Primary Blood Mononuclear Cells Research Articles

45 Senolytic Therapy Transiently Reduces Inflammatory Markers in Primary Blood Mononuclear Cells of Individuals with Early Alzheimer’s Disease: Exploring the Conserved Transcriptional Response to Adversity as a Biomarker for Disease State

OBJECTIVES/GOALS: Determine if the Conserved Transcriptional Response to Adversity transcriptomic profile established in primary blood mononuclear cells (PBMC) of chronically stress caregivers, is present in individuals with early Alzheimer’s disease. Chronic stress is a risk factor for Alzheimer’s, and may be an untapped biomarker for disease risk and pathology. METHODS/STUDY POPULATION: To collect preliminary data on the Conserved Transcriptional Response to Adversity profile in individuals with Alzheimer’s disease, we were able to utilize primary blood mononuclear cell samples from a small open label pilot study called Senolytic Therapy to Modulate the Progression of Alzheimer’s Disease, designed to clear stressed senescent cells. We hypothesized senolytics may beneficially reverse this stress profile. We developed a NanoString assay (measuring 19 inflammatory, 31 type-1 interferon, and 3 antibody synthesis genes) to compare these transcriptomic changes within 4 individuals measured at baseline, post-treatment with an intermittent 12-week senolytic therapy, and at an optional extended post-treatment follow-up time point > 3 months after their post treatment visit. RESULTS/ANTICIPATED RESULTS: There was relative downregulation of expression in transcription in 7 of 19 measured inflammatory genes (FOS, PTGS2, IL8, FOS, Il1b, JUNB, and JUN) in Alzheimer’s disease participants after receiving senolytic treatment (baseline vs. post-treatment). This is consistent with a decrease in the inflammatory arm of the Conserved Transcriptional Response to Adversity profile. These differences were not significant between baseline and the extended follow-up, indicative of a transient effect of senolytic. There were no changes in type 1 interferon or antibody synthesis genes. This data provides preliminary evidence for larger controlled studies to further establish this profile in Alzheimer’s disease, providing exciting evidence for transcript changes that may be reproducible with senolytic therapy. DISCUSSION/SIGNIFICANCE: Literature relevant to Alzheimer’s disease indicates global increases in inflammation paired with deficits in immune response, capturing some genes associated with the Conserved Transcriptional Response to Adversity. This profile may be a useful biomarker for prediction of disease severity or risk of dementia due to chronic stress.

Utilization of Aminoguanidine Prevents Cytotoxic Effects of Semen.

Studies of human semen in cell or tissue culture are hampered by the high cytotoxic activity of this body fluid. The components responsible for the cell damaging activity of semen are amine oxidases, which convert abundant polyamines, such as spermine or spermidine in seminal plasma into toxic intermediates. Amine oxidases are naturally present at low concentrations in seminal plasma and at high concentrations in fetal calf serum, a commonly used cell culture supplement. Here, we show that, in the presence of fetal calf serum, seminal plasma, as well as the polyamines spermine and spermidine, are highly cytotoxic to immortalized cells, primary blood mononuclear cells, and vaginal tissue. Thus, experiments investigating the effect of polyamines and seminal plasma on cellular functions should be performed with great caution, considering the confounding cytotoxic effects. The addition of the amine oxidase inhibitor aminoguanidine to fetal calf serum and/or the utilization of serum-free medium greatly reduced this serum-induced cytotoxicity of polyamines and seminal plasma in cell lines, primary cells, and tissues and, thus, should be implemented in all future studies analyzing the role of polyamines and semen on cellular functions.

Nondepolarizing Neuromuscular Blocking Agents Exacerbate Inflammation Response by Antagonizing α7 Nicotinic Acetylcholine Receptors

<p id="p00005">Studies have indicated that Nondepolarizing neuromuscular blocking agents (NMBA) can inhibit α7-nicotinic acetylcholine receptors (α7 nAChR). Our study aimed to determine whether NMBA can enhance the release of tumor necrosis factor TNF-α via α7 nAChR antagonism. In vitro studies showed that primary blood mononuclear cells were stimulated with lipopolysaccharide (LPS)+acetylcholine (Ach). NMBA had been added 5 min before ACh Pre-treat human macrophages, and the supernatant was collected to measure the concentration of TNF-α. In vivo studies indicated that tissue samples were collected from NMBAs pre-treated animal models. The content of TNF-α in blood supernatant as well as in serum, liver, and spleen samples was detected. It can be found that TNF-α release from macrophages was enhanced by NMBA. In addition, rats that were pre-treated with NMBA were also found to have higher levels of TNF-α. Therefore, NMBA can enhance TNF-α releasing from macrophage cells compromised by endotoxin, through actions involving α7 nAChR.

