Monocyte Cytotoxicity Research Articles

Cytotoxicity, pro-inflammatory cytokines gene expression and antioxidant activity of Acmella oleracea extract treated with active charcoal

This work reports for the first time the evaluation of the cytotoxicity and inflammatory potential of Acmella oleracea extract treated with active charcoal in THP-1 monocytes. A. oleracea flower ethanolic extract was treated with 4% activated charcoal (TCEE). Later, THP-1 human monocyte cytotoxicity assay was performed using resazurin fluorometric method. Gene expression of inflammatory cytokines in THP-1 cells were evaluated through RT-PCR by ΔΔCt method using IL-1β, IL-6, IL-8, and TNF genes primers. Finally, antioxidant assay was carried out with DPPH radical scavenging method. TCEE had a LD50 of 592.5 µg/mL and did not induce pro-inflammatory gene expression in THP-1 cells after 6 h of treatment. Lastly, TCEE (AA% of 69.4 ± 1.4%) and CEE (AA% of 63.0 ± 0.9%) showed moderate antioxidant activity. A. oleracea treated flower extract showed low cytotoxicity in THP-1 monocytes and does not induce inflammation in THP-1 cells, in addition to presenting antioxidant potential.

Both biofilm cytotoxicity and monocytes' adhesion may be used as estimators of enterococcal virulence.

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdańsk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m/z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m/z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection.

Trial in Progress: A Phase 1b Study of ELA026 in Patients with Secondary Hemophagocytic Lymphohistiocytosis

Background: ELA026 is a fully human, monoclonal immunoglobulin G1 (IgG1) signal regulatory protein (SIRP)-directed antibody. SIRP is a cell surface protein expressed on cells of the myeloid lineage and T cells, which are implicated in driving pathology in hemophagocytic lymphohistiocytosis (HLH). HLH is a rare, aggressive, and life-threatening syndrome of excessive immune activation. Patients typically present with fever, hepatosplenomegaly, and pancytopenia. The disease is associated with a massive systemic inflammatory response requiring immediate treatment. Left untreated, patients progress to a clinical hyperinflammatory state that can lead to multi-organ failure, circulatory shock, and death. Secondary HLH (sHLH), the most common form of HLH, is typically triggered by malignancy, autoimmune disease, or infection. Currently, there are no approved therapies for sHLH. The HLH-94 treatment protocol (etoposide, dexamethasone, and cyclosporin) is used with variable degrees of effectiveness. Preclinical studies with ELA026 have demonstrated its ability to induce both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of human monocytes and ADCC of human SIRP+ activated T lymphocytes. Administration of ELA026 to non-human primates causes a rapid, potent, and reversible reduction of circulating monocyte, granulocyte, and T cell counts. Following washout of ELA026, reconstitution of all affected cell types to predose levels is observed within hours/days, providing evidence that bone marrow hematopoiesis is unaffected. By reducing myeloid-derived antigen presenting cells (monocytes/macrophages) and interferon gamma-producing CD8+ T cells, ELA026 has the potential to halt the initiation and progression of the inflammatory process in sHLH. Objectives: Primary objectives are to evaluate the safety of ELA026 and identify a dose for Phase 2/3 testing. Secondary objectives are to determine the efficacy (best response by 4 weeks), characterize the pharmacokinetic (PK) profile and pharmacodynamic (PD) effects, and assess the immunogenicity of ELA026. Efficacy criteria were based on modified HLH-2004 criteria, similar to what were used in a prior pivotal trial for primary HLH (Locatelli F, et al. N Engl J Med. 2020;382(19):1811-22). Study Design: This Phase 1b, open-label, single-arm, multicenter study is investigating the safety, efficacy, PK, and PD of ELA026 following multiple intravenous (IV) doses administered in up to 24 adolescent or adult patients who are treatment naïve or who have relapsed/refractory sHLH regardless of underlying trigger (NCT05416307). Patients must be at least 12 years of age at the time of sHLH diagnosis. Patients are ineligible if they have received hemopoietic stem cell transplantation within 100 days or chimeric antigen receptor (CAR) T-cell therapy within 3 months prior to the first dose of ELA026. This study utilizes an adaptive 3+3 design, with an intra-patient dose-escalation phase and a fixed-dose phase (Figure 1). In Cohort 1, after a patient has received 3 daily IV doses at a pharmacologically active dose level, the dose will be escalated until that patient (1) demonstrates monocyte reduction and ferritin biomarker evidence of improvement, (2) develops dose-limiting toxicity; or (3) receives the maximum allowable dose. Biomarker evidence of improvement is defined as: (1) monocyte depletion ≥75% from baseline to the end of the dosing interval and (2) serum ferritin reduction ≥20% from baseline levels ≥3000 ng/mL (or if baseline ferritin levels are <3000 ng/mL, any decrease in these, along with improvement in (a) fasting triglyceride levels, (b) coagulation parameters, and/or (c) sIL2 receptor levels compared to baseline). At the completion of Cohort 1, a Data Monitoring Committee will determine the optimal fixed dose for Cohort 2 to confirm its safety and efficacy. Optional Cohorts 3 and 4 will explore less frequent dosing schedules. Subcutaneous administration also may be evaluated. Total treatment duration is 12 weeks. No formal statistical hypothesis is being tested. At the completion of Phase 1b, an appropriate dose, route of administration, and dose interval will be determined for a Phase 2/3 trial. The study is sponsored by Electra Therapeutics, Inc. Figure 1. Study SchemaFigure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Interleukin-33/ST2 Axis Enhances Lung-Resident CD14+ Monocyte Function in Patients with Non-Small Cell Lung Cancer

