scholarly journals Leukocyte cytokine responses in adult patients with mitochondrial DNA defects

2021 ◽  
Author(s):  
Kalpita R Karan ◽  
Caroline Trumpff ◽  
Marissa Cross ◽  
Kristin M Englestad ◽  
Anna L Marsland ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Large Scale ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Mitochondrial Diseases ◽  
Preliminary Evidence ◽  
Cytokine Responses ◽  
Mtdna Deletions ◽  
Immune Challenges

Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 174 -179% lower responses for IL-6 and IL-1β (pIL-6=0.031, pIL-1β=0.009). Moreover, IL-6 response to LPS in presence of GC was also blunted in cells from patients with mtDNA deletions (pIL-6=0.006), but not in leukocytes from patients with the m.3243A>G mutation. Overall, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and glucocorticoid sensitivity. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes.

Inter- and intraindividual variation of endotoxin- and β(1 → 3)-glucan-induced cytokine responses in a whole blood assay

2002 ◽  
Vol 18 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Inge M Wouters ◽  
Jeroen Douwes ◽  
Peter S Thorne ◽  
Dick Heederik ◽  
Gert Doekes
Keyword(s):  
Whole Blood ◽  
Large Scale ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Intraindividual Variation ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Within Subjects ◽  
Cytokine Responsiveness

Inflammatory airway responses to bioaerosols and to their active compounds, such as endotoxin and β(1 → 3)- glucan, vary between individuals. These differences may be explained by variation in cytokine responsiveness, which can be assessed by in vitro stimulation tests with isolated blood leukocytes or lung macrophages. In large- scale population studies, ex vivo induced cytokine production may also be tested with a more simple `whole blood assay’ (WBA). However, applicability of a WBA to characterize a subject’s responsiveness depends largely on its reproducibility. This study was conducted to: 1) assess the within- and between-subject variability in cytokine production in a WBA after stimulation with endotoxin or β(1 → 3)-glucan; and 2) to determine under which conditions this test is most discriminating between subjects and most reproducible within subjects. Blood was collected from 14 healthy volunteers, of whom 10 also participated on a second occasion. Each blood sample was used in two WBA tests; the first WBA was initiated two hours and the second 26 hours after venapuncture. The WBA test itself comprised overnight incubation with serial dilutions of endotoxin [lipopolysaccharide (LPS)] and curdlan (a β(1 → 3)-glucan), after which blood cell supernatant was collected. Interleukin(IL)-1, IL6, IL8 and tumor necrosis factor (TNF) were determined in the supernatant. In all individuals, a dose-dependent production of cytokines was observed for both LPS and curdlan. For all cytokines, variation between subjects was higher than within subjects, and this was most pronounced for IL1 and IL6. There was moderate-to-high correlation in the induced release of all four cytokines, and between cytokine release induced by LPS or curdlan. Optimal stimulation concentrations were 6.25 and 12.5 ng/mL for endotoxin and 12 500 and 25 000 ng/mL for curdlan. Cytokine production in WBA initiated 26 hours after venapuncture showed lower between-subject and larger within-subject variance, thus favoring an early initiation of the assay. In conclusion, measuring endotoxin-or glucan-induced cytokine production in a WBA initiated within two hours after venapuncture appears to be an effective method to determine a person’s cytokine responsiveness, at least in healthy naive subjects. Toxicology and Industrial Health 2002; 18: 15-27.

scholarly journals Mechanisms of replication and repair in mitochondrial DNA deletion formation

2020 ◽  
Vol 48 (20) ◽  
pp. 11244-11258
Author(s):  
Gabriele A Fontana ◽  
Hailey L Gahlon
Keyword(s):  
Dna Damage ◽  
Mitochondrial Dna ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Mitochondrial Dna Deletion ◽  
Deletion Formation ◽  
Dna Deletion ◽  
Mtdna Deletions ◽  
Mtdna Deletion

Abstract Deletions in mitochondrial DNA (mtDNA) are associated with diverse human pathologies including cancer, aging and mitochondrial disorders. Large-scale deletions span kilobases in length and the loss of these associated genes contributes to crippled oxidative phosphorylation and overall decline in mitochondrial fitness. There is not a united view for how mtDNA deletions are generated and the molecular mechanisms underlying this process are poorly understood. This review discusses the role of replication and repair in mtDNA deletion formation as well as nucleic acid motifs such as repeats, secondary structures, and DNA damage associated with deletion formation in the mitochondrial genome. We propose that while erroneous replication and repair can separately contribute to deletion formation, crosstalk between these pathways is also involved in generating deletions.

scholarly journals Erythromycin Inhibits Tumor Necrosis Factor Alpha and Interleukin 6 Production Induced by Heat-Killed Streptococcus pneumoniae in Whole Blood

