Comprehensive proteomics and meta-analysis of COVID-19 host response.

COVID-19 is characterised by systemic immunological perturbations in the human body, which can lead to multi-organ damage. Many of these processes are considered to be mediated by the blood. Therefore, to better understand the systemic host response to SARS-CoV-2 infection, we performed systematic analyses of the circulating, soluble proteins in the blood through global proteomics by mass-spectrometry (MS) proteomics. Here, we show that a large part of the soluble blood proteome is altered in COVID-19, among them elevated levels of interferon-induced and proteasomal proteins. Some proteins that have alternating levels in human cells after a SARS-CoV-2 infection in vitro and in different organs of COVID-19 patients are deregulated in the blood, suggesting shared infection-related changes.The availability of different public proteomic resources on soluble blood proteome alterations leaves uncertainty about the change of a given protein during COVID-19. Hence, we performed a systematic review and meta-analysis of MS global proteomics studies of soluble blood proteomes, including up to 1706 individuals (1039 COVID-19 patients), to provide concluding estimates for the alteration of 1517 soluble blood proteins in COVID-19. Finally, based on the meta-analysis we developed CoViMAPP, an open-access resource for effect sizes of alterations and diagnostic potential of soluble blood proteins in COVID-19, which is publicly available for the research, clinical, and academic community.

Targeted plasma proteomics reveals signatures discriminating COVID-19 from sepsis with pneumonia.

BACKGROUND: COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. METHODS: We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. RESULTS: We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. CONCLUSIONS: This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.

Plasma proteome alterations by MAPK inhibitors in BRAFV600-mutated metastatic cutaneous melanoma.

Approximately half of metastatic cutaneous melanomas (CM) harbor a mutation in the BRAF protooncogene, upregulating the mitogen-activated protein kinase (MAPK)-pathway. The development of inhibitors targeting the MAPK pathway (MAPKi), i.e., BRAF- and MEK-inhibitors (BRAFi and MEKi), have substantially improved the survival in BRAFV600E/K-mutated stage IV metastatic CM. However, most patients develop resistance to treatment and no predictive biomarkers exist in practice. This study aimed at discovering plasma proteome changes during treatment MAPKi in patients with metastatic (stage IV) CM. Matched plasma samples before (pre) and during treatment (trm) from 23 patients with stage IV CM, treated with BRAF-inhibitors (BRAFi) alone or BRAF- and MEK- inhibitors combined (BRAFi and MEKi), were collected and analyzed with targeted proteomics by proximity extension assays. Additionally, plasma from 9 patients treated with BRAFi and MEKi was analyzed with in-depth high-resolution isoelectric focusing liquid-chromatography mass-spectrometry proteomics. Alterations of plasma proteins involved in granzyme and interferon gamma pathways were detected in patients treated with BRAFi, and cell adhesion-, neutrophil degranulation-, and proteolysis pathways in patients treated with BRAFi and MEKi. Several proteins were associated with progression-free survival after MAPKi treatment. We show that the majority of the altered plasma proteins were traceable to BRAFV600E-mutant metastatic CM tissue at mRNA level in 154 patients from the TCGA, further strengthening their involvement in tumoral response to treatment. This wide screen of plasma proteins unravels proteins that may serve as predictive and/or prognostic biomarkers of MAPKi treatment, opening a window of opportunity for plasma biomarker discovery in MAPKi-treatment of BRAFV600-mutant metastatic CM.