Pseudolaric acid B induces mitotic arrest and apoptosis in both imatinib-sensitive and -resistant chronic myeloid leukaemia cells

The selective BCR-ABL tyrosine kinase inhibitor imatinib is one of the first-line therapies in the management of chronic myeloid leukaemia (CML). However, acquired resistance to this inhibitor, which is especially conferred by the T315I point mutation in BCR-ABL, impedes the efficacy of imatinib therapy. Therefore, the discovery and development of novel agents to overcome imatinib resistance is urgently needed. Pseudolaric acid B (PAB), a small molecule isolated from the traditional Chinese medicine Cortex pseudolaricis, has been reported to be a potential candidate for immune disorders and cancer treatment. However, its effects on CML and the involved molecular mechanism have not been reported. In the current study, by performing both in vitro and in vivo experiments in CML cells, we showed that PAB blocked the cell cycle at G2/M phase and subsequently activated the caspase pathway, cleaved the BCR-ABL protein and inhibited the BCR-ABL downstream pathways, ultimately leading to cell proliferation inhibition, cytotoxicity and apoptosis. These events were observed in both imatinib-sensitive and imatinib-insensitive CML cell lines. Moreover, PAB decreased the viability of primary blood mononuclear cells from CML patients and induced apoptosis in these cells. Our findings suggest that PAB could be used as a novel agent to sensitize imatinib-resistant CML.

Differential effects of performance-enhancing drugs 'Methamphetamine' and 'hCG' on ex-vivo cultured primary blood mononuclear cells of male athletes.

To bridge the knowledge gap, the present study aimed to investigate the effect of different doses of two widely used performance-enhancing drugs 'methamphetamine' (Meth) and 'human chorionic gonadotropin'(hCG) on ex-vivo cultured primary blood cells of young male Indian athletes. Primary blood mononuclear cells (PBMCs) were isolated and cultured to obtain pure T cells and monocyte-derived macrophages. Immunofluorescence, flow-cytometry, qRT-PCR, Western blot, ELISA and siRNA transfection studies were carried out to evaluate the effect of these two drugs on athletes' blood cells. Cell viability studies revealed that Meth at high doses was toxic for PBMCs and showed a significant negative impact on red blood cell fragility but hCG incubation did not result in any cytotoxicity or haemolysis. The current study also demonstrated that Meth incubation significantly affected T cell proliferation, percentage of regulatory T cells (Treg cells), Th17 cells, early activated T cells, ROS generation and mitochondrial dysfunction. On the other hand, hCG treatment upregulated the percentage of Treg cells. Within macrophage cells, Meth incubation upregulated MYD88 dependent TLR4 pathway and decreased the phagocytotic capability of the cells. Both hCG and Meth showed its potential action on alteration the pro/anti-inflammatory cytokine profiling but suppression of TLR4 pathway by RNA interference (TLR4 siRNA) suggested promising future treatment modalities. This study demonstrated the differential effects of Meth and hCG on immune cells of athlete's blood. Meth acted as an inflammation and T cell dysfunction inducing agent, while hCG acted as an anti-inflammatory immunosuppressive molecule.

Abstract TMP24: Soluble St2 Links Innate Inflammation to Secondary Injury After Stroke and Subarachnoid Hemorrhage