1998 ◽  
Vol 42 (7) ◽  
pp. 1605-1609 ◽  
Author(s):  
Marc J. Schultz ◽  
Peter Speelman ◽  
Sebastian Zaat ◽  
Sander J. H. van Deventer ◽  
Tom van der Poll
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tumor Necrosis Factor ◽  
Whole Blood ◽  
Tumor Necrosis ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT To determine the effects of penicillin and erythromycin on cytokine production induced by heat-killed Streptococcus pneumoniae (HKSP), we studied the effects of those drugs on cytokine production induced by S. pneumoniaein human whole blood in vitro and ex vivo. In whole blood in vitro, erythromycin, but not penicillin, caused a dose-dependent decrease in HKSP-induced production of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6), while the production of IL-10, IL-12, and gamma interferon was inhibited only at the highest erythromycin concentration tested (10−3 M). The production of TNF and IL-6 in whole blood obtained from healthy subjects after a 30-min infusion of erythromycin (1,000 mg) was lower after ex vivo stimulation with HKSP than that in blood drawn before the infusion. Inhibition of TNF contributed to erythromycin-induced inhibition of IL-6 synthesis. Inhibition of TNF and IL-6 production by erythromycin may have a negative impact on host defense mechanisms during pneumococcal pneumonia.

scholarly journals Human Newborn Monocytes Demonstrate Distinct BCG-Induced Primary and Trained Innate Cytokine Production and Metabolic Activation In Vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Asimenia Angelidou ◽  
Joann Diray-Arce ◽  
Maria-Giulia Conti ◽  
Mihai G. Netea ◽  
Bastiaan A. Blok ◽  
...  
Keyword(s):  
Cytokine Production ◽  
Colorimetric Assay ◽  
Age Groups ◽  
Metabolic Activation ◽  
Human Monocyte ◽  
Autologous Serum ◽  
Live Attenuated Vaccine ◽  
Cytokine Responses ◽  
The Impact

BackgroundNewborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells (“innate memory”). However, little is known regarding the impact of age on BCG-induced innate responses.ObjectiveEstablish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts.Design/MethodsHuman neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay.ResultsCytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults.ConclusionsBCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1892-1892
Author(s):  
Takuya Matsunaga ◽  
Ikuta Tanaka ◽  
Masayoshi Kobune ◽  
Yutaka Kawano ◽  
Maki Tanaka ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Large Scale ◽  
Culture Medium ◽  
Ex Vivo ◽  
Aggregation Assay ◽  
Three Phase ◽  
Cd34 Cells ◽  
Phase Culture

Abstract To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 106 CD34+ cells) was calculated to be 10.5 units (2 x 1011 PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.

scholarly journals Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

2016 ◽  
Vol 27 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Satish Kumar Tadi ◽  
Robin Sebastian ◽  
Sumedha Dahal ◽  
Ravi K. Babu ◽  
Bibha Choudhary ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Strand Break ◽  
Mitochondrial Disorders ◽  
Dna Lesions ◽  
End Joining ◽  
Dsb Repair

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

scholarly journals YY1-MEDIATED PROMOTER-SUPER ENHANCER INTERACTIONS REGULATE MSC MULTIPOTENCY DURING REPLICATIVE AGING

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S912-S913
Author(s):  
Luyang Sun ◽  
Brenna McCauley ◽  
Weiwei Dang
Keyword(s):  
Stem Cell ◽  
Conformational Changes ◽  
Large Scale ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Early Passage ◽  
Replicative Aging ◽  
Cell Functions ◽  
Super Enhancer

Abstract Mesenchymal stem cells (MSC) are versatile in stem cell therapy and regenerative medicine, due to its capacity of in-vitro expansion and multilineage differentiation. However, how the MSC’s functions and the underlying epigenomic features are affected by aging and cellular senescence remains poorly understood. Here we used culture expanded human MSC isolated from umbilical cord tissue as an ex vivo model to characterize the chromatin 3D conformational changes associated with aging and senescence and correlate these changes to the changes in MSC stem cell functions during aging. By comparing late passage (P15) vs early passage (P5) MSC, no fundamental changes were found on the large-scale chromatin spatial structures including compartment switching and TAD boundaries. However, when focused on super enhancers, which act as major cis-regulatory hubs associated with lineage-specific functions, a large number of significant promoter-super enhancer interaction changes were discovered. Further examination revealed that YY1, a key regulator of promoter-enhancer looping, was highly enriched in these promoters and their corresponding super-enhancers. The expression of the genes involved in such changes is positively correlated with the variation of promoter-super enhancer interaction, indicating that the spatial communications between promoter and super-enhancer positively regulate gene expression. GO-term and TFBS enrichment analysis showed that these genes involved in cell cycle, adipocyte and osteoblast differentiation. Collectively, these results suggest that YY1-mediated promoter-super enhancer looping is an important regulatory mechanism for MSC multipotency during replicative aging.

