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The effects of glutamine supplementation on markers of apoptosis and autophagy in sickle cell disease peripheral blood mononuclear cells.
Walter, PB, Hohman, LS, Rokeby, A, Lum, JJ, Hagar, R, Lavrisha, L, Saulys, A, Kuypers, FA, Vichinsky, E, Morris, CR
Complementary therapies in medicine. 2022;:102856
Abstract
OBJECTIVES L-Glutamine was FDA-approved for sickle cell disease (SCD) in 2017, yet the mechanism(s)-of-action are poorly understood. This study investigates the potential activation of autophagy as a previously unexplored mechanism-of-benefit. DESIGN Prospective, open-label, 8-week, phase-2 trial of oral L-glutamine (10 g TID) in patients with SCD at risk for pulmonary hypertension identified by Doppler-echocardiography by an elevated tricuspid-regurgitant-jet-velocity (TRV)≥ 2.5 m/s. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD patients at baseline, two, four, six and eight weeks of glutamine therapy, and from controls at baseline; BAX (pro-apoptotic marker) and LC3-II/LC3-I (autophagy marker) were measured via western blot analysis to assess apoptosis and autophagy respectively. SETTING Comprehensive SCD Center in Oakland, California. RESULTS Patients with SCD (n = 8) had a mean age of 44 ± 16, 50% were male; 63% Hb-SS, and mean TRV= 3.1 ± 0.7 m/s. Controls' mean age (n = 5) was 32 ± 12% and 57% were male; all were Hb-AA with a mean TRV= 1.8 ± 0.6. At baseline, SCD-PBMCs had 2-times higher levels of BAX and LC3-I versus controls (both p = 0.03). Levels of BAX expression increased by 300% after 8-weeks of glutamine supplementation (p = 0.005); LC3-I protein levels decreased while LC3-II levels increased by 70%, giving a significant increase in the LC3-II/LC3-I ratio (p = 0.02). CONCLUSION PBMCs from glutamine-supplemented SCD patients have upregulated apoptotic and autophagy proteins. The parallel increase in BAX and the LC3-II / LC3-I ratio with glutamine supplementation suggest a possible role of autophagic cell death. The increase in apoptotic markers provide insight into a possible mechanism used by peripheral PBMCs during glutamine supplementation in patients with SCD.
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Immunomonitoring of Tacrolimus in Healthy Volunteers: The First Step from PK- to PD-Based Therapeutic Drug Monitoring?
In 't Veld, AE, Grievink, HW, Saghari, M, Stuurman, FE, de Kam, ML, de Vries, APJ, de Winter, BCM, Burggraaf, J, Cohen, AF, Moerland, M
International journal of molecular sciences. 2019;(19)
Abstract
Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless, toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose, tacrolimus concentration, and its effect on the target cell, we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus, after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71, p = 0.009), while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35, p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFNγ, as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose, when maximum tacrolimus levels were observed. Moreover, the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion, our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations, and that PHA-induced cytokine production (IL-2 and IFNγ) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell.
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Effect of Infla-Kine supplementation on the gene expression of inflammatory markers in peripheral mononuclear cells and on C-reactive protein in blood.
Mikirova, NA, Kesari, S, Ichim, TE, Riordan, NH
Journal of translational medicine. 2017;(1):213
Abstract
BACKGROUND Chronic inflammation is a predisposing factor to numerous degenerative diseases including cancer, heart failure and Alzheimer's disease. Infla-Kine is a natural supplement comprised of a proprietary blend of Lactobacillus fermentum extract, burdock seed (arctigenin), zinc, alpha lipoic acid, papaya enzyme and an enhanced absorption bio-curcumin complex (BCM-95®). METHODS Infla-Kine was administered twice daily to 24 health volunteers for 4 weeks. Quantitative RT-PCR was used to assess mRNA transcripts of IL-1b, IL8, IL-6, NF-κB, and TNF-α from peripheral blood mononuclear cells (PBMC). C reactive protein (CRP) was measured from serum. Additionally, quality of life questionnaires were employed to assess general feeling of well-being. Assessments were made before treatment and at conclusion of treatment (4 weeks). RESULTS As compared to pre-treatment, after 4 weeks, a statistically significant reduction of IL8, IL-6, NF-κB, and TNF-α transcripts was observed in PBMC. Furthermore, reduction of IL-1b transcript and serum CRP was observed but did not reach statistical significance. Quality of life improvements were most prevalent in muscle and joint pains. CONCLUSIONS Overall, our data demonstrate that twice daily administration of Infla-Kine for 4 weeks reduces inflammatory markers and quality of life in healthy volunteers.