Background: We recently found that soluble ST2 (sST2) is a plasma marker that predicts neurological outcome after ischemic stroke and aneurysmal subarachnoid hemorrhage. We sought to extend these findings by studying sST2 associations with markers of secondary brain injury and peripheral innate immunity. Methods: We studied 241 acute ischemic stroke (AIS) patients with sST2 level measured on admission (7.4 ± 3.7 hrs after stroke onset) and at least one follow-up brain imaging study, obtained at a mean of 2.2 ± 1.4 days after stroke onset. A subgroup (n=147) had a second sST2 measurement at a mean of 2.1 ± 1.7 days after stroke onset. A separate cohort of 110 subarachnoid hemorrhage (SAH) patients were studied who had plasma sST2 measured at approximately 3.5 ± 1.2 days after ictus. Markers of secondary brain injury included edema after AIS (midline shift), and delayed cerebral ischemia (DCI) or epileptiform discharges on EEG after SAH. The relationships between sST2 level and outcome measures were assessed. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry to phenotype the peripheral immune response. Results: In the AIS cohort, baseline plasma sST2 level was associated with the presence of midline shift (46.1 vs. 41.8 ng/mL, p=0.017), which further increased at 48 hr after stroke onset (50.6 vs. 38.4 ng/mL, p=0.006). In the SAH cohort, median sST2 in patients who developed new epileptiform abnormalities was higher compared to patients who did not (114.8 ng/ml vs. 74.7 ng/ml, p=0.024). Higher median sST2 concentration was also observed in those patients with DCI (90.8 vs 53.7ng/mL, p=0.003). In patients with high sST2, flow cytometry identified a peripheral monocyte population with decreased expression of CD14 (4.27x105 ± 2950 A.U. vs. 5.64x105 ± 1290 A.U., p&lt;0.001), and increased expression of CD16 (39,960 ± 272 A.U. vs. 34,869 ± 183 A.U., p&lt;0.001). Conclusion: Plasma sST2 independently predicts edema after stroke, and DCI and epileptiform abnormalities after subarachnoid hemorrhage. Elevated sST2 is also associated with changes in peripheral innate immune cells. These data suggest that sST2 links the innate immune response to secondary brain injury.

Soluble ST2 links inflammation to outcome after subarachnoid hemorrhage.

To investigate whether soluble growth stimulation expressed gene 2 (sST2), a prognostic marker in cardiovascular and inflammatory disorders, is associated with neurological injury after aneurysmal subarachnoid hemorrhage (SAH). We studied SAH patients from 2 independent cohorts. Outcome assessments included functional status at 90 days using the modified Rankin Scale (mRS), mortality, and delayed cerebral ischemia (DCI). The relationships between sST2 plasma level and outcome measures were assessed in both cross-sectional and longitudinal analysis. Primary blood mononuclear cells from SAH patients and elective aneurysm controls were analyzed by multiparameter flow cytometry. In the discovery cohort, sST2 predicted 90-day mRS 3-6 (C index = 0.724, p < 0.001) and mortality in Kaplan-Meier analysis (p < 0.001). The association with functional status was independent of age, sex, World Federation of Neurosurgical Societies score, modified Fisher score, treatment modality, and cardiac comorbidities (adjusted odds ratio = 2.28, 95% confidence interval = 1.04-5.00, p = 0.039). Higher sST2 concentration was observed in those patients with DCI (90.8 vs 53.7ng/ml, p = 0.003). These associations were confirmed in a replication cohort. In patients with high sST2, flow cytometry identified decreased expression of CD14 (4.27 × 105 ± 2,950 arbitrary unit (AU) vs 5.64 × 105 ± 1,290 AU, p < 0.001), and increased expression of CD16 (39,960 ± 272 AU vs 34,869 ± 183 AU, p < 0.001). Plasma sST2 predicts DCI, functional outcome, and mortality after SAH, independent of clinical and radiographic markers. Elevated sST2 is also associated with changes in CD14+ CD16+ monocytes. ANN NEUROL 2019;86:384-394.

De novo prediction of cell-type complexity in single-cell RNA-seq and tumor microenvironments.

Recent single-cell transcriptomic studies revealed new insights into cell-type heterogeneities in cellular microenvironments unavailable from bulk studies. A significant drawback of currently available algorithms is the need to use empirical parameters or rely on indirect quality measures to estimate the degree of complexity, i.e., the number of subgroups present in the sample. We fill this gap with a single-cell data analysis procedure allowing for unambiguous assessments of the depth of heterogeneity in subclonal compositions supported by data. Our approach combines nonnegative matrix factorization, which takes advantage of the sparse and nonnegative nature of single-cell RNA count data, with Bayesian model comparison enabling de novo prediction of the depth of heterogeneity. We show that the method predicts the correct number of subgroups using simulated data, primary blood mononuclear cell, and pancreatic cell data. We applied our approach to a collection of single-cell tumor samples and found two qualitatively distinct classes of cell-type heterogeneity in cancer microenvironments.

In Vivo Choroidal Neovascularization and Macrophage Studies Provide Further Evidence for a Broad Role of Prostacyclin in Angiogenesis.

