Peripheral Blood Mononuclear Cell Lysates Research Articles

Immune status of people living in the Tande-Tande sub-village (Indonesia), an area with high indoor radon concentration.

On Earth, there are significant variations in terms of exposure to naturally occurring radiation among different areas. Radon, a naturally-occurring radioactive gas that is the primary cause of lung cancer in nonsmokers and the second most prevalent cause among smokers, poses a considerable risk. Indoor radon, in particular, constitutes the most substantial source of natural radiation to which individuals are exposed. This study assessed the immune status of a population chronically exposed to high indoor radon concentration in Indonesia. Fifty-seven subjects from the Tande-Tande sub-village (high indoor radon concentration area) were compared to fifty-three participants living in the Topoyo village (low concentration area). We contrasted the immunological conditions of these two populations by measuring levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-10 in serum. Moreover, we also measured levels of the nuclear factor kappa B (NF-κB), superoxide dismutase (SOD), glutathione peroxidase (GPX), and protein kinase B in its phosphorylated (pAkt) and non-phosphorylated form (Akt) in peripheral blood mononuclear cells (PBMCs) of a subset of participants (31 from each population). TNF-α, IFN-γ, and IL-4 levels in Tande-Tande sub-village inhabitants were significantly lower than those in the control group living in the Topoyo village (p = 0.001, p = 0.017, and p = 0.002). The concentration of IL-10 also tended to be lower in people living in the high indoor radon concentration area, but it did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.106). Protein levels of NF-κB, pAkt, and Akt in Tande-Tande sub-village inhabitants also did not differ significantly between Tande-Tande sub-village inhabitants and Topoyo inhabitants (p = 0.234, p = 0.210, and p = 0.657). Similarly, activities of SOD and GPX did not differ significantly between the two populations (p = 0.569 and p = 0.949). Overall, despite their chronic exposure to high indoor radon concentrations, our study revealed no increase in the levels of TNF-α, IFN-γ, IL-10, IL-4, SOD, and GPX in the inhabitants of Tande-Tande sub-village compared with people living in the Topoyo village. Furthermore, our study demonstrated no activation in the Akt pathway, as indicated by the pAkt/Akt ratio observed in PBMC lysates of individuals residing in the Tande-Tande sub-village.

Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di-and triphosphate anabolites in peripheral blood mononuclear cells

Quantitation of intracellular tri-phosphate anabolite, GSK1-TP, was required to understand drug efficacy of a proprietary molecule, GSK1; while quantitation of the di-phosphate, GSK1-DP, provided an indicator of potential GSK1-TP instability during sample processing and storage. A novel derivatization approach with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hexylamine was developed to mitigate the challenges associated with the analysis of GSK1-DP and GSK1-TP, rendering them more amendable to reversed-phase LC-MS/MS detection. Extensive effort was spent to minimize the analyte loss and cell counts variability during peripheral blood mononuclear cells (PBMC) collection and best practices were thoroughly discussed. A solution of 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid proved to be an effective method of analyte stabilization and recovery. Methods were developed for simultaneous analysis of GSK1-DP and GSK1-TP with a limit of quantitation of 2.0 ng/mL in dog and rat PBMC lysate, with a subsequent improvement to 0.1 ng/mL for the analysis of GSK1-TP in human PBMC lysate. All three assays were found to be robust over the PBMC concentration range of 1–25 × 106 lysed cells/mL. This novel methodology could alleviate some challenges associated with the bioanalysis of intracellular phosphorylated anabolites such as PBMC collection variability, analyte instability, poor chromatographic retention, high carryover, matrix effect and insufficient assay sensitivity.

Restricted Activation of the NF-κB Pathway in Individuals with Latent Tuberculosis Infection after HIF-1α Blockade.

