Association of Altered Plasma Lipidome with Disease Severity in COVID-19 Patients.

The severity of COVID-19 is linked to an imbalanced immune response. The dysregulated metabolism of small molecules and bioactive lipids has also been associated with disease severity. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyze over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). This is the third publication in a series, and it reports the results of comprehensive lipidome profiling using targeted LC-MS/MS. We identified 1076 lipid features across 25 subclasses, including glycerophospholipids, sterols, glycerolipids, and sphingolipids, among which 531 lipid features were dramatically changed in the plasma of intensive care unit (ICU) patients compared to patients in the ward. Patients in the ICU showed 1.3-57-fold increases in ceramides, (lyso-)glycerophospholipids, diglycerides, triglycerides, and plasmagen phosphoethanolamines, and 1.3-2-fold lower levels of a cyclic lysophosphatidic acid, sphingosine-1-phosphates, sphingomyelins, arachidonic acid-containing phospholipids, lactosylceramide, and cholesterol esters compared to patients in the ward. Specifically, phosphatidylinositols (PIs) showed strong fatty acid saturation-dependent behavior, with saturated fatty acid (SFA)- and monosaturated fatty acid (MUFA)-derived PI decreasing and polystaturated (PUFA)-derived PI increasing. We also found ~4000 significant Spearman correlations between lipids and multiple clinical markers of immune response with |R| ≥ 0.35 and FDR corrected Q < 0.05. Except for lysophosphatidic acid, lysophospholipids were positively associated with the CD4 fraction of T cells, and the cytokines IL-8 and IL-18. In contrast, sphingosine-1-phosphates were negatively correlated with innate immune markers such as CRP and IL-6. Further indications of metabolic changes in moderate COVID-19 disease were demonstrated in recovering ward patients compared to those at the start of hospitalization, where 99 lipid species were altered (6 increased by 30-62%; 93 decreased by 1.3-2.8-fold). Overall, these findings support and expand on early reports that dysregulated lipid metabolism is involved in COVID-19.

Targeted proteomics identifies circulating biomarkers associated with active COVID-19 and post-COVID-19.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the highly infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). There is an urgent need for biomarkers that will help in better stratification of patients and contribute to personalized treatments. We performed targeted proteomics using the Olink platform and systematically investigated protein concentrations in 350 hospitalized COVID-19 patients, 186 post-COVID-19 individuals, and 61 healthy individuals from 3 independent cohorts. Results revealed a signature of acute SARS-CoV-2 infection, which is represented by inflammatory biomarkers, chemokines and complement-related factors. Furthermore, the circulating proteome is still significantly affected in post-COVID-19 samples several weeks after infection. Post-COVID-19 individuals are characterized by upregulation of mediators of the tumor necrosis (TNF)-α signaling pathways and proteins related to transforming growth factor (TGF)-ß. In addition, the circulating proteome is able to differentiate between patients with different COVID-19 disease severities, and is associated with the time after infection. These results provide important insights into changes induced by SARS-CoV-2 infection at the proteomic level by integrating several cohorts to obtain a large disease spectrum, including variation in disease severity and time after infection. These findings could guide the development of host-directed therapy in COVID-19.

Severe COVID-19 Is Characterised by Perturbations in Plasma Amines Correlated with Immune Response Markers, and Linked to Inflammation and Oxidative Stress.

The COVID-19 pandemic raised a need to characterise the biochemical response to SARS-CoV-2 infection and find biological markers to identify therapeutic targets. In support of these aims, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The first publication in a series reports the results of quantitative LC-MS/MS profiling of 56 amino acids and derivatives. A comparison between samples taken from ICU and ward patients revealed a notable increase in ten post-translationally modified amino acids that correlated with markers indicative of an excessive immune response: TNF-alpha, neutrophils, markers for macrophage, and leukocyte activation. Severe patients also had increased kynurenine, positively correlated with CRP and cytokines that induce its production. ICU and ward patients with high IL-6 showed decreased levels of 22 immune-supporting and anti-oxidative amino acids and derivatives (e.g., glutathione, GABA). These negatively correlated with CRP and IL-6 and positively correlated with markers indicative of adaptive immune activation. Including corresponding alterations in convalescing ward patients, the overall metabolic picture of severe COVID-19 reflected enhanced metabolic demands to maintain cell proliferation and redox balance, alongside increased inflammation and oxidative stress.