ABSTRACT Interleukin-33 (IL-33) binds to its cognate receptor suppression of tumorigenicity 2 (ST2), leading to critical modulatory roles in immune responses during inflammation and cancers. The aim of this study was to investigate the role of IL-33/ST2 signaling in monocyte function in non-small cell lung cancer (NSCLC). Sixty-two NSCLC patients and nineteen controls were enrolled. IL-33 levels and ST2 expression were measured in peripheral blood and bronchoalveolar lavage fluid (BALF) by ELISA and flow cytometry. HLA-DR expression by CD14+ monocytes, granzyme B and proinflammatory cytokine secretion were also investigated in lipopolysaccharide-stimulated cells. CD14+ monocytes purified from BALF in the tumor site were stimulated with IL-33 in vitro, and co-cultured with a lung cancer cell line A549 cells. The cytotoxicity of monocytes with IL-33 stimulation was then assessed. IL-33 levels were lower in the peripheral blood and tumor microenvironment of NSCLC patients. There was no significant difference in peripheral ST2 expression between NSCLC patients and controls. Soluble ST2 levels were increased but membrane-bound ST2 expression in CD14+ monocytes was decreased in tumor microenvironment of NSCLC patients. There were no remarkable differences in either HLA-DR expression or proinflammatory cytokine secretion by circulating CD14+ monocytes between NSCLC patients and controls. CD14+ monocytes in the tumor microenvironment revealed a dysfunctional phenotype, which presented as lower HLA-DR expression and reduced granzyme B and proinflammatory cytokines. A higher concentration of IL-33 stimulation promoted tumor-resident CD14+ monocyte-induced target cell death. The present study indicates that IL-33/ST2 signaling pathway might enhance the activity of tumor-resident CD14+ monocytes in NSCLC.

Synergistic antioxidant effects of natural compounds on H2O2-induced cytotoxicity of human monocytes.

Natural compounds are endowed with a broad spectrum of biological activities, including protection against Toxins. Most of them are known for their antioxidant and radical scavenging activities. However, the synergistic combination of these natural molecules is not well studied. Therefore, the present study aims first to investigate the effect of four potent natural molecules [rosmarinic acid (Ros-A), ellagic acid (Ella-A), curcumin (Cur), and syringic acid (Syr-A)] on H2O2 -induced cell cytotoxicity and oxidative stress on the human monocytes (THP-1) and then to evaluate their combined action effect. Optimal combinations of these molecules were predicted using an augmented mixture design approach. In the first, as preliminary antioxidant activities screening, two in vitro assays were adopted to assess the single radicals scavenging activity of these natural compounds, DPPH• and ABTS• + tests. Based on the results obtained, the multitude of optimal formulas proposed by the mixture design study led to choosing four potent compositions (comp) in addition to ellagic acid, proposed as the most efficient when applied alone. The different molecules and mixtures were used to assess their cytoprotective effect on THP-1 cells in the presence and absence of H2O2. The most potent Comp-4, as well as the molecules forming this mixture, were exploited in a second experiment, aiming to understand the effect on oxidative stress via antioxidant enzyme activities analysis in the H2O2-induced oxidative stress in the THP-1 cell line. Interestingly, the natural molecules used for THP-1 cells treatment exhibited a significant increase in the antioxidant defense and glyoxalase system as well as suppression of ROS generation evaluated as MDA content. These results indicate that the natural compounds tested here, especially the synergistic effect of Cur and Ros-A (Comp-4), could serve as cytoprotective and immunostimulant agents against H2O2-induced cytotoxicity THP-1 cells, which makes them interesting for further investigations on the molecular mechanisms in preclinical animal models.