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Improvement in asymmetric dimethylarginine and oxidative stress in patients with limb salvage after autologous mononuclear stem cell application for critical limb ischemia.
Madaric, J, Valachovicova, M, Paulis, L, Pribojova, J, Mateova, R, Sebekova, K, Postulkova, L, Madaricova, T, Bucova, M, Mistrik, M, et al
Stem cell research & therapy. 2017;(1):165
Abstract
BACKGROUND Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, acts as an inhibitor of angiogenesis and is associated with an increased risk of cardiovascular mortality. Administration of stem cells may affect endogenous mechanisms that regulate ADMA production and metabolism. The aim of the present study was to analyze ADMA concentration and changes in oxidative stress in patients with advanced critical limb ischemia (CLI) after bone marrow-derived mononuclear cell (BM-MNC) therapy. METHODS Fifty patients (age 64 ± 11 years, 44 males, 6 females) with advanced CLI (Rutherford category 5 or 6) not eligible for revascularization were treated by intramuscular (n = 25) or intra-arterial (n = 25) injection of 40 ml BM-MNC concentrate. Patients with limb salvage and improved wound healing after 6 months were considered responders to cell therapy. The concentrations of markers of oxidative stress and angiogenesis were analyzed before, and at 3 and 6 months after BM-MNC delivery. RESULTS At 6-month follow-up, four patients died of reasons unrelated to stem cell therapy. Among the survivors, 80% (37/46) showed limb salvage and improved wound healing. At 6 months follow-up, ADMA concentration significantly decreased in patients with limb salvage (1.74 ± 0.66 to 0.90 ± 0.49 μmol/L, p < 0.001), in parallel with decreased tumor necrosis factor (TNF)-α (2.22 ± 0.16 to 1.94 ± 0.38 pg/ml, p < 0.001), and increased reduced glutathione (6.96 ± 3.1 to 8.67 ± 4.2 μmol/L, p = 0.02), superoxide dismutase activity (168 ± 50 to 218 ± 37 U/L, p = 0.002), and coenzyme Q10 concentration (468 ± 182 to 598 ± 283 μg/L, p = 0.02). The number of delivered BM-MNCs significantly correlated with the decrease in ADMA concentration at 3 months (p = 0.004, r = -0.48) and the decrease in TNF-α concentration at 6 months (p = 0.03, r = -0.44) after cell delivery. ADMA or TNF-α improvement did not correlate with the number of applied CD34+ cells, C-reactive protein concentration, leukocyte count, or the dose of atorvastatin. CONCLUSIONS The therapeutic benefit of BM-MNC therapy is associated with reduced ADMA levels and oxidative stress. Regulation of the ADMA-nitric oxide axis and improved antioxidant status may be involved in the beneficial effects of stem cell therapy. TRIAL REGISTRATION The study was approved and retrospectively registered by ISRCTN registry, ISRCTN16096154 . Registered on 26 July 2016.
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High insulin and leptin increase resistin and inflammatory cytokine production from human mononuclear cells.
Tsiotra, PC, Boutati, E, Dimitriadis, G, Raptis, SA
BioMed research international. 2013;:487081
Abstract
Resistin and the proinflammatory cytokines, such as TNF- α , IL-6, and IL-1 β , produced by adipocytes, and macrophages, are considered to be important modulators of chronic inflammation contributing to the development of obesity and atherosclerosis. Human monocyte-enriched mononuclear cells, from ten healthy individuals, were exposed to high concentrations of insulin, leptin, and glucose (alone or in combination) for 24 hours in vitro. Resistin, TNF- α , IL-6, and IL-1 β production was examined and compared to that in untreated cells. High insulin and leptin concentrations significantly upregulated resistin and the cytokines. The subsequent addition of high glucose significantly upregulated resistin and TNF- α mRNA and protein secretion, while it did not have any effect on IL-6 or IL-1 β production. By comparison, exposure to dexamethasone reduced TNF- α , IL-6, and IL-1 β production, while at this time point it increased resistin protein secretion. These data suggest that the expression of resistin, TNF- α , IL-6, and IL-1 β from human mononuclear cells, might be enhanced by the hyperinsulinemia and hyperleptinemia and possibly by the hyperglycemia in metabolic diseases as obesity, type 2 diabetes, and atherosclerosis. Therefore, the above increased production may contribute to detrimental effects of their increased adipocyte-derived circulating levels on systemic inflammation, insulin sensitivity, and endothelial function of these patients.