Plasma Oxylipins and Their Precursors Are Strongly Associated with COVID-19 Severity and with Immune Response Markers.

COVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The second publication in a series reports the results of quantitative LC-MS/MS profiling of 63 small lipids including oxylipins, free fatty acids, and endocannabinoids. Compared to samples taken from ward patients, intensive care unit (ICU) patients had 2−4-fold lower levels of arachidonic acid (AA) and its cyclooxygenase-derived prostanoids, as well as lipoxygenase derivatives, exhibiting negative correlations with inflammation markers. The same derivatives showed 2−5-fold increases in recovering ward patients, in paired comparison to early hospitalisation. In contrast, ICU patients showed elevated levels of oxylipins derived from poly-unsaturated fatty acids (PUFA) by non-enzymatic peroxidation or activity of soluble epoxide hydrolase (sEH), and these oxylipins positively correlated with markers of macrophage activation. The deficiency in AA enzymatic products and the lack of elevated intermediates of pro-resolving mediating lipids may result from the preference of alternative metabolic conversions rather than diminished stores of PUFA precursors. Supporting this, ICU patients showed 2-to-11-fold higher levels of linoleic acid (LA) and the corresponding fatty acyl glycerols of AA and LA, all strongly correlated with multiple markers of excessive immune response. Our results suggest that the altered oxylipin metabolism disrupts the expected shift from innate immune response to resolution of inflammation.

Blood eosinophil count and GOLD stage predict response to maintenance azithromycin treatment in COPD patients with frequent exacerbations.

INTRODUCTION: Maintenance treatment with macrolides are useful in preventing COPD exacerbations. We investigated which characteristics of COPD patients with frequent exacerbations predicted the best response to maintenance treatment with azithromycin. METHODS: This study was part of the COLUMBUS trial, a prospective randomized, double-blind, placebo-controlled study in 92 COPD patients with frequent exacerbations. During the 1-year treatment period, follow-up data were collected for spirometry, mMRC scores, sputum cultures and blood inflammatory markers. RESULTS: In the azithromycin group a significant lower number of exacerbations per patient was observed in patients with the following characteristics: baseline blood eosinophil count ≥2.0% (x̄ = 1.26), compared to an eosinophil count < 2.0% (x̄ = 2.50; p = 0.02), GOLD stage 1-2 (x̄ = 1.06), versus GOLD stage 4 (x̄ = 2.62; p = 0.02) and GOLD group C (x̄ = 0.45) compared to group D (x̄ = 2.18; p < 0.01). Moreover, the number of hospitalizations was significantly lower in patients, with a blood eosinophil count ≥2.0% (x̄ = 0.26) compared to an eosinophil count < 2.0% (x̄ = 0.90; p = 0.01) and in GOLD stages 1-2 (x̄ = 1.06) compared to stage 4 (x̄ = 2.62; p = 0.04). CONCLUSION: In conclusion, azithromycin maintenance treatment appears to be effective in COPD patients with frequent exacerbations, who are either classified in GOLD stage 1-2 or GOLD C and those with a blood eosinophil count of ≥2.0%.

Usability of femoral head bone marrow to verify reference ranges for the assessment of myelodysplasia by flow cytometry.

