatory dialog” with colon cancer cells expressed by an increased production of pro-inflammatory cytokines by PBMC. The aim of the current study was to examine the model of cell-to-cell interaction between PBMC and prostate cancer cells from two lines – androgen resistant (PC-3) and androgen-dependent (LNCaP). For that purpose, cancer cells from both lines were incubated with PBMC, and cytokine secretion by PBMC was evaluated. The results showed a cell-concentration dependent increase in secretion of the pro-inflammatory cytokine IL-6 by PBMC induced by cells from both lines, whereas generation of IL-1β and the anti-inflammatory cytokine IL-10 were found to be increased after incubation with PC-3 cells only. The secretion of IL-10 was slightly lower following incubation of PBMC with supernatants derived from PC-3 cells. The results of the study support the possibility that prostate cancer cell-induced cytokine production by PBMC, and particularly IL-6, are involved in prostate cancer development. The discrepancy between the effect of the two prostate cancer cell lines on cytokine secretion by PBMC may be due to their different androgen dependency.

Expression of Toll-like receptors and their detection of nuclear self-antigen leading to immune activation in JSLE

Toll-like receptors (TLRs) essential in the functioning of the immune system have been implicated in the development of autoimmunity. TLR3, 7, 8 and 9 are capable of recognizing nucleic autoantigens typical of SLE. Their expression correlates positively with disease activity in adult-onset SLE. This study aimed to determine the role of TLRs in JSLE and whether apoptotic neutrophils are a source of nuclear autoantigen being detected through TLR3, 7, 8 and 9, leading to an inflammatory response. TLR3, 7, 8 and 9 mRNA and protein expression were measured in peripheral blood mononuclear cells (PBMCs) in JSLE patients compared with JIA and non-inflammatory controls. Activation of the TLRs by JSLE serum-induced apoptotic neutrophils was detected by measuring IFN-α mRNA and protein expression, and confirmed using myeloid differentiation factor 88 (MyD88) and TIR domain-containing adapter-inducing IFN-β (TRIF) inhibitors. JSLE patients have increased TLR3, 8 and 9 mRNA and protein expression compared with controls (P < 0.05). Incubation of PBMCs with apoptotic neutrophils demonstrated a dose-response relationship for IFN-α mRNA expression. Inhibition of TLR signalling by blocking MyD88 and TRIF signalling decreased IFN-α mRNA expression in PBMCs incubated with apoptotic neutrophils (P < 0.05). This study demonstrated significantly increased TLR expression in JSLE compared with controls. Our data indicate that apoptotic neutrophils trigger TLR activation through their presentation of autoantigens. The role of TLRs in this inflammatory response was demonstrated by a dose-response relationship to apoptotic neutrophil concentration and confirmed by a decrease in IFN-α production after inhibition of TLR signalling.

Coagulation Activation Is Associated with Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Dependent Reactive Oxygen Species Generation in Hemodialysis Patients

This study investigated on (i) the role of gp91(phox)/NOX2 in reactive oxygen species (ROS) generation in hemodialysis (HD) patients, and (ii) the link between clotting activation and ROS production in this setting. The study was performed on peripheral blood mononuclear cells (PBMCs) isolated from HD patients randomized to polysulphon/polyamide (S-group, n=30) or ethylene-vinyl-alcohol (EVAL) membrane (E-group, n=30) treatment and from healthy subjects (control group, n=15). ROS generation was increased in PBMCs of HD patients compared with healthy subjects. S-group showed higher levels of intracellular ROS generation than control, whereas E-group did not. In addition, S-group displayed an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity compared with E-group and healthy subjects. A further increase in NADPH activity shortly after HD treatment was observed only in S-group. The plasma levels of the prothrombin fragment F1+2, a marker of in vivo clotting activation, were significantly higher in S-group than in E-group. Moreover, a heightened thrombin generation was recorded in the plasma of S-group. Intracellular ROS production correlated with NADPH oxidase activity and coagulation priming in HD patients. The in vitro validation study demonstrated that incubation of PBMCs with activated FX induced a significant increase in intracellular ROS production, superoxide generation, and gp91(phox)/NOX2 expression. The pivotal role of NADPH oxidase in the upregulation of ROS in HD patients makes this enzyme a potential target for therapeutic intervention in the treatment of HD-related oxidative stress. The EVAL membrane, by reducing clotting activation, inhibits gp91(phox)/NOX2-related ROS production in HD patients.