Tuberculous granuloma formation is mediated by hypoxia-inducible factor 1 alpha (HIF-1α), and is essential for establishing latent tuberculosis infection (LTBI) and its progression to active tuberculosis (TB). Here, we investigated whether HIF-1α expression and adjacent mechanisms were associated with latent or active TB infection. Patients with active TB, individuals with LTBI, and healthy controls were recruited, and the expression of cytokine genes IL15, IL18, TNFA, IL6, HIF1A, and A20 in peripheral blood mononuclear cells (PBMCs) and serum vitamin D (25(OH)D3) levels were evaluated. Additionally, nuclear factor kappa B (NF-κB) and tumor necrosis factor-alpha (TNF-α) levels were analyzed in PBMC lysates and culture supernatants, respectively, after HIF-1α blockade with 2-methoxyestradiol. We observed that IL-15 expression was higher in individuals with LTBI than in patients with active TB, while IL-18 and TNF-α expression was similar between LTBI and TB groups. Additionally, serum 25(OH)D3 levels and expression of IL-6, HIF1A, and A20 were higher in patients with active TB than in individuals with LTBI. Moreover, PBMCs from individuals with LTBI showed decreased NF-κB phosphorylation and increased TNF-α production after HIF-1α blockade. Together, these results suggest that under hypoxic conditions, TNF-α production and NF-κB pathway downregulation are associated with the LTBI phenotype.

Interleukin-1 Beta in Peripheral Blood Mononuclear Cell Lysates as a Longitudinal Biomarker of Response to Antidepressants: A Pilot Study.

Interleukin-1 beta (IL1β) is primarily produced by monocytes in the periphery and the brain. Yet, IL1β protein levels have to date been investigated in major depressive disorder (MDD) and antidepressant response using either plasma or serum assays although with contradictory results, while mononuclear cell assays are lacking despite their extensive use in other contexts. In this pilot study, we comparatively assessed IL1β in mononuclear lysates and plasma in depressed MDD patients over treatment and healthy controls (HC). We recruited 31 consecutive adult MDD inpatients and 25 HC matched on age, sex, and BMI. Twenty-six patients completed an 8-week follow-up under treatment. IL1β was measured in both lysates and plasma in patients at baseline (T0) and at study end (T1) as well as in HC. We calculated ΔIL1β(%) for both lysates and plasma as IL1β percent changes from T0 to T1. Seventeen patients (65.4% of completers) were responders at T1 and had lower baseline BMI than non-responders (p = 0.029). Baseline IL1β from either plasma or lysates could not efficiently discriminate between depressed patients and HC, or between responders and non-responders. However, the two response groups displayed contrasting IL1β trajectories in lysates but not in plasma assays (response group by time interactions, p = 0.005 and 0.96, respectively). ΔIL1β(%) in lysates predicted response (p = 0.025, AUC = 0.81; accuracy = 84.6%) outperforming ΔIL1β(%) in plasma (p = 0.77, AUC=0.52) and was robust to adjusting for BMI. In conclusion, ΔIL1β(%) in mononuclear lysates may be a longitudinal biomarker of antidepressant response, potentially helpful in avoiding untimely switching of antidepressants, thereby warranting further investigation.

Impairment of Annexin A2-Mediated Cell Surface Fibrinolysis in Nonalcoholic Steatohepatitis Cirrhosis with Portal Vein Thrombosis

The annexin A2 (A2) complex assembles tissue plasminogen activator and plasminogen on cell surfaces, resulting in efficient plasmin generation, and is markedly reduced in some patients with thrombophilia. Nonalcoholic steatosis (NASH) cirrhosis is the leading cause of cirrhosis in developed countries, and patients with NASH cirrhosis are at increased risk of portal vein thrombosis (PVT). However, the exact mechanism for PVT in NASH cirrhosis is poorly understood. Our objective was to determine the potential contribution of A2 to NASH thrombogenesis.Using semiquantitative immunoblot analysis, we examined A2 expression in peripheral blood mononuclear cell (PBMC) lysates from obese controls and NASH subjects with or without cirrhosis, and in human umbilical vein endothelial cells (HUVECs) treated with platelet-poor plasma (PPP) from patients with varying degrees of NASH cirrhosis. We also analyzed formalin-fixed liver tissue from patients with or without steatosis or NASH cirrhosis for A2 expression by immunofluorescence. Finally, we evaluated A2-dependent PBMC surface plasmin generation using a fluorogenic assay, and systemic fluid-phase fibrinolysis via plasmin-anti-antiplasmin (PAP) complex and D-dimer ELISAs.Total A2 expression in PBMC lysates did not differ among subjects with a range of severity of NASH cirrhosis versus obese controls. Src kinase-dependent tyrosine phosphorylation of A2, which is critical for its translocation to the cell surface, decreased in HUVECs after co-culture with NASH Child-Turcotte-Pugh C PPP despite no change in total A2. Immunofluorescence of liver tissue from patients with NASH cirrhosis who developed PVT revealed an 85% decrease in microvascular A2 expression compared to NASH cirrhosis patients without PVT. Similarly, PBMC surface plasmin generation decreased as NASH severity worsened, even though d-dimer and PAP complex level increased with worsening disease severity, indicating more activated systemic fibrinolysis in decompensated NASH cirrhosis.Despite preservation of total A2 and acceleration of systemic fibrinolysis in NASH, A2-mediated cell surface fibrinolysis decreased as NASH cirrhosis worsened. We show for the first time that impaired A2-related fibrinolysis may reflect inhibition of A2 phosphorylation by NASH plasma, and that reduction of A2 expression in the hepatic microvasculature may contribute to PVT in these subjects. DisclosuresDesancho: Sanofi-Genzyme: Membership on an entity's Board of Directors or advisory committees.