BACKGROUND: A flow cytometry score (FCS) based on four parameters has been proposed for analysis of myelodysplastic syndromes patients with <5% blasts. We evaluated whether bone marrow aspirate samples isolated from femoral heads could be used for verification of cutoff values of the individual parameters score (IPS), contributing to the FCS and compared the applicability of FCS parameters in two centers. STUDY DESIGN AND METHODS: Bone marrow cells were obtained from femoral heads of patients who underwent hip replacement surgery. The score of the 4 individual parameters of the cell samples were obtained independently in two centers, using their own facilities and methods. Flow cytometry data files were subsequently exchanged and reanalyzed in the other center. The resulting four data sets were compared to assess reproducibility and outcomes in both centers. RESULTS: Twenty-nine of 40 bone marrow samples contained sufficient cells for analysis. Proposed cutoff values for 3 of 4 individual parameters were appropriate. All 29 samples showed a positive individual parameters score (IPS:1) in 1 of the 4 obtained data sets. Most differences in IPS scores were a result of reanalyzing the data file in the other center, rather than data acquisition. FCS: ≥2 were observed in 11 samples. CONCLUSION: Bone marrow samples from femoral heads are appropriate for verification of the proposed reference cutoff values of the individual parameters. Proposed reference cutoff values needed to be adjusted for reliable interpretation. Standardization of both data acquisition and data analysis is necessary for obtaining uniform results.

Azithromycin maintenance treatment in patients with frequent exacerbations of chronic obstructive pulmonary disease (COLUMBUS): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Macrolide resistance is an increasing problem; there is therefore debate about when to implement maintenance treatment with macrolides in patients with chronic obstructive pulmonary disease (COPD). We aimed to investigate whether patients with COPD who had received treatment for three or more exacerbations in the previous year would have a decrease in exacerbation rate when maintenance treatment with azithromycin was added to standard care. METHODS: We did a randomised, double-blind, placebo-controlled, single-centre trial in The Netherlands between May 19, 2010, and June 18, 2013. Patients (≥18 years) with a diagnosis of COPD who had received treatment for three or more exacerbations in the previous year were randomly assigned, via a computer-generated randomisation sequence with permuted block sizes of ten, to receive 500 mg azithromycin or placebo three times a week for 12 months. Randomisation was stratified by use of long-term, low-dose prednisolone (≤10 mg daily). Patients and investigators were masked to group allocation. The primary endpoint was rate of exacerbations of COPD in the year of treatment. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00985244. FINDINGS: We randomly assigned 92 patients to the azithromycin group (n=47) or the placebo group (n=45), of whom 41 (87%) versus 36 (80%) completed the study. We recorded 84 exacerbations in patients in the azithromycin group compared with 129 in those in the placebo group. The unadjusted exacerbation rate per patient per year was 1·94 (95% CI 1·50-2·52) for the azithromycin group and 3·22 (2·62-3·97) for the placebo group. After adjustment, azithromycin resulted in a significant reduction in the exacerbation rate versus placebo (0·58, 95% CI 0·42-0·79; p=0·001). Three (6%) patients in the azithromycin group reported serious adverse events compared with five (11%) in the placebo group. During follow-up, the most common adverse event was diarrhoea in the azithromycin group (nine [19%] patients vs one [2%] in the placebo group; p=0·015). INTERPRETATION: Maintenance treatment with azithromycin significantly decreased the exacerbation rate compared with placebo and should therefore be considered for use in patients with COPD who have the frequent exacerbator phenotype and are refractory to standard care. FUNDING: SoLong Trust.