HDAC Inhibition with Valproate Improves Direct Cytotoxicity of Monocytes against Mesothelioma Tumor Cells.

Simple SummaryTumor-associated macrophages and monocyte myeloid-derived immunosuppressive cells are associated with bad prognosis in malignant pleural mesothelioma (MPM). This study shows that peripheral blood monocytes can, nevertheless, be cytotoxic for MPM tumor cells. This cytotoxic activity that involves direct cell-to-cell contact can be improved with a lysine deacetylase inhibitor (VPA), opening new prospects for further improvement of still unsatisfactory MPM therapies.The composition of the tumor microenvironment (TME) mediates the outcome of chemo- and immunotherapies in malignant pleural mesothelioma (MPM). Tumor-associated macrophages (TAMs) and monocyte myeloid-derived immunosuppressive cells (M-MDSCs) constitute a major fraction of the TME. As central cells of the innate immune system, monocytes exert well-characterized functions of phagocytosis, cytokine production, and antibody-dependent cell-mediated cytotoxicity (ADCC). The objective of this study was to evaluate the ability of monocytes to exert a direct cytotoxicity by cell-to-cell contact with MPM cells. The experimental model is based on cocultures between human blood-derived monocytes sorted by negative selection and mesothelioma cell lines. Data show (i) that blood-derived human monocytes induce tumor cell death by direct cell-to-cell contact, (ii) that VPA is a pharmacological enhancer of this cytotoxic activity, (iii) that VPA increases monocyte migration and their aggregation with MPM cells, and (iv) that the molecular mechanisms behind VPA modulation of monocytes involve a downregulation of the membrane receptors associated with the M2 phenotype, i.e., CD163, CD206, and CD209. These conclusions, thus, broaden our understanding about the molecular mechanisms involved in immunosurveillance of the tumor microenvironment and open new prospects for further improvement of still unsatisfactory MPM therapies

Immune Modulating Brevetoxins: Monocyte Cytotoxicity, Apoptosis, and Activation of M1/M2 Response Elements Is Dependent on Reactive Groups.

Brevetoxins are a suite of marine neurotoxins that activate voltage-gated sodium channels (VGSCs) in cell membranes, with toxicity occurring from persistent activation of the channel at high doses. Lower doses, in contrast, have been shown to elicit neuroregeneration. Brevetoxins have thus been proposed as a novel treatment for patients after stroke, when neuron regrowth and repair is critical to recovery. However, findings from environmental exposures indicate that brevetoxins may cause inflammation, thus, there is concern for brevetoxins as a stroke therapy given the potential for neuroinflammation. In this study, we examined the inflammatory properties of several brevetoxin analogs, including those that do and do not bind strongly to VGSCs, as binding has classically indicated toxicity. We found that several analogs are toxic to monocytes, while others are not, and the degree of toxicity is not directly related to VGSC binding. Rather, results indicate that brevetoxins containing aldehyde groups were more likely to cause immunotoxicity, regardless of binding affinity to the VGSC. Our results demonstrate that different brevetoxin family members can elicit a spectrum of apoptosis and necrosis by multiple possible mechanisms of action in monocytes. As such, care should be taken in treating “brevetoxins” as a uniform group, particularly in stroke therapy research.