In-depth plasma proteomics reveals increase in circulating PD-1 during anti-PD-1 immunotherapy in patients with metastatic cutaneous melanoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have significantly improved the outcome in metastatic cutaneous melanoma (CM). However, therapy response is limited to subgroups of patients and clinically useful predictive biomarkers are lacking. METHODS: To discover treatment-related systemic changes in plasma and potential biomarkers associated with treatment outcome, we analyzed serial plasma samples from 24 patients with metastatic CM, collected before and during ICI treatment, with mass-spectrometry-based global proteomics (high-resolution isoelectric focusing liquid chromatography-mass spectrometry (HiRIEF LC-MS/MS)) and targeted proteomics with proximity extension assays (PEAs). In addition, we analyzed plasma proteomes of 24 patients with metastatic CM treated with mitogen-activated protein kinase inhibitors (MAPKis), to pinpoint changes in protein plasma levels specific to the ICI treatment. To detect plasma proteins associated with treatment response, we performed stratified analyses in anti-programmed cell death protein 1 (anti-PD-1) responders and non-responders. In addition, we analyzed the association between protein plasma levels and progression-free survival (PFS) by Cox proportional hazards models. RESULTS: Unbiased HiRIEF LC-MS/MS-based proteomics showed plasma levels' alterations related to anti-PD-1 treatment in 80 out of 1160 quantified proteins. Circulating PD-1 had the highest increase during anti-PD-1 treatment (log2-FC=2.03, p=0.0008) and in anti-PD-1 responders (log2-FC=2.09, p=0.005), but did not change in the MAPKis cohort. Targeted, antibody-based proteomics by PEA confirmed this observation. Anti-PD-1 responders had an increase in plasma proteins involved in T-cell response, neutrophil degranulation, inflammation, cell adhesion, and immune suppression. Furthermore, we discovered new associations between plasma proteins (eg, interleukin 6, interleukin 10, proline-rich acidic protein 1, desmocollin 3, C-C motif chemokine ligands 2, 3 and 4, vascular endothelial growth factor A) and PFS, which may serve as predictive biomarkers. CONCLUSIONS: We detected an increase in circulating PD-1 during anti-PD-1 treatment, as well as diverse immune plasma proteomic signatures in anti-PD-1 responders. This study demonstrates the potential of plasma proteomics as a liquid biopsy method and in discovery of putative predictive biomarkers for anti-PD-1 treatment in metastatic CM.

Evaluating the diagnostic utility of new line immunoassays for myositis antibodies in clinical practice: a retrospective study.

BACKGROUND: Myositis-associated antibodies (MAA) and myositis-specific antibodies (MSA) are detected in patients with idiopathic inflammatory myopathies (IIM); their role as diagnostic biomarkers is however still debated. The aim of our study was to assess the utility of MAA/MSA assessed by new line immunoassays in detecting myositis among neuromuscular patients. METHODS: We retrospectively analysed sera samples obtained from patients tested for myositis antibodies with the "Euroline: Autoimmune Inflammatory Myopathies 16Ag" and "myositis profile 3" kits (Mi-2, TIF1γ, MDA5, NXP2, SAE1, Jo-1, SRP, PL-7/12, EJ, OJ, Ro-52, Ku, PM-Scl75/100). First symptom, CK, EMG, muscle biopsy and diagnosis were also analysed. Using logistic regression analysis, two diagnostic models were built to evaluate the diagnostic power of MAA/MSA in distinguishing myositis patients from controls and other myopathies. RESULTS: 1229 patients were identified. 141 patients had a bioptic confirmed IIM; other diagnoses included: myopathy (n = 357), other neuromuscular diseases (n = 144) and no neuromuscular diseases (n = 587). The specificity was 95% for MSA and 89% for MAA, the sensitivity 20% and 22%, respectively. MAA showed no use in differentiating myositis patients from controls, whereas MSA had limited effect (OR = 5.165), compared to other variables as EMG (OR = 47.755) or CK > 2000 U/L (OR = 45.307). MSA were, however, the most useful parameter differentiating IIM from non-IIM patients (OR = 7.259), better than CK > 2000 U/L (OR = 4.033) and MAA (OR = 2.737). CONCLUSIONS: Line immunoassays for myositis antibodies show high specificity but low sensitivity. Their usefulness as diagnostic biomarkers widely depends on the clinical settings. Our study suggests that MSA/MAA should be used for confirmatory and differential diagnosis rather than for screening purposes in inflammatory myopathies.

CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: A systematic review.

INTRODUCTION: The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment. METHODS AND DATA ACQUISITION: We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD. RESULTS: We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review. DISCUSSION: Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.

Safety and efficacy of short- and long-term inspiratory muscle training in late-onset Pompe disease (LOPD): a pilot study.

