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- Carlsson, Henrik, et al.
(författare)
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Measurement of hydroxychloroquine in blood from SLE patients using LC-HRMS : Evaluation of whole blood, plasma and serum as sample matrices
- 2020
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Ingår i: Arthritis Research & Therapy. - : BioMed Central. - 1478-6362. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Hydroxychloroquine (HCQ) is the standard of care in the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other inflammatory rheumatic diseases and potentially for the treatment in COVID-19 patients. Determination of HCQ for therapeutic drug monitoring (TDM) can be performed in whole blood (WB), serum, and plasma. Direct comparisons of WB, serum, and plasma levels of HCQ in patients with SLE have not previously been reported. We describe a method for the determination of HCQ in human blood using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices. Methods A method for the determination of HCQ in human blood using LC-HRMS was developed, validated, and applied for the determination of HCQ levels in WB, serum, and plasma from 26 SLE patients. The reproducibility of the method, in the three matrices, was evaluated using quality control samples and repeated preparations and measurements of patient samples. The performance of the developed method for HCQ measurement in serum was further evaluated by comparison with two previously reported extraction methods. Results The performance of the presented method demonstrated high accuracy and precision. A large range of HCQ concentrations was observed for the SLE patients in all three matrices (WB, serum, and plasma). The mean levels in WB were approximately two-fold the levels in serum and plasma (813 ng/mL compared to 436 ng/mL and 362 ng/mL, respectively). Spiked quality controls showed high reproducibility for all matrices (coefficient of variation, CV, approx. 5%), whereas in patient samples, equally high-precision was only found using WB as the matrix (CV 3%). The CV for serum and plasma was 14% and 39%, respectively. Two alternative methods applied to serum samples did not demonstrate improved precision. Conclusions A LC-HRMS method for the measurement of HCQ in human blood was developed and validated. Whole blood was found to be the superior sample matrix in terms of sample reproducibility. Thus, whole blood samples should be used for HCQ analysis when patients are monitored for HCQ treatment effects. The assay is in clinical use to monitor levels of HCQ in patients.
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| 2. |
- Hjorton, Karin, 1974-
(författare)
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The activation and regulation of plasmacytoid dendritic cells in SLE : and possible therapeutic interventions
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Systemic Lupus Erythematosus (SLE) is an autoimmune disease, characterized by the presence of anti-nuclear antibodies and the formation of nucleic acid containing immune complexes (ICs), which can cause organ damage by deposition in tissues. ICs strongly trigger the plasmacytoid dendritic cells (pDCs) to produce interferon (IFN)-α, which potently activates the immune system. An activated type I IFN system is seen in a majority of SLE patients. Natural killer (NK) cells enhance the IC triggered response of pDCs. Other NK cell alterations are described in SLE. Standard SLE treatment, hydroxychloroquine (HCQ), reduces flare risks, and HCQ concentration measurement can optimize its dosing. However, new treatments are needed.In paper I, we screened for autoantibodies to lectin-like NK cell receptors. In 3.4% of SLE patient sera, autoantibodies to CD94/NKG2A and CD94/NKG2C were found, which interfered with HLA-E mediated regulation of NK cell cytotoxicity, and facilitated elimination of target cells expressing expressing these receptors. Autoantibody levels correlated with SLE disease activity and with a more severe disease phenotype.In paper II, we found that RNA-IC triggered proinflammatory cytokine production in immune cells from healthy blood donors and SLE patients. After RNA-IC stimulation of pDCs, RNA sequencing detected 975 differentially expressed genes, connected to cytokine pathways, cell regulation and apoptosis. An IRAK4i had a broader inhibitory effect on RNA-IC triggered cytokine production and pro-inflammatory pathways than HCQ.In paper III we showed that RNA-IC induced type III IFN production in a subset of pDCs (3%) which also produced type I IFN. Type III IFN production by pDCs was enhanced by NK and B cells, as well as by IFN-λ2, IFN-α2b, interleukin (IL)-3, IL-6 and GM-CSF. Type III IFN production by RNA-IC stimulated immune cells of SLE patients was detected in a minority. IFN-α2b and GM-CSF increased the proportion of responders to RNA-IC from 11 to 33%.In paper IV a LC-HRMS method, was evaluated for HCQ concentration measurement in whole blood (WB), serum and plasma from 26 SLE patients. The levels in WB were approximately 2-fold compared to serum and plasma, and correlated with weekly HCQ-dose. Large inter-individual variations were observed, despite equal doses. The WB matrix showed superior reproducibility in patient samples (CV<5%).These findings add to the knowledge of how cytokine production by pDCs in SLE is regulated, and support a role for NK cells in the pathogenesis of SLE. Moreover WB was the superior matrix for HCQ measurement in SLE patients.
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| 3. |
- Hjorton, Karin, 1974-, et al.
(författare)
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The regulation and pharmacological modulation of immune complex induced type III IFN production by plasmacytoid dendritic cells
- 2020
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Ingår i: Arthritis Research & Therapy. - : Springer Science and Business Media LLC. - 1478-6362. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivePatients with systemic lupus erythematosus (SLE) have an ongoing interferon (IFN) production due to an activation of plasmacytoid dendritic cells (pDCs), which can be triggered to type I IFN synthesis by RNA containing immune complexes (RNA-IC). Considering emerging data suggesting a role of type III IFN in the SLE disease process, we asked if RNA-IC can induce type III IFN production in pDC and how this production can be regulated.MethodsPeripheral blood mononuclear cells (PBMCs) or immune cell subsets were isolated from healthy blood donors or SLE patients and stimulated with IC containing U1 snRNP and SLE-IgG (RNA-IC). Hydroxychloroquine (HCQ) and an interleukin receptor 1-associated kinase 4 inhibitor (IRAK4i) were added to cell cultures. Cytokine mRNA levels were determined with a microarray and protein levels with immunoassays. Single-cell RNA sequencing of pDCs using ddSEQ technology was performed.ResultsType III IFN mRNA and protein was induced in RNA-IC-stimulated pDC-NK and pDC-B cell co-cultures. A subset of activated pDCs (3%) expressed both type III and type I IFN mRNA. IFN-λ2, IFN-α2b, interleukin (IL)-3, IL-6, or granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced IFN-λ1/3 production 2–5-fold. HCQ and an IRAK4i blocked the RNA-IC-triggered IFN-λ1/3 production (p < 0.01). IFN-α2b and GM-CSF increased the proportion of SLE patients producing IFN-λ1/3 in response to RNA-IC from 11 to 33%.ConclusionsType III IFN production is triggered by RNA-IC in pDCs in a TLR-MyD88-dependent manner, enhanced by NK and B cells as well as several pro-inflammatory cytokines. These results support a contributing role for both type I and type III IFNs in SLE, which needs to be considered when targeting the IFN system in this disease.
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