Abstract 338: Peripheral blood monocyte count prediction for anti-HER2 combination therapy: Outcomes in HER2-positive metastatic breast cancer patients

Abstract Background: Breast cancer is the commonest cancer in Saudi Arabia and worldwide. Nineteen percent of breast cancers are HER2-positive. Both trastuzumab and pertuzumab inhibit cancer cell proliferation by blocking HER2 activation and HER2-mediated downstream signals. They also induce extracellular antibody-dependent cell-mediated cytotoxicity (ADCC), exerted by immune cells expressing surface CD16 Fcγ receptors (FcγRs), such as NK cells subset of human blood monocytes. Inside tissue, monocytes differentiate to macrophages and potentiate anti-cancer innate immunity. Increased macrophages' infiltration in the tumor tissue was associated with enhanced efficacy of trastuzumab antitumor efficacy in mouse xenograft tumor models. In Metastatic HER2-positive breast cancer, increased M1 polarized macrophages improved survival and were independent prognostic predictors. Herceptin significantly increased peripheral blood monocyte (PBMC) cytotoxicity against HER-2 positive cancer cells. This study aims to determine the impact of peripheral blood monocyte (PBMC) count on response rate (RR) and progression-free survival (PFS) in metastatic HER2-positive breast cancer patients treated with trastuzumab and pertuzumab. Method: A retrospective chart review at Princess Norah Oncology Center (PNOC) that included histopathology-proven metastatic HER2-positive breast cancer patients receiving first-line anti-HER2 systemic therapy from January 2008 to May 2018. Younger patients (&amp;lt; 18 years old) and those with cranial and immeasurable metastases were excluded. Before each chemotherapy cycle, peripheral blood monocyte counts (PBMC) were checked, and values were classified as high, low, or normal according to hospital lab reference ranges. Chi-square and Kaplan-Meier curves were applied to estimate response rate (RR) and progression-free survival (PFS). Results: Of 307 identified cases, 81 were analyzed. The median age was 62 years, 73% were postmenopausal, and 70% had grade 3 tumors. All had visceral metastases, treated with chemotherapy, along with an anti-HER2 agent. We found improved RR with high PBMC before the second cycle (P = 0.025) and increased PFS for high PBMC before the third cycle (P = 0.048). Conclusion(s): Peripheral blood monocyte count before cycle 2 and 3 might predict a favorable outcome of chemotherapy anti-HER2 combination in metastatic HER2-positive breast cancer. Citation Format: Amer Naeem Radwi, Reyadh Khalid Albeladi, Shahrazad Abdulsalam Alzahrani, Raghad Abdulrahman Alahmadi, Haneen Abdulrahman Aljohani, Dalia Ismaiel Ahmed. Peripheral blood monocyte count prediction for anti-HER2 combination therapy: Outcomes in HER2-positive metastatic breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 338.

PI3K-Delta Inhibitors Induce Primary Monocyte Cytotoxicity but Do Not Alter Monocyte Differentiation

It is well appreciated that an altered tumor microenvironment is a hallmark of cancer, including in chronic lymphocytic leukemia (CLL) where monocyte-derived cells known as “nurse-like cells” (NLC) in the lymph node microenvironment support CLL cell viability, survival, and resistance to drug-induced apoptosis. CLL directed therapy may include conventional chemotherapy, immunotherapy, targeted kinase inhibitors, and/or Bcl2 inhibitors. Of these options, kinase inhibitors such as PI3K-delta inhibitors are an effective therapy that have been found to disrupt NLC-CLL cell interaction in vitro and induce initial lymphocytosis thought to be related to this disrupted cellular interaction. We previously demonstrated that the PI3K-delta inhibitors idelalisib and umbralisib (TGR-1202) induce cytotoxicity, apoptosis, and inhibition of pAKT in primary CLL cells. We now investigate the effect of idelalisib and umbralisib on monocytes by evaluating cytotoxicity and cytokine-induced differentiation. We isolated monocytes from blood obtained from normal donors using negative selection (RosetteSep monocyte). For cytotoxicity experiments, primary purified monocytes were incubated ± M-CSF (10 ng/mL) ± PI3K-delta inhibitor (at 1.25 to 20 μM) for three days, and cell viability was measured with the MTS reagent after three hours. For differentiation experiments, primary purified monocytes were incubated first with M-CSF (10 ng/mL) for three days, then washed and incubated ± IL-10 (20 ng/mL) ± PI3K-delta inhibitor (10 μM) for three days. To judge monocyte differentiation, we used flow cytometry to measure expression of CD14, CD206, CD163, CD124, CD80, and CD86. We also visually assessed monocyte appearance using phase microscopy with these conditions. Both idelalisib and umbralisib induced concentration dependent cytotoxicity (N = 3), but cytotoxicity was higher with idelalisib (62% cytotoxicity at 1.25 μM) compared to umbralisib (24% cytotoxicity at 1.25 μM). The addition of M-CSF reduced PI3K-delta inhibitor-induced cytotoxicity of primary monocytes. For example, 1.25 μM idelalisib induced 62% cytotoxicity without M-CSF, compared with 5% cytotoxicity with M-CSF. Staged cytokine incubation of monocytes with M-CSF and IL-10 induced the expression of M2 differentiation markers (CD163 and CD124) and reduced expression of the M1 differentiation marker (CD86). Addition of idelalisib or umbralisib did not significantly affect monocyte surface marker expression compared to no drug (N = 4). Even though idelalisib and umbralisib were cytotoxic for monocytes, these drugs did not affect morphologic changes induced by IL-10 (marked spreading and shape change). Thus, PI3K-delta inhibitors do not appear to affect cytokine-induced monocyte differentiation, but they do induce monocyte cytotoxicity (idelalisib > umbralisib). The addition of M-CSF, which contributes to M2 differentiation, blocks these effects. Together, our results suggest that depleting monocyte or NLC number may contribute to the anti-CLL therapeutic effects of PI3K-delta inhibitors. In addition, a higher level of monocyte cytotoxicity with idelalisib compared to umbralisib could potentially contribute to the higher infectious side effect profile with idelalisib seen in clinical use, as monocyte derived cells are a key component of the innate immune system. The anti-monocyte effect of PI3K-delta inhibitors could provide rationale for using these agents in other malignancies in which monocyte-derived cells are an integral part of the tumor microenvironment. DisclosuresFriedman:TG Therapeutics: Research Funding. Maryanski:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.