The purpose of these studies was (1) to investigate the ability of human M1 phenotype macrophages to secrete vascular endothelial growth factor (VEGF) and the influence of prostacyclin receptor (IP) stimulation (2) to evaluate the contribution of the proangiogenic prostanoid prostacyclin to experimental choroidal neovascularization Methods: Human macrophages derived from primary blood mononuclear cells were functionally biased toward the M1 phenotype by using tumor necrosis factor α (TNFα). Experimental choroidal neovascularization was produced by laser photocoagulation. Antagonist drugs RO-3244794 (IP antagonist) and GW 627368 (EP4 antagonist) were administered according to an optimal dosing regimen that was predetermined by bioavailability studies. IP receptor stimulation had diametrically opposed effects on VEGF release compared with reported data on cytokine/chemokine secretion from human macrophages. For example, the IP agonist cicaprost stimulated VEGF secretion although it inhibits monocyte chemoattractant protein-1 (MCP-1) secretion: both would favor a proangiogenic effect. The IP receptor antagonist RO-3244794 produced an ∼20% statistically significant reduction in the neovascularized lesion area in the choroidal neovascularization model, which was a similar level to that produced by the EP4 antagonist GW 627368. Combining the 2 drugs produced a statistically significant reduction in neovascularization but only of slightly greater magnitude than that obtained with each antagonist administered alone. IP receptor stimulation potently and highly efficaciously promoted VEGF release from human M1 macrophages, indicating a possible contribution of the M1 macrophage subtype to VEGF-induced choroidal neovascularization. Studies in living animals suggest that prostacyclin and its target IP receptor contribute to choroidal neovascularization, although to a more modest extent than might have been expected.

Chemotherapy-induced genotoxic damage to bone marrow cells: long-term implications.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow (BM) are vitally important in forming the micro-environment supporting haematopoiesis after myeloablative chemotherapy. MSCs are known to be damaged phenotypically and functionally by chemotherapy; however, to the best our knowledge, the persistence of genotoxic effects of chemotherapy on the BM micro-environment has not been studied. We therefore aimed to evaluate genotoxic effects of chemotherapy on the BM both in vitro and in vivo, using the comet and micronucleus assays, focussing on the persistence of DNA lesions that may contribute to complications in the patient. The MSC cell line (HS-5) and primary cord blood mononuclear cells (CBMNCs: a source of undamaged DNA) were exposed to the chemotherapeutic agent cyclophosphamide (CY) within a physiologically relevant in vitro model. CY treatment resulted in significant increases in CBMNC DNA damage at all time points tested (3-48 h exposure). Similarly, HS-5 cells exposed to CY exhibited significant increases in DNA damage as measured by the comet assay, with increased numbers of abnormal cells visible in the micronucleus assay. In addition, even 48 h after removal of 48-h CY treatment, DNA damage remains significantly increased in treated cells relative to controls. In patients treated with chemotherapy for haematological malignancy, highly significant increases in damaged DNA were seen in BM cells isolated from one individual 1 year after completion of therapy for acute leukaemia compared with pretreatment (P < 0.001). Similarly, two individuals treated 7 and 17 years previously with chemotherapy exhibited significant increases of damaged DNA in MSC compared with untreated age- and sex-matched controls (P < 0.05). Unlike haematopoietic cells, MSCs are not replaced following a stem cell transplant. Therefore, long-term damage to MSC may impact on engraftment of either allogeneic or autologous transplants. In addition, persistence of DNA lesions may lead to genetic instability, correlating with the significant number of chemotherapy-treated individuals who have therapy-related malignancies.

PGE2 enhanced TNFα-mediated IL-8 induction in monocytic cell lines and PBMC

Background & purposeRecent studies suggested a role of prostaglandin E2 (PGE2) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNFα-induced IL-8 expression was studied in monocytic cell lines. Experimental approachIL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. Key resultsIn monocytic cell lines THP-1, MonoMac and U937 PGE2 had only a marginal impact on IL-8 induction but strongly enhanced TNFα-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNFα and PGE2 than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNFα-induced IL-8 mRNA and protein formation to the same extent as PGE2. In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE2 enhanced TNFα-induced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNFα-mediated IL-8 mRNA induction by PGE2 was mimicked by a PKA-activator. Furthermore in these cells PGE2 induced expression of transcription factor C/EBPß, enhanced NF-κB activation by TNFα and inhibited TNFα-mediated AP-1 activation. PGE2 and TNFα synergistically activated transcription factor CREB, induced C/EBPß expression and enhanced the activity of an IL-8 promoter fragment containing -223 bp upstream of the transcription start site. Conclusions and implicationsThese findings suggest that a combined stimulation of TNFα and PGE2/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PKA/CREB/C/EBPß as well as NF-κB signal chains.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1β via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

Rationally Designed TLR4 Ligands for Vaccine Adjuvant Discovery.

ABSTRACTAdjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates.

Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

ABSTRACTThe human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro. Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most prevalent human cell-tropic FeLV variant, FeLV-B. We found that blood cells are uniquely resistant to infection with FeLV-B due to the activity of cellular enzymes that mutate the viral genome. A second block, which appears to suppress viral gene expression after the viral genome has integrated into the host cell genome, was identified. Since cells derived from other normal human cell types are fully supportive of FeLV replication, innate resistance of blood cells could be critical in protecting against cross-species infection.

Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model.

Cell biology differs between traditional cell culture and 3-dimensional (3-D) systems, and is modulated by the extracellular matrix. Experimentation in 3-D presents challenges, especially with virulent pathogens. Mycobacterium tuberculosis (Mtb) kills more humans than any other infection and is characterised by a spatially organised immune response and extracellular matrix remodelling. We developed a 3-D system incorporating virulent mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction. Infection in 3-D led to greater cellular survival and permitted longitudinal analysis over 21 days. Key features of human tuberculosis develop, and extracellular matrix integrity favours the host over the pathogen. We optimised multiparameter readouts to study emerging therapeutic interventions: cytokine supplementation, host-directed therapy and immunoaugmentation. Each intervention modulates the host-pathogen interaction, but has both beneficial and harmful effects. This methodology has wide applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimes and vaccination approaches.

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.

In Vitro Validation of Cancer Immunotherapy Reagents Using an Impedance‐Based Platform

Immunotherapy is emerging as one of the most promising approaches in cancer treatment, allowing specific targeting of cancer cells from immune cells, mediated by surface marker expression. In vitro characterization of the efficacy and potency of cancer immunotherapy reagents is a necessary step before moving to animal models and Phase I and II clinical studies. In this study we report the validation and optimization of protocols for analyzing the efficacy of reagents designed to stimulate the immune response against cancer cells using an impedance‐based and label‐free platform. The platform allows detection of cell adhesion and cell death trough monitoring the change in conductance of microelectrodes embedded in the bottom of 96‐wells cell culture plates. Because cell adhesion alters the conductivity of the electrodes, any change in cell morphology, cell adhesion or cell number is detected as variation of an impedance‐related parameter named Cell index (Ci). The platform avoids the use of any chemical label or sample processing, allowing continuous monitoring of cell activity over prolonged time periods. Furthermore, the untouched cells are still available for secondary assays that can better clarify the biological mechanism involved in the response.To mimic tumors that are target of immunotherapy efforts we utilized several carcinoma cell lines whit different level of expression of the EpCAM membrane protein. Such antigen is overexpressed in many tumors and is currently under evaluation in clinical trials. In PC3 prostate cancer cells we measured cell cytotoxicity of Primary Blood Mononuclear Cells (PBMCs) mediated by an EpCAM/CD3 Bispecific T cell Engager (BiTE) antibody. The use of the impedance‐based platform allowed measuring the full kinetic of the cytotoxic activity under different effector:target ratios and BiTE concentrations. Apoptosis of tumor cells was confirmed trough flow cytometer analysis. We also demonstrated the relation between amount of PBMCs cytotoxic activity and EpCAM surface expression trough testing MCF7 (high EpCAM expressor) and HeLa (low EpCAM expressor).To evaluate cancer immunotherapy protocols in lymphomas and leukemia, we developed a protocol where lymphomas cells are immobilized on electrodes‐coated plates and the T‐Lymphoblast cell line TALL‐104 is used as cytotoxic effector.Taken together our results demonstrate high sensitivity of the impedance‐based approach in measuring cytotoxic activity in sophisticated high throughput set up. Furthermore, the availability of continuous recording of the Cell Index over the temporal scale allows better characterization, comparing to alternative end point assays, of construct efficacy.