DSB Repair Status Predicts Radiation-Induced Severe Side-Effects in Breast and Prostate Cancers Before the Beginning of Treatment

<h3>Purpose/Objective(s)</h3> Late adverse events following radiotherapy (RT) are tissue complications present in 5 to 10% of RT patients. Deficiencies in DSB (double strand break) repair are associated with a large number of diseases including immunodeficiency and susceptibility to radiation-induced adverse effects. To quantify the patients DSB repair capacities can predict individual radiosensitivity and discriminate patients at high risk of developing radiotoxicity in order to improve their therapeutic management. <h3>Materials/Methods</h3> A functional fluorescent multiplexed DSB repair assay, NEXT-SPOT, was used to profile simultaneously four DSB repair pathways (HR, NHEJ, SSA and alt-EJ), in peripheral blood mononuclear cells (PBMC) lysates, from a cohort of patients undergoing RT. Patients with either non-metastatic prostate or breast cancers were enrolled and patients' characteristics (sex, age, BMI), lifestyle habits (smoking and drinking), and concomitant treatments were collected. Radiation-induced adverse events were recorded for at least 6 months after the beginning of RT. The balance between the DSB repair mechanisms in the patients' PBMC collected before and early during the treatment course was assessed. Impact of different parameters (radiation dose, received treatments, life style) on the DSB repair capacities was evaluated, and the ability of the test to predict radiation-induced adverse effects was determined using Random Forest and Boruta algorithms. <h3>Results</h3> The DSB repair profile was established before and after few fractions of RT for 149 and 50 patients with breast and prostate cancer, respectively. Radiation-induced cutaneous and gastro-intestinal/genitourinary adverse events were considered, in breast and prostate cancer patients, respectively. The DSB repair activities were compared between patients with and without grade ≥3 adverse events, occurring > 6 months after the beginning of RT. Significant differences were detected on the blood sample taken before the beginning of the treatment. HR and NHEJ were significantly upregulated in patients who developed late grade ≥3 adverse events (<i>P</i> = 0.0005 and 0.0054 respectively). Conversely, the relative contributions of SSA and alt-EJ were down-regulated in these patients (<i>P</i> = 0.0022 and 0.0034, respectively). The best biomarker combination defined with the Random Forest and Boruta algorithms predicted the appearance of severe late adverse events with 100% sensibility and 77% specificity using the blood sample taken before RT. <h3>Conclusion</h3> With this clinical proof-of-concept we identified blood radiotoxicity biomarkers based on the comprehensive analysis of DSB repair activities. This assay enabled to detect simultaneously defects in all DSB repair pathways and identified patients with an increased risk of late severe radiotoxicity following radiotherapy. These promising results require confirmation on a larger cohort.