Dysregulatory processes of the cellular link of the immune system in the dynamics of common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a variant of primary immunodeficiency in which inhibition of antibody production is formed due to disorders of intercellular interaction affecting cellular elements of both innate and adaptive immune responses. A feature of CVID is the late start and variability of clinical minifestation. These arguments determine the purpose of the study: to identify the dynamics of changes in the cellular parameters of the adaptive and innate immune response depending on the duration and severity of the infectious manifestation of CVID. In this regard, a retrospective analysis of medical histories and dynamic observation of fifteen patients with CVID were carried out. Selection of specific parameters of cellular indices of factors of innate resistance and adaptive immunity was carried out on the basis of systemic-functional approach of immunodiagnostics. It is shown that in patients with CVID -mediated hypogammaglobulinemia and infectious phenotype of clinical manifestation, enhancement of quantitative and functional potentials of T-link effector cells of adaptive immunity is recorded against the background of reduction of number of regulatory T-helpers. With a more severe clinical course of the disease, the number of CD3+HLA DR + limphocytes is lower than with a more favorable version, there is a tendency to decrease the number of these cells, as well as the number of peripheral Treg with an increase in the length of the disease. Cellular components of innate immunity are characterized by a decrease in neutrophil activity, inhibition of antigen-presenting monocyte activity, the number and cytotoxicity of natural killers. At the same time, the tendency to decrease the cytolytic potential of NK with an increase in the length of illness and statistically significant differences depending on the severity of the manifestation of the infectious phenotype of CVID was recorded. The obtained results determine the importance of evaluating the cellular link of the immune system in patients with CVID, including as a prognostic criterion for the severity of the course.

Production of a cellular product consisting of monocytes stimulated with Sylatron\xae (Peginterferon alfa-2b) and Actimmune\xae (Interferon gamma-1b) for human use

BackgroundMonocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood.MethodsCounter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction.ResultsBoth iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs.ConclusionLarge scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

Production of autologous monocytes stimulated ex vivo with peg-interferon alfa 2b and interferon gamma 1b for intraperitoneal administration in phase I clinical trial.