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A short-term diet and exercise intervention ameliorates inflammation and markers of metabolic health in overweight/obese children.
Izadpanah, A, Barnard, RJ, Almeda, AJ, Baldwin, GC, Bridges, SA, Shellman, ER, Burant, CF, Roberts, CK
American journal of physiology. Endocrinology and metabolism. 2012;(4):E542-50
Abstract
The present study was designed to examine the effects of short-term diet and exercise on markers of metabolic health, serum-stimulated production of inflammatory biomarkers from cultured monocytes and adipocytes, and serum lipomics. Twenty-one overweight/obese children (9 boys and 12 girls, age 13.0 ± 0.5 yr, BMI 33.0 ± 1.8 kg/m(2)) were placed on a 2-wk ad libitum, high-fiber, low-fat diet and daily exercise regimen. Fasting serum samples were taken pre- and postintervention for determination of cytokines, metabolic risk markers, and lipomics. Monocytes and adipocytes were incubated with pre- and postintervention serum to investigate changes in cytokine secretion. Correlative associations were calculated, followed by hierarchical clustering to determine relationships between fatty acid (FA) species and clinical biomarkers. Despite remaining overweight/obese, interleukin (IL)-6, IL-8, TNFα, PAI-1, resistin, amylin, leptin, insulin, and IL-1ra decreased and adiponectin increased. Culture studies indicated decreases in monocyte secretion of IL-6, TNFα, and IL-1β and adipocyte secretion of IL-6. Lipomic analysis revealed a decrease in total lipids and decreases in saturated FAs and an increase in 18:1/18:0. In general, Pearson's correlations revealed that inflammatory markers are negatively associated with a cluster of polyunsaturated FAs and positively correlated with several saturated FAs. These results indicate significant modification of multiple indices of metabolic health with short-term rigorous lifestyle modification in overweight/obese children prior to obesity reversal.
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Increased proinflammatory and oxidant gene expression in circulating mononuclear cells in older adults: amelioration by habitual exercise.
Gano, LB, Donato, AJ, Pierce, GL, Pasha, HM, Magerko, KA, Roeca, C, Seals, DR
Physiological genomics. 2011;(14):895-902
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Abstract
We tested the hypothesis that peripheral blood mononuclear cells (PBMC) of older adults demonstrate a proinflammatory/-oxidative gene expression profile that can be improved by regular aerobic exercise. PBMC were isolated from young (n = 25, 18-33 yr) and middle-aged/older (n = 40, 50-76 yr) healthy adults. The older adults had greater mRNA expression (real-time RT-PCR) of the proinflammatory/-oxidant transcription factor nuclear factor-κB (1.58-fold, P < 0.05) and receptor for advanced glycation end products (1.12-fold, P < 0.05), the proinflammatory cytokines tumor necrosis factor-α (1.90-fold, P < 0.05) and monocyte chemoattractant protein-1 (1.47-fold, P < 0.05), and the oxidant-producing enzymes nicotinamide adenine dinucleotide phosphate-oxidase (0.91-fold, P < 0.05) and inducible nitric oxide synthase (2.60-fold, P < 0.05). In 11 subjects (58-70 yr), maximal oxygen consumption (+11%) and exercise time (+19%) were increased (both P < 0.001), and expression of the above proinflammatory/-oxidative genes was or tended to be decreased in PBMC after vs. before 2 mo of aerobic exercise (brisk walking ∼6 days/wk, 50 min/day, 70% of maximal heart rate). Expression of interleukin-6 was not different with age or exercise intervention. Age group- and exercise intervention-related differences in gene expression were independent of other factors. PBMC of healthy older adults demonstrate increased expression of several genes associated with inflammation and oxidative stress, which is largely ameliorated by habitual aerobic exercise. This proinflammatory/-oxidative gene signature may represent a therapeutic target for lifestyle and pharmacological prevention and treatment strategies.