Development and validation of assays for the quantification of β-D-N4-hydroxycytidine in human plasma and β-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates

The novel antiviral prodrug molnupiravir is under evaluation for the treatment of SARS-CoV-2. Molnupiravir is converted to β-D-N4-hydroxycytidine (NHC), which is the primary form found in systemic circulation. β-D-N4-hydroxycytidine-triphosphate (NHCtp) is the bioactive anabolite produced intracellularly. Sensitive and accurate bioanalytical methods are required to characterize NHC and NHCtp pharmacokinetics in clinical trials. Human K2EDTA plasma or peripheral blood mononuclear cell (PBMC) lysates were spiked with NHC (plasma) or NHCtp (PBMC), respectively. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or lysate dilution, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations. NHC can be quantified in plasma with a lower limit of quantification (LLOQ) of 1 ng/mL; the primary linearity of the assay is 1–5000 ng/mL. Assay precision and accuracy were ≤ 6.40% and ≤ ± 6.37%, respectively. NHC is unstable in whole blood and has limited stability in plasma at room temperature. The calibration range for NHCtp in PBMC lysates is 1–1500 pmol/sample, and the assay has an LLOQ of 1 pmol/sample. Assay precision and accuracy were ≤ 11.8% and ≤± 11.2%. Ion suppression was observed for both analytes; isotopically-labeled internal standards showed comparable ion suppression, resulting in negligible (<5%) relative matrix effects. Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the quantification of NHC in plasma and NHCtp in PBMC lysates. The described methods are appropriate for use in clinical trials.

PACSIN2 rs2413739 Polymorphism and Thiopurine Pharmacokinetics: Validation Studies in Pediatric Patients

Thiopurines such as thioguanine (TG), mercaptopurine (MP) and its prodrug azathioprine (AZA) are key antileukemic and immunosuppressive drugs in the treatment of several pediatric diseases such as acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD): their therapeutic action is affected by the enzyme thiopurine-S-methyltransferase (TPMT) whose encoding gene is polymorphic and whose enzymatic activity is also trans -regulated by the intronic single nucleotide polymorphism (SNP) rs2413739 (T>C) in the PACSIN2 gene.This study was aimed at validating the impact of PACSIN2 rs2413739 on thiopurine pharmacokinetic and pharmacodynamic parameters in two Italian pediatric cohorts: the first cohort was multicentric, consisted of 297 pediatric patients newly diagnosed with ALL (median (IQ) age: 4.8 (3.0-9.2) years; male: 54.9%) treated according to the AIEOP-BFM ALL 2009 protocol; the second consisted of 119 pediatric IBD patients (median age (interquartile range (IQ)): 15.1 (12.3-16.9) years; male: 52.1%) treated continuously with daily weight-based AZA therapy for at least three months before enrollment at the Gastroenterology Unit of the Pediatric Hospital IRCCS “Burlo Garofolo” in Trieste. Moreover, the role of rs2413739 polymorphism on TPMT and PACSIN2 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) of adult healthy donors.The presence of the PACSIN2 rs2413739 T allele significantly reduced the TPMT activity only in the ALL cohort (p=0.026, linear mixed-effect model, multivariate analysis considering the TPMT SNP rs1142345 and rs1800460), without affecting MP metabolites levels in patients' erythrocytes: the PACSIN2 rs2413739 genetic effect on TPMT activity was particularly evident within TPMT heterozygotes (linear mixed-effect model, p=0.041). Interestingly, TPMT enzymatic function was significantly higher in ALL patients undergoing a long-term thiopurine-based therapy compared to IBD patients (p=0.032, linear mixed-effect model). In IBD patients, treatment response to AZA was significantly affected by PACSIN2 genotype: AZA effectiveness was reduced in patients carrying the T allele of the SNP rs2413739 (linear mixed effects, p=0.018; genotyping results: CC in 19, CT in 29 and TT in 20 patients): indeed, a clinically active disease was observed in ~40% of TT patients, in 30% of those with the CT genotype and in 20% of the CC subjects. For ALL patients, clinical data are still under collection and are not available at the time of preparation of the abstract.To evaluate the role of PACSIN2 SNP rs2413739 on TPMT and PACSIN2 protein expression, 20 adult healthy donors were genotyped for the SNP of interest, and Western-blot analysis against human TPMT and human PACSIN2 were performed on their PBMCs lysates. No significant contribution of the polymorphism was found on TPMT and PACSIN2 protein expression in this small population. In contrast, a significant direct correlation between the TPMT and PACSIN2 levels was observed (linear mixed effect, p = 0.045), suggesting a putative coordination in their protein expression in PBMCs.These results support the role of PACSIN2 polymorphism on TPMT activity, although this effect may be tissue-specific or dependent on the patients' clinical condition. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Inflammasome Activation in Peripheral Blood Mononuclear Cells of Hemodialysis Treated Patients with Glomerulonephritis.