In vitro Immunoreactivity towards Lectin-rich or Viscotoxin-rich Mistletoe (Viscum album L.) Extracts Iscador Applied to Healthy Individuals

The aim of the study was to investigate how exposure to mistletoe extracts in vivo may influence cellular immune reactions by peripheral blood mononuclear cells (PBMC). 47 healthy individuals were exposed for twelve weeks either to Iscador Quercus special (IQ; rich in mistletoe lectin [ML]) (n = 16), to Iscador Pinus (IP; poor in ML but rich in viscotoxin) (n = 15), or to placebo (physiological saline) (n = 16). PBMC were isolated before exposure, during exposure in week 4, 8 and 12, as well as 13 weeks after last exposure (week 25) and incubated for 7 days with IQ or IP. Proliferative response and cytokine release were determined (type 1 cytokines: interferon [IFN]-gamma, tumor necrosis factor [TNF]-gamma, type 2 cytokines: interleukin [IL]-5, IL-13, macrophage/ monocyte-cytokines: IL-1, TNFalpha). IP- and IQ incubation of PBMC from non-exposed individuals lead to a significant increase of proliferation and production of all cytokines. There was a significant increase of IQ-induced TNFalpha (but not IL-1) production by PBMC in week 25 in individuals receiv-type 2-cytokines IL-5 and IL-13 significantly decreased during the observation period (p < 0.05 compared to placebo in week 8). These data indicate that IP seems to contain an antigen, which decreases type 2 reactivity. Furthermore, a component present preferentially in IQ seems to activate the production of the monocyte/ macrophage-related cytokine TNFalpha by PBMC. Intriguingly, also in individuals receiving placebo injections immunological alterations were detected during the observation period.

B-Cell Depletion, Remissions of Malignancy, and Cytokine-Associated Toxicity in a Clinical Trial of T Cells Genetically-Engineered to Express An Anti-CD19 Chimeric Antigen Receptor

Abstract 167▪FN2▪This icon denotes a clinically relevant abstractNew therapies are needed for chemotherapy-resistant B-cell malignancies. Adoptive transfer of T cells genetically-engineered to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19 is a promising new approach for treating B-cell malignancies. We are conducting a clinical trial in which patients receive infusions of autologous T cells that are transduced with gamma-retroviruses encoding an anti-CD19 CAR. The CAR is made up of the variable regions of an anti-CD19 antibody, a portion of the CD28 molecule, and a portion of the CD3-zeta molecule. Our clinical protocol consists of cyclophosphamide plus fludarabine chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of high-dose IL-2. We have treated 8 patients on this clinical trial. Four of the patients had chronic lymphocytic leukemia (CLL), and 4 patients had B-cell lymphoma. Anti-CD19-CAR-transduced T cells that specifically recognized CD19-expressing target cells were produced for all patients. The total number of cells administered to each patient ranged from 0.5×107 to 5.5×107 cells per kg of bodyweight. A mean of 54% of the administered cells expressed the anti-CD19 CAR. One patient with CLL obtained a complete remission that is ongoing 15 months after treatment. Five patients obtained partial remissions that are ongoing in 3 patients. One patient with CLL had stable disease. One patient with lymphoma died of culture-proven influenza A pneumonia and is not evaluable for lymphoma response. A striking depletion of CD19+ B-lineage cells occurred in 4 of 8 patients. This B-cell depletion lasted for up to 15 months, and it is ongoing in 3 of 4 patients. Because of the long duration of B-cell depletion, it cannot be attributed to the chemotherapy that the patients received. For example, a patient with follicular lymphoma had a normal level of polyclonal blood B cells before treatment on our protocol. Six months after treatment, he had a blood B cell count of 1/microliter (normal range 61–321 B cells/microliter). A patient with CLL had a regression of adenopathy in the first 32 days after chemotherapy and CAR-transduced T cell administration. Interestingly, this adenopathy continued to substantially regress between 33 and 133 days after chemotherapy ended. CAR-transduced cells were detected in the blood of all 8 patients by quantitative PCR. The percentage of peripheral blood mononuclear cells (PBMC) containing the CAR gene varied widely, but in 2 patients the CAR gene was detected in greater than 0.1% of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 20% of all blood T cells. Patients had significant toxicity during the first 10 days after CAR-transduced T cell infusion. The most prominent toxicity was hypotension. Sharp increases in serum interferon gamma (IFN) and tumor necrosis factor (TNF) occurred in 4 of 8 patients during the first 10 days after cell infusion. In the 4 patients with prominent elevations in inflammatory cytokines, peak serum IFN levels ranged from 865 to 2305 pg/mL, and peak TNF levels ranged from 49 to 118 pg/mL. As measured by an objective sequencial organ failure assessment score, the 4 patients with prominent elevations in IFN and TNF had more severe clinical toxicity during the first 10 days after cell infusion than the other 4 patients who did not have prominent elevations of IFN and TNF. We analyzed PBMC of three patients with elevations of serum IFN and TNF by using an ex vivo assay consisting of a 6-hour incubation of PBMC with target cells followed by intracellular cytokine staining. In all three patients, we detected T cells that produced IFN and TNF in a CD19-specific manner in PBMC samples collected after anti-CD19-CAR-transduced T cell infusions but not in PBMC samples collected before CAR-transduced T cell infusions. This indicates that anti-CD19-CAR-transduced T cells were a source of the elevated serum cytokines. In multiple patients receiving infusions of anti-CD19-CAR-transduced T cells, we have demonstrated elimination of CD19+ B-lineage cells, regressions of malignancy, elevated levels of serum inflammatory cytokines, and CD19-specific T cells that produce inflammatory cytokines ex vivo. These results demonstrate that CAR-expressing T cells can specifically eliminate targeted cells and cause significant cytokine-mediated toxicity in humans. Disclosures:No relevant conflicts of interest to declare.