scholarly journals Peripheral Benzodiazepine Binding Sites in Platelets of Patients Affected by Mitochondrial Diseases and Large Scale Mitochondrial DNA Rearrangements

2002 ◽  
Vol 8 (12) ◽  
pp. 841-846 ◽  
Author(s):  
Claudia Martini ◽  
Beatrice Chelli ◽  
Laura Betti ◽  
Marina Montali ◽  
Michelangelo Mancuso ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Binding Sites ◽  
Large Scale ◽  
Mitochondrial Diseases ◽  
Dna Rearrangements ◽  
Benzodiazepine Binding ◽  
Benzodiazepine Binding Sites

scholarly journals Deep sequencing of mitochondrial DNA and characterization of a novel POLG mutation in a patient with arPEO

2020 ◽  
Vol 6 (1) ◽  
pp. e391
Author(s):  
Carola Hedberg-Oldfors ◽  
Bertil Macao ◽  
Swaraj Basu ◽  
Christopher Lindberg ◽  
Bradley Peter ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Muscle Biopsy ◽  
Deep Sequencing ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Late Onset ◽  
Novel Mutation ◽  
Progressive External Ophthalmoplegia ◽  
Oxidase Deficiency

ObjectiveTo determine the pathogenicity of a novel POLG mutation in a man with late-onset autosomal recessive progressive external ophthalmoplegia using clinical, molecular, and biochemical analyses.MethodsA multipronged approach with detailed neurologic examinations, muscle biopsy analyses, molecular genetic studies, and in vitro biochemical characterization.ResultsThe patient had slowly progressive bilateral ptosis and severely reduced horizontal and vertical gaze. Muscle biopsy showed slight variability in muscle fiber size, scattered ragged red fibers, and partial cytochrome c oxidase deficiency. Biallelic mutations were identified in the POLG gene encoding the catalytic A subunit of POLγ. One allele carried a novel mutation in the exonuclease domain (c.590T>C; p.F197S), and the other had a previously characterized null mutation in the polymerase domain (c.2740A>C; p.T914P). Biochemical characterization revealed that the novel F197S mutant protein had reduced exonuclease and DNA polymerase activities and confirmed that T914P was inactive. By deep sequencing of mitochondrial DNA (mtDNA) extracted from muscle, multiple large-scale rearrangements were mapped and quantified.ConclusionsThe patient's phenotype was caused by biallelic POLG mutations, resulting in one inactive POLγA protein (T914P) and one with decreased polymerase and exonuclease activity (F197S). The reduction in polymerase activity explains the presence of multiple pathogenic large-scale deletions in the patient's mtDNA.

Ex vivo zidovudine (AZT) treatment of CD34+ bone marrow progenitors causes decreased steady state mitochondrial DNA (mtDNA) and increased lactate production

2004 ◽  
Vol 23 (4) ◽  
pp. 173-185 ◽  
Author(s):  
L D Lewis ◽  
S Amin ◽  
C I Civin ◽  
P S Lietman
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Steady State ◽  
Nuclear Dna ◽  
Ex Vivo ◽  
Lactate Production ◽  
Nuclear Dna Content ◽  
Content Ratio ◽  
The Mean

Haematopoietic suppression is one of the dose-limiting side effects of chronic zidovudine (AZT) therapy. We tested the hypothesis that AZT would reduce mitochondrial DNA (mtDNA) content in haematopoietic progenitors causing impaired haematopoiesis and mitochondrial dysfunction. We studied the effects of AZT 0 / 50 M in vitro, on normal human CD34 / haematopoietic progenitor cells cultured ex vivo for up to 12 days. The mean AZT IC50 for granulocyte (phenotype CD15 / /CD14 /) and erythroid (phenotype glycophorin / /CD45 /) cell proliferation was 2.5 M (SD9 / 0.7) and 0.023 M (SD9 / 0.005), respectively. In myeloid-rich cell cultures, the mean lactate content of the media, compared to untreated controls, increased by 86% (SD9 / 23) at 10 M AZT and in erythroid-rich cultures it increased by 134% (SD9 / 24) in the presence of 0.5 M AZT. In myeloid-rich cultures the AZT IC50 for the reduction in the mitochondrial/nuclear DNA content ratio was 5.6 M, whereas in erythroid rich cultures this AZT IC50 was B / 0.0005 M. AZT produced concentration-dependent inhibition of CD34 / progenitor proliferation into both myeloid and erythroid lineages; erythropoiesis was more sensitive than myelopoiesis. Concurrently, AZT reduced steady state mtDNA content, while increasing lactate production. These findings support the hypothesis that mtDNA is one of the intracellular targets involved in the pathogenesis of AZT-associated bone marrow progenitor cell toxicity.

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