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Effects of increasing docosahexaenoic acid intake in human healthy volunteers on lymphocyte activation and monocyte apoptosis.
Mebarek, S, Ermak, N, Benzaria, A, Vicca, S, Dubois, M, Némoz, G, Laville, M, Lacour, B, Véricel, E, Lagarde, M, et al
The British journal of nutrition. 2009;(6):852-8
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Dietary intake of long-chain n-3 PUFA has been reported to decrease several markers of lymphocyte activation and modulate monocyte susceptibility to apoptosis. However, most human studies examined the combined effect of DHA and EPA using relatively high daily amounts of n-3 PUFA. The present study investigated the effects of increasing doses of DHA added to the regular diet of human healthy volunteers on lymphocyte response to tetradecanoylphorbol acetate plus ionomycin activation, and on monocyte apoptosis induced by oxidized LDL. Eight subjects were supplemented with increasing daily doses of DHA (200, 400, 800, 1600 mg) in a TAG form containing DHA as the only PUFA, for 2 weeks each dose. DHA intake dose-dependently increased the proportion of DHA in mononuclear cell phospholipids, the augmentation being significant after 400 mg DHA/d. The tetradecanoylphorbol acetate plus ionomycin-stimulated IL-2 mRNA level started to increase after ingestion of 400 mg DHA/d, with a maximum after 800 mg intake, and was positively correlated (P < 0.003) with DHA enrichment in cell phospholipids. The treatment of monocytes by oxidized LDL before DHA supplementation drastically reduced mitochondrial membrane potential as compared with native LDL treatment. Oxidized LDL apoptotic effect was significantly attenuated after 400 mg DHA/d and the protective effect was maintained throughout the experiment, although to a lesser extent at higher doses. The present results show that supplementation of the human diet with low DHA dosages improves lymphocyte activability. It also increases monocyte resistance to oxidized LDL-induced apoptosis, which may be beneficial in the prevention of atherosclerosis.
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Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones.
Fuchs, D, Vafeiadou, K, Hall, WL, Daniel, H, Williams, CM, Schroot, JH, Wenzel, U
The American journal of clinical nutrition. 2007;(5):1369-75
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BACKGROUND The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. OBJECTIVE The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. DESIGN The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were collected from 10 volunteers after 8 wk of receiving daily 2 placebo cereal bars and after a subsequent 8 wk of intervention with 2 cereal bars each providing 25 mg of isoflavones. The proteome of the cells was visualized after 2-dimensional gel electrophoresis, and peptide mass fingerprinting served to identify proteins that by the intervention displayed altered protein concentrations. RESULTS Twenty-nine proteins were identified that showed significantly altered expression in the mononuclear blood cells under the soy-isoflavone intervention, including a variety of proteins involved in an antiinflammatory response. Heat shock protein 70 or a lymphocyte-specific protein phosphatase and proteins that promote increased fibrinolysis, such as alpha-enolase, were found at increased intensities, whereas those that mediate adhesion, migration, and proliferation of vascular smooth muscle cells, such as galectin-1, were found at reduced intensities after soy extract consumption. CONCLUSION Proteome analysis identified in vivo markers that respond to a dietary intervention with isoflavone-enriched soy extract in postmenopausal women. The nature of the proteins identified suggests that soy isoflavones may increase the antiinflammatory response in blood mononuclear cells that might contribute to the atherosclerosis-preventive activities of a soy-rich diet.
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Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions.
Smith, JG, Joseph, HR, Green, T, Field, JA, Wooters, M, Kaufhold, RM, Antonello, J, Caulfield, MJ
Clinical and vaccine immunology : CVI. 2007;(5):527-37
Abstract
The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (-20 degrees C) or repeated cycling between more optimal storage temperatures (less than -130 degrees C and -70 degrees C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample-specific acceptance criteria (AC). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.