Recently, it has been found that abnormal activation of inflammasomes, the intracellular multiprotein complexes, plays an important role in the pathogenesis and the development of inflammatory diseases. To determine whether the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is involved in chronic inflammatory condition reported in glomerulonephritic- hemodialysis (HD) patients, we investigated the mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18, NLRC4, and P2X7 in human peripheral blood mononuclear cells (PBMCs) collected from 28 glomerulonephritic-HD patients. To confirm the mRNA quantification results, we investigated the IL-1ß content and Caspase 1 activity in serum and PBMC lysates, respectively. Compared with PBMCs derived from healthy subjects, genes encoding proinflammatory cytokines such as IL-1β and IL-18 as well as NLRP3, ASC, CASP-1 were markedly overexpressed in those derived from patients. Moreover, there was no significant difference between the expression level of P2X 7 mRNA in PBMCs isolated from glomerulonephritis-HD patients and controls. The serum level of active IL1-β and cell lysate CASP-1 activity were up-regulated in patients compared to controls. We also revealed that PBMCs isolated from glomerulonephritis-HD patients had elevated mRNA levels of NLRC4 compared to controls, suggesting the priming of NLRC4 inflammasome. These results revealed that the NLRP3-ASC-caspase-1 axis might have a role in increased inflammation severity reported in glomerulonephritic patients undergoing hemodialysis. These findings provide new insights into molecular mechanisms underlying chronic inflammation in HD- glomerulonephritic patients. Additionally, the NLRP3 inflammasome pathway can be attractive as a potential therapeutic target for complication avoidance in HD- glomerulonephritic patients.

Aptamer-based approaches to detect nucleolin in prostate cancer

We have investigated the expression of nucleolin (NCL) in liquid biopsies of prostate cancer (PCa) patients and healthy controls to determine its correlation with tumor prognosis. To detect NCL we used a modified AS1411 aptamer designated by AS1411-N5. In presence of NCL, AS1411-N5 increases the fluorescence by assuming a G-quadruplex (G4) structure, while in the absence of NCL the fluorescence signal remains quenched. The structural characterization of AS1411-N5 was performed by biophysical studies, which demonstrated the formation of G4 parallel conformation in the presence of 100 mM K+ and the ability to recognize NCL with high affinity (KD = 138.1 ± 5.5 nM). Furthermore, the clinical relevance of NCL in PCa liquid biopsies was assessed by using an NCL-based ELISA assay. The protein was measured in the peripheral blood mononuclear cells (PBMCs) cell lysate of 158 individuals, including PCa patients and healthy individuals. The results depicted a remarkable increase of NCL levels in the PBMC's lysate of PCa patients (mean of 626.1 pg/mL whole blood) when compared to healthy individuals (mean of 198.5 pg/mL whole blood). The ELISA results also provided evidence for the usefulness of determining NCL levels in advanced PCa stages. Furthermore, a microfluidic assay showed the ability of AS1411-N5 in recognizing NCL in spiked human plasma samples.

Blood-based kinase activity profiling: a potential predictor of response to immune checkpoint inhibition in metastatic cancer

BackgroundMany cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may...

Granzyme B in peripheral blood mononuclear cells as a measure of cell-mediated immune response in paraneoplastic neurological syndromes and malignancy