5 Background: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes are cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha and Interferon Gamma. We previously showed that monocytes stimulated with both interferons (IFNs) result in synergistic killing of ovarian cancer cells. We aimed to develop this cell therapy product for clinical application. Methods: Counter flow elutriation was performed on healthy donors. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without cryopreservation. All 5 fractions from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets. This procedure is now implemented in a clinical trial where 8 patients completed the apheresis and elutriation process for a total of 13 runs. Results: Iterative monocyte isolation using counter flow elutriation with or without cryopreservation can yield over 2 billion monocytes for each subject with high purity. We show that monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. Flow cytometric analysis shows expected heterogeneity in the collected monocytes. In each case, the elutriation run met pre-set release criteria for both total monocyte number and purity requirements for the therapeutic product. Conclusions: We demonstrated that large scale isolation of monocytes can be achieved with elutriation from either healthy donors or patients with advanced, chemotherapy resistant ovarian cancer. Monocytes can be cryopreserved and maintain viability and cytotoxic function in the final cell therapy product prior to infusion. We translated these observations to a currently accruing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex-vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. (Protocol NCT00327067). Clinical trial information: NCT02948426.

Fc Gamma Receptor 3A Polymorphism and Risk for HIV-Associated Cryptococcal Disease

ABSTRACTCryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4+ T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4+ T cell decline, and nadir CD4+ T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation.

Quantitation and Human Monocyte Cytotoxicity of the Polymerization Agent 1-Hydroxycyclohexyl Phenyl Ketone (Irgacure 184) from Three Brands of Aqueous Injection Solution

In this study, levels of the photoinitiator 1-hydroxycyclohexyl phenyl ketone (1-HCHPK) in aqueous injection solutions were analyzed by GC-MS. In our previous studies, photoinitiators such as 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MTMP) were detected in intravenous (i.v.) injection bag solution, and they were found to be cytotoxic to human monocytes. Therefore, we hypothesized that 1-HCHPK might display similarly cytotoxicity. The purpose of this study was to quantitate the amount of contaminants from plastic containers such as those used for peripheral parenteral nutrition and to determine the cytotoxicity of such extracts on human monocytes. The sample extraction procedure for GC-MS analysis involved a liquid-phase extraction. The solvent was evaporated under a stream of nitrogen at 50°C to yield a residue, which was dissolved in n-hexane and injected into a GC-MS. Normal human peripheral blood mononuclear cells (PBMC), isolated from the buffy coat by centrifugation, were suspended in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay, cells (1×10(4)) were treated with 1-HCHPK for 24 h or 48 h at 37°C. From the GC-MS analysis, 6.13-8.32 µg/mL of 1-HCHPK was found in 20 mL vials of water for injection solution. In the MTT assay, 1-HCHPK decreased cell viability for both the 24 h and 48 h incubation periods. In conclusion, our findings suggest that 1-HCHPK could promote adverse events in patients. Future studies will clarify the possible health risks of photoinitiator accumulation in human cells.

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.

Designing a plague vaccine utilizing α-gal modified antigens (52.6)

Abstract Background: F1-V from Yersinia pestis has been shown to be a protective antigen against disease. We modified the F1-V antigen with α-galactose residues and immunized α galactosyl transferase (α-GT) KO mice in order to mimic the same defect in humans that results in circulating antibodies against α-gal epitopes enhancing immune responsiveness. Aim: To demonstrate that α-gal modified F1-V induces a more robust immune response than unmodified F1-V. Methods: α-GT KO mice were injected subcutaneously with different doses of α-gal-F1-V or F1-V. All mice were boosted with F1-V at 36 dpi and necropsied on day 40 in order to evaluate in vitro recall response of lymphocytes and to measure anti-F1-V antibody titers. Results: Mean IgG titer values were 50,000 and 10,000 for mice immunized with the α-gal-F1-V or F1-V, respectively. The relative avidity of the antibody response was also higher for the α-gal-F1-V immunized mice. Moreover, lymphocytes from α-gal-F1-V immunized mice demonstrated enhanced proliferation in response to F1-V compared to cells from F1-V immunized mice. Conclusions: α-gal modified F1-V increases both the avidity and amount of anti-F1-V produced following vaccination. As a functional assay, α-gal-F1-V antisera efficiently opsonized Y. pestis, facilitated phagocytosis by human monocytes and prevented Y. pestis-induced monocyte cytotoxicity.

In vitro effects of leukocyte interferon on the cytotoxic potential of human monocytes.