Influence of macrolide maintenance therapy and bacterial colonisation on exacerbation frequency and progression of COPD (COLUMBUS): study protocol for a randomised controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by progressive development of airflow limitation that is poorly reversible. Because of a poor understanding of COPD pathogenesis, treatment is mostly symptomatic and new therapeutic strategies are limited. There is a direct relationship between the severity of the disease and the intensity of the inflammatory response. Besides smoking, one of the hypotheses for the persistent airway inflammation is the presence of recurrent infections. Macrolide antibiotics have bacteriostatic as well as anti-inflammatory properties in patients with cystic fibrosis and other inflammatory pulmonary diseases. There is consistent evidence that macrolide therapy reduces infectious exacerbations, decreases the requirement for additional antibiotics and improves nutritional measures. Because of these positive effects we hypothesised that maintenance macrolide therapy may also have beneficial effects in patients with COPD who have recurrent exacerbations. The effects on development of bacterial resistance to macrolides due to this long-term treatment are unknown. Until now, studies investigating macrolide therapy in COPD are limited. The objective of this study is to assess whether maintenance treatment with macrolide antibiotics in COPD patients with three or more exacerbations in the previous year decreases the exacerbation rate in the year of treatment and to establish microbial resistance due to the long-term treatment. METHODS/DESIGN: The study is set up as a prospective randomised double-blind placebo-controlled single-centre trial. A total of 92 patients with COPD who have had at least three exacerbations of COPD in the previous year will be included. Subjects will be randomised to receive either azithromycin 500 mg three times a week or placebo. Our primary endpoint is the reduction in the number of exacerbations of COPD in the year of treatment. DISCUSSION: We investigate whether long-term therapy with macrolide antibiotics can prevent exacerbations in patients with COPD. Additionally, our study aims to assess the effect of long-term use of macrolide on the development of antimicrobial resistance and on inflammatory parameters related to COPD. We believe this study will provide more data on the effects of macrolide treatment in patients in COPD and will add more knowledge on its working mechanisms. TRIAL REGISTRATION: http://www.clinicaltrials.gov NCT00985244.

Improving diagnosis of adult-type hypolactasia in patients with abdominal complaints.

BACKGROUND: Adult-type hypolactasia is caused by genetic lactase non-persistence. It is the most common cause of lactose intolerance, which results in gastrointestinal symptoms after ingestion of dairy products. Currently, lactose intolerance is investigated by the hydrogen breath test (HBT), which is considered the preferred diagnostic test. Adult-type hypolactasia may also be diagnosed by genotyping. The single nucleotide polymorphism -13910C>T, which is located upstream of the lactase gene (LCT), is tightly associated with lactase persistence. Several other variants, mostly in non-European populations, can also lead to lactase persistence. This study investigated the accuracy of a modified, recently proposed algorithm which includes genotyping for the diagnosis of adult-type hypolactasia in a patient population with unexplained abdominal complaints. METHODS: In 126 patients with unexplained abdominal symptoms or who were suspected to have adult-type hypolactasia, LCT genotyping by melting curve analysis on a LightCycler was performed. Those patients with CC(-13910) genotype (indicating loss of lactase expression) were directly referred to a dietician for a lactose-free diet. Those identified as CT(-13910) or TT(-13910) genotype underwent a HBT. Those who tested positive for hydrogen were also referred to a dietician for a lactose-free diet. The response to diet modification was recorded. RESULTS: Genotype prevalences were: CC(-13910): 43 (34.1%); CT(-13910): 48 (38.1%); TT(-13910): 33 (26.2%); TG-13915: 2 (1.6%). Eleven of 48 (23%) patients with CT(-13910)-genotype and 1/33 (3%) patients with TT(-13910)-genotype had a positive hydrogen breath test. They all improved after a lactose-free diet. Four of 43 (9%) patients with CC(-13910)-genotype still had symptoms after a lactose-free diet. CONCLUSIONS: The results show that lactase-genotype testing can be used as a first step to diagnose lactose intolerance in a patient population with unexplained abdominal complaints. It accurately identifies the group of patients sensitive to lactose, those who need further breath testing and those in whom adult-type hypolactasia can be excluded with high probability without performing a HBT. This algorithm would save hydrogen breath testing in more than 50% of the patients who present with unexplained abdominal symptoms.

Hyperhomocysteinemia and functional cobalamin deficiency due to granulocytosis-induced alterations in the cobalamin-binding protein.

Serum cobalamin and homocysteine levels were studied in patients with chronic myelogenous leukemia (CML) and in stem cell donors treated with granulocyte-colony stimulating factor (G-CSF). Cytoreductive treatment in patients with CML resulted in a decrease of cobalamin and homocysteine levels. In stem cell donors cobalamin and homocysteine levels increased after G-CSF administration. The increase of homocysteine level was accompanied by a decrease in the serum levels of the cobalamin-binding protein transcobalamin. We hypothesize that the increased homocysteine levels in patients with CML and donors treated with G-CSF may be the result of a functional methylcobalamin deficiency due to decreased transcobalamin levels.