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Cellular Bioenergetic and Metabolic Changes in Patients with Autism Spectrum Disorder.
Gevezova, M, Minchev, D, Pacheva, I, Sbirkov, Y, Yordanova, R, Timova, E, Kotetarov, V, Ivanov, I, Sarafian, V
Current topics in medicinal chemistry. 2021;(11):985-994
Abstract
BACKGROUND Although Autism Spectrum Disorder (ASD) is considered a heterogeneous neurological disease in childhood, a growing body of evidence associates it with mitochondrial dysfunction explaining the observed comorbidities. INTRODUCTION The aim of this study is to identify variations in cellular bioenergetics and metabolism dependent on mitochondrial function in ASD patients and healthy controls using Peripheral Blood Mononuclear Cells (PBMCs). We hypothesized that PBMCs may reveal the cellular pathology and provide evidence of bioenergetic and metabolic changes accompanying the disease. METHODS PBMC from children with ASD and a control group of the same age and gender were isolated. All patients underwent an in-depth clinical evaluation. A well-characterized cohort of Bulgarian children is selected. Bioenergetic and metabolic studies of isolated PBMCs are performed with a Seahorse XFp analyzer. RESULTS Our data show that PBMCs from patients with ASD have increased respiratory reserve capacity (by 27.5%), increased maximal respiration (by 67%) and altered adaptive response to oxidative stress induced by DMNQ. In addition, we demonstrate а strong dependence on fatty acids and impaired ability to reprogram cell metabolism. The listed characteristics are not observed in the control group. These results can contribute to a better understanding of the underlying causes of ASD, which is crucial for selecting a successful treatment. CONCLUSION The current study, for the first time, provides a functional analysis of cell bioenergetics and metabolic changes in a group of Bulgarian patients with ASD. It reveals physiological abnormalities that do not allow mitochondria to adapt and meet the increased energetic requirements of the cell. The link between mitochondria and ASD is not yet fully understood, but this may lead to the discovery of new approaches for nutrition and therapy.
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2.
Quantitation of intracellular triphosphate metabolites of antiretroviral agents in peripheral blood mononuclear cells (PBMCs) and corresponding cell count determinations: review of current methods and challenges.
Xiao, D, Ling, KHJ, Custodio, J, Majeed, SR, Tarnowski, T
Expert opinion on drug metabolism & toxicology. 2018;(8):781-802
Abstract
Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system and the target cells for human immunodeficiency virus, type 1 (HIV-1) infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular pharmacokinetic (PK) of the TPs and its correlation with plasma PK, the drug effect, and dose determination. Areas covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantitative analysis of the nucleoside reverse transcriptase inhibitor-triphosphate (NRTI-TP) metabolites, and analogs. Expert opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, and an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.
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3.
THP-1 cell line: an in vitro cell model for immune modulation approach.
Chanput, W, Mes, JJ, Wichers, HJ
International immunopharmacology. 2014;(1):37-45
Abstract
THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.
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4.
Comparison of in vitro tests for antioxidant and immunomodulatory capacities of compounds.
Becker, K, Schroecksnadel, S, Gostner, J, Zaknun, C, Schennach, H, Uberall, F, Fuchs, D
Phytomedicine : international journal of phytotherapy and phytopharmacology. 2014;(2):164-71
Abstract
Oxidative stress is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies like cardiovascular and neurodegenerative diseases, as well as of infections and malignant tumors. These pathological conditions involve an overwhelming production of reactive oxygen species (ROS), which are released as part of an anti-proliferative strategy during pro-inflammatory immune responses. Moreover, ROS themselves are autocrine forward regulators of the immune response. Most of the beneficial effects of antioxidants are considered to derive from their influence on the immune system. Due to their antioxidant and/or radical scavenging nature, phytochemicals, botanicals and herbal preparations can be of great importance to prevent oxidation processes and to counteract the activation of redox-regulated signaling pathways. Antioxidants can antagonize the activation of T-cells and macrophages during the immune response and this anti-inflammatory activity could be of utmost importance for the treatment of above-mentioned disorders and for the development of immunotolerance. Herein, we provide an overview of in vitro assays for the measurement of antioxidant and anti-inflammatory activities of plant-derived substances and extracts, by discussing possibilities and limitations of these methods. To determine the capacity of antioxidants, the oxygen radical absorbance capacity (ORAC) assay and the cell-based antioxidant activity (CAA) assay are widely applied. To examine the influence of compounds on the human immune response more closely, the model of mitogen stimulated human peripheral blood mononuclear (PBMC) cells can be applied, and the production of the inflammatory marker neopterin as well as the breakdown of the amino acid tryptophan in culture supernatants can be used as readout to indicate an immunomodulatory potential of the tested compound. These two biomarkers of immune system activation are robust and correlate with the course of cardiovascular, neurodegenerative and malignant tumor diseases, but also with the normal aging process, and they are strongly predictive. Thus, while the simpler ORAC and CAA assays provide insight into one peculiar chemical aspect, namely the neutralization of peroxyl radicals, the more complex PBMC assay is closer to the in vivo conditions as the assay comprehensively enlights several properties of immunomodulatory test compounds.
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5.
Transcription factor NF-kappaB as a potential biomarker for oxidative stress.
van den Berg, R, Haenen, GR, van den Berg, H, Bast, A
The British journal of nutrition. 2001;:S121-7
Abstract
There is increasing interest in the involvement of transcription factors, such as of the transcription factor NF-kappaB (nuclear factor-kappaB), in the pathogenesis of various diseases. NF-kappaB is involved in the control of the transcription of a variety of cellular genes that regulate the inflammatory response by the production of cytokines, chemokines, cell adhesion molecules and acute phase proteins. The involvement of NF-kappaB is especially of interest as it is activated by oxidative stress and its activation can be modulated by antioxidant compounds. The activation of NF-kappaB can be determined by the electromobility shift assay (EMSA) with a NF-kappaB binding-site-specific probe. EMSA can also be used on human mononuclear cells isolated from peripheral blood, which could make the assay applicable for clinical trials. The critical steps of the EMSA are discussed, addressing some pitfalls of the assay. The procedure that can be used to express NF-kappaB activity in human subjects is evaluated. This offers the possibility to use NF-kappaB as a functional biomarker of oxidative stress as illustrated by several examples of in vitro and in vivo studies.
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6.
Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.
Rodriguez Orengo, JF, Santana, J, Febo, I, Diaz, C, Rodriguez, JL, Garcia, R, Font, E, Rosario, O
Puerto Rico health sciences journal. 2000;(1):19-27
Abstract
Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.
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7.
Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.
Tsurudome, M, Ito, Y
Critical reviews in immunology. 2000;(3):167-96
Abstract
Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.