The in vitro cytotoxicity of human monocytes was enhanced by leukocyte interferon (IFN-alpha). In contrast to lymphokine activation which is obtained gradually under long-term contact with monocytes, maximal activation of effector cells was obtained within 4 hours of culture with IFN-alpha prior to medium exchange and target cell addition. The degree of activation was clearly dose-dependent, reaching a plateau level of cytolysis at 400 U/ml of IFN-alpha. In cytostasis no such plateau was demonstrable. IFN-alpha enhanced the level of cytolysis mediated by human monocytes without inducing any change in the kinetics of lysis, regardless of whether IFN-alpha was used for activation of monocytes prior to or simultaneously with the cytolysis assay. The direct effect of IFN-alpha on K-562 target cells was sparse. The incorporation of thymidine was modestly depressed in the presence of IFN-alpha. Exposure of K-562 cells to IFN-alpha did not have any effect on the spontaneous isotope leakage from prelabelled target cells. The exposure of K-562 cells to IFN-alpha prior to the start of co-culture with monocytes did not change the level of cytolysis reached, either with non-activated or with IFN-alpha activated monocytes, and irrespective of the in vitro age of the monocytes. The effect of IFN-alpha thus seems to be mediated via the effector cell population and not via target cell modulation.

The Effects of the Herbal Enzyme Bromelain Against Breast Cancer Cell Line GI101A

Bromelain is a proteinase derived from the stem of pineapple and has been studied for its anti‐inflammatory, antithrombotic, and antimetastatic properties. Bromelain has also been known to significantly reduce local tumor growth and to raise the impaired cytotoxicity of monocytes in the immune system against tumor cells. The goal of this project is to advance the mechanistic knowledge of herbal remedies and to confirm the already known antimetastatic properties of Bromelain. The MTS assay method was used 24 hours after Bromelain treatment to detect the cell death. The data show that after 1 μM of Bromelain treatment, the population of GI101A cells is significantly reduced by up to 70%. Using the M30 Apoptosense assay, levels of the protein cytokeratin 18 (CK18) were measured to detect any apoptotic activity. After 10 μL of Bromelain treatment, CK18 levels increased and a large number of apoptotic cell bodies were observed. The antitumor effects of Bromelain are mainly involved in cancer cell division by the induction of apoptosis.

BNP directly immunoregulates the innate immune system of cardiac transplant recipients in vitro

Background The innate immune system plays an important role in cardiac allograft rejection. BNP has frequently been reported to elevate during acute cardiac rejection, yet the explanation behind this phenomenon is unclear. We hypothesized that BNP might interact with the innate immune system in cardiac transplant recipients and devised a series of in vitro culture experiments to explore this phenomena. Methods PBMCs were isolated from whole blood of (total n = 40) cardiac transplant recipients. Short (24h, n = 20) and long term (72h, n = 20) co-cultures of innate cells in the presence or absence of BNP were performed. BNP was added at two specific concentrations and compared to placebo control. Innate cells were immunophenotyped using flow cytometry. Results BNP dose dependently reduced the total number of monocytes, B cells and NK cells. Furthermore, BNP co-culture impaired NK cell cytotoxicity and adhesion of non-classical monocytes (via down-regulation of CD11c). Discussion BNP has an additional physiological role of moderating components of the innate immune system. Although speculative, this could be beneficial to cardiac transplant recipients as the innate immune system is involved in allograft rejection. Further investigation is required to elucidate the mechanism behind how BNP affects immune cells and whether the same effects are consistent with the adaptive immune system.

Lactoferrin-inducible monocyte cytotoxicity for K562 cells and decay of natural killer lymphocyte cytotoxicity

Monocyte-enriched and lymphocyte-enriched fractions of peripheral blood from three healthy volunteers were obtained by percoll density gradient centrifugation. The cytotoxic activity of each fraction against 51Cr-labelled K562 cells was quantified in a 2-h assay using freshly isolated cells of each fraction and cells of each fraction which had been incubated with and without lactoferrin in complete medium for 18 h before performing the assay. We have thereby shown that cytotoxicity was not demonstrable in the lymphocyte fraction (containing 7.3 +/- 2% large granular lymphocytes) after 18 h in medium, whereas the cytotoxicity of the monocyte fraction (containing 3 +/- 0.4% large granular lymphocytes) was still significantly increased (P less than or equal to 0.01) and that lactoferrin had no effect on lymphocyte fraction cytotoxicity while producing an 11-fold increase in the cytotoxicity of the monocyte fraction. It is therefore possible to perform a relatively simple test of monocyte cytotoxicity using lactoferrin as a stimulant in a 2-h 51Cr-labelled K562 assay system by allowing 18 h to elapse for lymphocyte natural killer cytotoxicity to decay.