BackgroundParaneoplastic neurological syndromes (PNS) may coexist with ovarian or lung cancers. Some tumors coexisting with PNS are smaller and have a better prognosis than tumors without PNS. PNS may constitute an opportunity to observe a natural immune antitumor response. We aimed to investigate a cytotoxic immune response by measuring granzyme B (GrB) in peripheral blood mononuclear cells (PBMC) in patients affected with ovarian or lung malignancy, with and without accompanying PNS.MethodsWe enrolled patients with: nonmalignant lesions (n = 21), ovarian cancer (n = 19), lung cancer (n = 57), and PNS (n = 30). PBMC were isolated by density gradient centrifugation with Ficoll–Paque. We evaluated the expression of GrB in PBMC lysates by ELISA and normalized to protein content as measured by the Lowry method.ResultsGrB levels in PBMC in the group with malignant tumors—median 1650 pg/mg protein (interquartile range 663–3260 pg/mg) and in patients with PNS—median 1890 pg/mg protein (range 1290–2640 pg/mg) was lower than in control group with nonmalignant lesions—median 5240 pg/mg protein (range 2160–7440 pg/mg), p = 0.0003 and p = 0.0038, respectively. The differences in GrB levels in PBMC between these groups were independent of epidemiological factors—age, sex, body mass index (BMI), and the number of immune cells, as confirmed by multiple regression analysis. Within the group of patients with malignancy and PNS, GrB levels in PBMC were elevated if onconeural antibodies were detected (2610; 2390–3700 pg/mg protein) as compared to patients without antibodies (1680; 970–1880 pg/mg protein, p = 0.035). GrB in PBMC was higher if the malignancy was diagnosed at the low (3060; 2120–5220 pg/mg protein) as compared to the high stage (1330; 348–2140, p = 0.00048). In patients with lung cancer, the expression of GrB in PBMC was lower (1430; 635–2660 pg/mg protein) than in the group with ovarian cancer (2580; 1730–3730, p = 0.02).ConclusionThe cytotoxic response measured in peripheral blood by GrB in PBMC is impaired both in the course of malignancy and PNS. Levels of GrB in PBMC were higher if onconeural antibodies were detected. Tracking reactive immune responses, such as GrB in PBMC may have diagnostic and monitoring value in malignancy and PNS.

An increase in p62/NBR1 levels in melioidosis patients of Sri Lanka exhibit a characteristic of potential host biomarker.

Introduction Melioidosis, caused by Burkholderia pseudomallei , in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei , host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.

Early detection of Ebola virus proteins in peripheral blood mononuclear cells from infected mice

BackgroundDetection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48–72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins.MethodsA mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs.ResultsUsing quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point.ConclusionsWhile mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.

Uric acid increases IL-1β secretion and Caspase-1 activation in PBMCs of Behçet’s disease patients: The in vitro immunomodulatory effect of xanthine oxidase inhibitor Allopurinol

Behçet’s disease (BD) is a multisystem disease, which shares some features with other diseases belonging to the autoinflammatory disorders panel. Recent studies have postulated that IL-1β/Caspase-1 may play a cardinal role in autoinflammatory diseases. In this study, we aimed to (i) elucidate the mechanism underlying the involvement of xanthine oxidase (XO) and Uric Acid (UA) in BD (ii) study the direct effects of UA and XO inhibitor “Allopurinol” on nitric oxide (NO) and caspase-1-mediated IL-1β release in peripheral blood mononuclear cells (PBMCs) of BD patients. In this context, plasma of BD patients and healthy controls (HC) were used to measure XO activity, UA, advanced oxidized proteins products (AOPP) and NO levels. In Addition, PBMCs of BD patients and HC were treated or not with either UA or Allopurinol. Then we quantified NO and IL-1β levels, and Caspase-1 Activity in the supernatants and lysates of PBMCs, respectively. We showed that plasma levels of XO activity, UA, AOPP and NO are significantly increased in BD patients compared to those of HC. Interestingly, a significant positive correlation between XO and UA was observed in BD patients. Additionally, while UA has markedly increased NO, IL-1β, and Caspase-1 activity levels in PBMCs of BD patients, Allopurinol has exerted an immunomodulatory effect resulting in reduced NO, IL-1β and Caspase-1 levels in PBMCs of BD patients particularly during the active stages. Collectively, our results indicate a potential clinical use of XO as a tool for assessing BD activity, and suggest that the in-vitro immunomodulatory effect of Allopurinol may have a promising therapeutic value in BD management.