Hydroxytyrosol, the phenolic compound of olive oil protects human PBMC against oxidative stress and DNA damage mediated by 2,3,7,8-TCDD

The protective effect of hydroxytyrosol (HT), a strong antioxidant compound from extra virgin olive oil, against TCDD induced toxicity was investigated in human peripheral blood mononuclear cells (PBMC). PBMC (1×106cellsmL−1) were divided into four groups and were incubated in a CO2 incubator (5% CO2) for 12h with vehicle, TCDD (10nM), TCDD+HT (10nM+100μM) and HT alone (100μM) respectively. To clarify the role of HT against TCDD induced cytotoxicity, oxidative stress and the levels of antioxidant enzymes were assessed. Incubation of PBMC with TCDD significantly decreased cell viability, catalase (CAT) and glutathione peroxidase (GPx) and increased the levels of superoxide dismutase (SOD), glutathione reductase (GR) and oxidative stress markers such as lipid peroxidation products (LPO), protein carbonyl content (PCC) and reactive oxygen species (ROS). Whereas, HT had an effective antioxidant property as observed by the increased cell viability, normalization of antioxidant enzymes and decreased levels of LPO, PCC and ROS in PBMC co-treated with HT and TCDD. Apoptosis detection and comet assay results shows that HT, by acting as an antioxidant, prevents the damage to DNA induced by TCDD. In addition light microscopic and histopathological observations revealed that the cells are apoptotic and degenerated during TCDD treatment, whereas cells showed intact morphology during co-treatment with HT. On the whole, the results reveal that HT exerts a promising antioxidant potential in protecting the PBMC against TCDD induced oxidative stress, which might be due to the presence of catechol moiety in its structure.

Determination of optimal incubation time for the production of acute phase cytokines ex vivo by peripheral blood mononuclear cells from renal transplant recipients

Levels of acute phase cytokines secreted ex vivo by peripheral blood mononuclear cells (PBMCs) have been shown to be associated with clinical conditions or histologic lesions in renal transplant recipients. One of the limiting factors for the potential use of this assay as a diagnostic tool is the incubation time needed to measure adequate cytokine levels. Here, we validated that shorter time periods than the usual 48 h are sufficient for the production of acute phase cytokines. Cytokine levels were measured with the Luminex platform. We observed that, in contrast to cytokines associated with adaptive immunity, cytokines such as IL-1β, IL-6 and TNF-α are measurable as early as 2 h following incubation at a concentration of 1.5 million PBMC/150 μL. Levels obtained in the 2 h cultures have good correlations with the levels obtained after 48 h of culture for IL-1β and TNF-α (R = 0.79, P = 0.004 and R = 0.92, P < 0.001 respectively). We conclude that same-day incubation of PBMCs and measurement of these cytokines following blood collection in transplant recipients is feasible. It provides a rationale for further studies using shorter incubation times for ex vivo cellular assay measuring acute phase cytokine levels.

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data.Isolated human PBMC were exposed for 20h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10μg/ml) for 48h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively.All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.