IMPS-15PDT-TREATED GBM CELLS INCREASE EFFECTOR FUNCTIONS OF HUMAN CD8+ T-CELLS

OBJECTIVE: Photodynamic therapy (PDT)-induced modulation of the adaptive immune response might account for long-term survival observed in glioblastoma (GBM) patients after PDT. We here analyze the impact of PDT-treated GBM cells on the effector functions of primary human T cells. METHODS: After incubation with 5-aminolevulinic acid (5-ALA) for 4 hours, the human glioblastoma cell line U87 was subjected either to PDT (3 Joule/cm2) or temozolomide (350 µM). PDT and temozolomide doses were equivalent regarding the evoked cell death rates. CD3/CD28-activated primary blood mononuclear cells (PBMCs) obtained from healthy donors were co-cultivated with PDT- or temozolomide-treated U87 or untreated controls for 48 hours. Thereafter, CD4+ and CD8 + T cells were separated by magnetic cell separation. Expression of cytokines, cytotoxicity and activation markers was measured by quantitative RT-PCR (qPCR) and flow cytometry. Cytotoxicity was determined by ELISPOT and CalceinAM fluorescence assays. Differences between groups were analyzed using Student's t test (parametric distribution) or Mann-Whitney U test (non-parametric distribution). Values are means ± SEM. RESULTS: In vitro activated primary human CD4 + T cells did not exhibit changes in their cytokine profiles upon co-culturing with PDT-treated U87 cells. In CD8+ T cells, however, significantly increased expression levels of Interferon-gamma (qPCR, 1.8-fold ± 0.34; n = 14, p < 0.001) and Perforin (qPCR, 1.6-fold ± 0.14, n = 14, p < 0.001; Perforin-ELISPOT 1.3-fold ± 0.11, n = 8, p = 0.003) were seen. Cytotoxic activity was enhanced by 13 ± 4.4% (CalceinAM fluorescence of supernatants, n = 5, p = 0.017). Flow cytometry revealed a significant decrease of the co-inhibitory T cell marker CTLA4 (23 ± 3.5%, n = 5, p < 0.001). Following temozolomide treatment, CD8+ cytotoxic activity was not enhanced (n = 4, p = 0.7). CONCLUSION: 5-ALA PDT but not temozolomide treatment of glioblastoma cells increases the cytotoxic activity of human CD8+ T cells. This might contribute to long-term tumor control in a considerable number of glioblastoma patients after PDT.

Transcriptional profiling and biological pathway analysis of human equivalence PCB exposure in vitro: Indicator of disease and disorder development in humans

Background and aimsOur earlier gene-expression studies with a Slovak PCBs-exposed population have revealed possible disease and disorder development in accordance with epidemiological studies. The present investigation aimed to develop an in vitro model system that can provide an indication of disrupted biological pathways associated with developing future diseases, well in advance of the clinical manifestations that may take years to appear in the actual human exposure scenario. MethodsWe used human Primary Blood Mononuclear Cells (PBMC) and exposed them to a mixture of human equivalence levels of PCBs (PCB-118, -138, -153, -170, -180) as found in the PCBs-exposed Slovak population. The microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR Taqman Low Density Array (TLDA) was done to further validate the selected 6 differentially expressed genes of our interest, viz., ARNT, CYP2D6, LEPR, LRP12, RRAD, TP53, with a small population validation sample (n=71). ResultsOverall, we revealed a discreet gene expression profile in the experimental model that resembled the diseases and disorders observed in PCBs-exposed population studies. The disease pathways included endocrine system disorders, genetic disorders, metabolic diseases, developmental disorders, and cancers, strongly consistent with the evidence from epidemiological studies. InterpretationThese gene finger prints could lead to the identification of populations and subgroups at high risk for disease, and can pose as early disease biomarkers well ahead of time, before the actual disease becomes visible.

In vitro exposure of human blood mononuclear cells to active vitamin D does not induce substantial change to DNA methylation on a genome-scale

It is well-established that vitamin D impacts gene regulation via vitamin D response elements (VDREs) across the genome. Recent evidence, primarily at a locus-specific level, suggests that alterations to DNA methylation may also be a relevant mechanism through which vitamin D regulates gene expression. Given the intense interest in vitamin D, particularly as an immune modifier, we sought to examine the impact of vitamin D exposure on the immune cell methylome in vitro. We exposed primary human blood mononuclear cells with up to 100nM calcitriol for up to 120h, and measured genome-scale DNA methylation response using the Illumina Infinium HumanMethylation450 beadchip array. We observed that, while the expression of known vitamin D responsive genes was clearly altered by calcitriol exposure, substantial genome-scale changes to DNA methylation were not induced. Our data suggests that, over the exposure period measured, changes to DNA methylation may not be a predominant mechanism through which vitamin D impacts gene expression in human immune cells.