Markedly Reduced Expression of Annexin A2 Is Associated with Hypofibrinolysis and Provoked and Unprovoked Venous Thromboembolism

Introduction: Recent evidence indicates that plasma hypofibrinolysis, as measured by clot lysis times (CLT) at or above the 90th percentile, can double the risk of venous thromboembolism (VTE). To our knowledge, there are no studies on the role of cell surface fibrinolysis in thrombosis. Annexin A2 (A2) is a calcium dependent, phospholipid binding protein that forms a heterotetramer with S100A10 (A2-S100A10)2 on the surface of endothelial cells (ECs). This complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), and increases the catalytic efficiency of tPA-dependent cell surface plasmin generation by 60-fold. In mouse studies, global knockout of the annexin A2 gene (Anxa2) is associated with fibrin accumulation and impaired clearance of arterial thrombi. In addition, there are several examples of the regulatory role of A2 in fibrinolysis in human diseases such as antiphospholipid antibody syndrome, cerebral venous thrombosis, and sickle cell disease. In the current study, we aimed to explore the potential role of the A2 system and cell surface based fibrinolysis in the development of VTE. Methods: Study subjects included patients 18-65 years old with history of VTE and healthy controls. Subjects were classified as having provoked or unprovoked VTE based on the presence or absence of identifiable environmental risk factors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected in EDTA tubes, and used as a surrogate for ECs. Assays performed on PBMCs included determination of the rate of tPA-dependent cell surface plasmin generation using a fluorogenic substrate and analysis of CLT in the presence of phospholipid vesicles. Because A2 accounts for approximately 50% of cell surface based plasmin generation on both ECs and PBMCs, we analyzed total A2 protein expression relative to GAPDH in whole cell lysates of PBMCs using semi-quantitative western blotting. Additional assays included quantitative RT-PCR and ANXA2 gene sequencing. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results: Overall, 116 subjects with VTE (76 with provoked and 40 with unprovoked VTE) and 87 healthy controls were studied between September 2010 and May 2019. Plasma based clot lysis assays revealed that the mean CLT for subjects with VTE was significantly longer than that for healthy controls (37.5 versus 30.7 minutes, p=0.0001). Additionally, the mean rate of cell surface plasmin generation was significantly reduced in subjects with VTE as compared with healthy controls (0.33 RFU/min2 versus 0.49 RFU/min2, p=0.0021). Moreover, none of the 60 healthy controls (0%), but 18 of the 107 subjects with thrombosis (17%) had cell surface plasmin generating capacity in the lowest 10th percentile. In protein expression studies, we observed that none of the 21 healthy controls (0%), but 5 of 41 subjects with thrombosis (12%) had A2 expression in the lowest 10th percentile for the group; 4 of 18 (22%) of those with unprovoked VTE and 1 of 23 (4%) of those with provoked VTE fell into the lowest decile for protein expression. For plasmin generating capacity, 8 of 36 (22%) of subjects with unprovoked VTE and 10 of 71 (14%) of those with provoked VTE occupied the lowest decile. Probing of western blots of samples obtained on two separate occasions several months apart with A2 epitope-specific antibodies revealed abnormalities within either the tPA binding N-terminal tail or C-terminal core domain in proximity to the plasminogen binding site. Neither quantitative RT-PCR nor ANXA2 gene sequencing of selected samples revealed abnormalities in either mRNA or genomic DNA that could explain the reduced A2 expression. Conclusion: These data confirm findings previously reported by Lisman that plasma hypofibrinolysis is associated with VTE and may represent an independent risk factor for VTE. Additionally, we demonstrate for the first time that impaired cell surface based fibrinolysis and aberrations in A2 protein expression are associated with both provoked and unprovoked VTE, and may represent a novel risk factor for thrombosis. Possible explanations for reduced A2 expression include dysfunctional translation of mRNA into protein or post-translational proteolysis of the translated protein. Compromise of the A2-based fibrinolytic system may represent a previously unrecognized contributor to thrombophilia in VTE. Disclosures Ruisi: BMY: Equity Ownership; EMD, subsidiary of Merck KGaA: Employment. De Sancho:Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Chemical proteomics reveals target selectivity of clinical Jak inhibitors in human primary cells

Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.

Validation of monoclonal anti-PKC isozyme antibodies for flow cytometry analyses in human T cell subsets and expression in cord blood T cells

T cells from neonates (cord blood) with a tendency to develop allergic diseases express low PKCζ levels. More extensive investigations into PKC isozyme levels in T cell subsets and changes during neonatal T cell maturation are hampered by limitations of Western blot analyses. We have undertaken to validating the specificity of commercially available antibodies marketed for flow cytometry to measure PKCα, βI, βII, δ, ε, η, θ, ζ, ι/λ and μ. Western blot analyses of human peripheral blood mononuclear cell (PBMC) lysates demonstrated that some antibodies were unsuitable for flow cytometry assays. A panel of antibodies with the desirable specificity and reliability in the flow cytometry assay were identified using both PBMC and whole blood assays. The results showed that all PKC isozymes were expressed in CD4+ and CD8+ T cells, monocytes and neutrophils. Murine lymphocytes showed similar patterns of expression. A major finding was that 35.2% and 38.5% of cord blood samples have low PKCζ (≤the 5th percentile of adult levels) in the CD4+ and CD8+ subsets, respectively, consistent with the incidence of allergy development in the population. Furthermore, these low PKCζ levels ‘normalised’ within 24 h after initiation of maturation of these cells in culture, providing a ‘window of opportunity’ for altering PKCζ levels.

Identification of autophagy markers in clinical cases of sepsis

Abstract Apoptotic cell death plays a key role in sepsis-induced immunosuppression and organ dysfunction. Autophagy may serve as a compensatory protective mechanism in sepsis owing to its potential role in reversing cell death-induced immunosuppression. The goal of our study was to investigate for the presence of autophagy markers in septic patients. Our preliminary study design included patients diagnosed with, and treated for, sepsis in clinical settings (N =13). Healthy volunteers served as control subjects (N = 10). Markers of autophagy were assessed in serum, peripheral blood mononuclear cells (PBMCs) and PBMC lysates derived from patients and healthy controls. Samples were analyzed by ELISA and flow cytometry methods. Data and statistical analyses were performed using GraphPad Prism v7.0. Results from flow cytometry analysis showed significantly elevated levels of autophagy markers in PBMCs derived from septic patients compared to that observed in PBMCs from control subjects (p &amp;lt; 0.01 from unpaired t-test analysis). Results from ELISA experiments showed significantly lower levels of the autophagy marker in patient serum samples when compared to that seen in control serum samples (p &amp;lt; 0.01 from unpaired t-test analysis). ELISA analysis showed a concomitant increase in the same autophagy marker in PBMC lysates obtained from patients with sepsis compared to that observed in PBMC lysates from control subjects. Preliminary results serve as a basis for us to investigate the role of autophagy in the prediction of clinical outcomes in sepsis.

Resveratrol ameliorates gouty inflammation via upregulation of sirtuin 1 to promote autophagy in gout patients.

Resveratrol exerts an anti-inflammatory effect on collagen-induced arthritis and osteoarthritis in rats via activation of sirtuin 1 (SIRT1). Autophagy can be induced by resveratrol and leads to amelioration of interleukin-1 beta (IL-1β) release in vitro. We aimed to determine the anti-inflammatory mechanisms of resveratrol in patients with gout. Blood samples were obtained from patients with acute gout, intercritical gout (IG) and healthy controls (HC). The mRNA and protein levels of SIRT1 and nuclear factor-kappa B (NF-kB) p65 were determined in peripheral blood mononuclear cells (PBMCs) lysate from these patients. In the in vitro experiment, SIRT1, autophagy-related genes (beclin-1 and microtubule-associated protein 1 light-chain 3) and key genes involved in the gouty inflammatory pathway, including NF-κB p65, NLR family pyrin domain containing 3 (NLRP3), caspase-1 and IL-1β, were determined in PBMCs lysate or plasma from IG patients exposed to monosodium urate (MSU) crystals with or without resveratrol. The mRNA and protein levels of SIRT1 were downregulated in PBMCs from gout patients in comparison with HC. In the in vitro experiment, the protein levels of SIRT1 were downregulated in PBMCs from IG patients exposed to MSU crystals and were restored by resveratrol in a dose-dependent manner. Furthermore, high doses of resveratrol ameliorated the release of the inflammatory cytokine IL-1β. In addition, the mRNA levels of NLRP3 and NF-κB p65 were regulated by resveratrol, but caspase-1 and IL-1β were not. Furthermore, resveratrol promoted MSU-induced autophagy in PBMCs from patients with gout. These findings suggest that resveratrol ameliorates gouty inflammation via upregulation of SIRT1 to promote autophagy in patients with gout.