BACKGROUND: In patients with late-onset Pompe disease, progressive respiratory muscle weakness with predominantly diaphragmatic involvement is a frequent finding at later stages of the disease. Respiratory muscle training (RMT) is an established therapy option for patients with several neuromuscular disorders including Duchenne muscular dystrophy. Forced voluntary muscle contractions of inspiration and/or expiration muscles enhance ventilation by increasing respiratory coordination, endurance, and strength. Efficacy of RMT in LOPD is rarely examined, and the clinical studies performed are difficult to compare because of different training programs and protocols. This impedes a useful statement and recommendation about the safety and efficacy of respiratory muscle training. METHODS: We conducted a monocentric unblinded single-arm pilot study in patients with LOPD to evaluate the safety and efficacy of inspiratory muscle training (IMT). The primary objective was to determine the efficacy of a 6-week repetitive IMT with a gradual increase of inspiratory resistance, measured by MIP (maximum inspiratory pressure) in the upright position. For statistical analysis, we used an A-B-C single subject design. The 6-week training-period A was followed by a 6-week non-training period B and an optional training period of 40 weeks in period C. The total study duration for the periods A, B and C was 52 weeks. Throughout the study, spirometry assessments (FCV, FEV1) and measurements of respiratory strength (MIP, MEP) were performed at defined time points, as well as capillary oximetry and capnometry, motor function test and patient's questionnaires for quality of life and dyspnea, measured by St. George's Respiratory Questionnaire (SGRQ) and MMRC-Dyspnea scale. For the cross-sectional comparison, a paired two-sided t test, and for the longitudinal comparison, a two-sample, two-sided t test were used. When data were not normally distributed, a Wilcoxon-Mann-Whitney test was added. Finally, the annual decline in FVC and FEV1 before and after IMT was compared. FINDINGS: 11 subjects were included in this pilot study. Overall, IMT was well tolerated. In four subjects, a total of six adverse events related to the study procedures were noticed. Training compliance was excellent in the first weeks of training, but declined continuously in the extension period. There was a significant increase in our primary outcome measure MIP within the 6-week period of frequent IMT with a mean of 15.7% (p =0.024; d =0.402). A significant increase was also seen after week 52 by a mean of + 26.4% (mean + 13.4 cmH2O, p =0.001, d =0.636). In the 6-week non-training interim-period (period B), the values remained stable, and there was no clinically meaningful decline in secondary outcome measures. The increase in MIP did not have any effect on secondary outcome measures like spirometry tests (FVC, FEV1), capillary blood gas analysis, motor function tests, patient's perceived quality of life or any significant change in dyspnea score. CONCLUSIONS: Frequent IMT improves MIP and thereby stabilizes and decelerates the decline of the diaphragm strength. The gradual increase of inspiratory resistance is well tolerated without any increase of side effects, as long as IMT is supervised and resistance is individually adjusted to the patient's perceived grade of exhaustion. Although we could not detect a significant impact on secondary outcome measures, IMT should be offered to all LOPD patients, especially to those who demonstrate a progressive decline in respiratory muscle function or are unable to receive ERT.

How to Interpret Abnormal Findings of Spirometry and Manometry in Myotonic Dystrophies?

BACKGROUND/AIM: Pulmonary function tests are used for screening respiratory insufficiency in patients with myotonic dystrophy (DM). We analysed the agreement between two different approaches in assessment of abnormal findings of forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP), in DM patients. METHODS: We used Cohen's κ- and Bangdiwala's B- statistic to compare the agreement between different cut-off values recommended by experts (ENMC) and the cut-off values based on the reference range (RR). We further analysed their sensitivity (Sn) and specificity (Sp) in detecting symptoms associated with respiratory insufficiency. RESULTS: The observed agreement was: 1) for FVC: κ= -0.002, B = 0.406; 2) for FEV1: κ= 0.944, B = 0.946; 3) for MIP: κ= 0.625, B = 0.674; and 4) for MEP: κ= 0.241, B = 0.373. Overall, RR cut-off values showed higher sensitivity, whereas the ENMC values showed higher specificity in detecting symptoms of respiratory involvement. CONCLUSIONS: The two approaches showed perfect agreement in assessment of FEV1, substantial agreement for MIP, and weak agreement for FVC and MEP. RR is an established method of assessment for spirometry and should be favoured because it takes variability within the population into account. Further development and validation of regression equations for RR calculations of predicted maximal respiratory pressures, with corresponding lower limits of normal, is required.The B statistic is more robust in assessing agreement between two diagnostic methods, resolving the issue of the κ paradox.

The disease burden of Multiple Sclerosis from the individual and population perspective: Which symptoms matter most?

BACKGROUND: MS symptoms affect many functional domains. Knowing the specific impact of symptoms on health-related quality of life (HRQoL) is vital for successful disease and symptom management in MS. We aimed at investigating how specific MS symptoms contribute to the disease burden in individuals and from a population perspective. METHODS: We included 855 Swiss Multiple Sclerosis Registry participants with a relapsing-remitting form (RRMS) or a progressive form (PMS). HRQoL was measured with the EuroQol 5-Dimension EQ-5D-index and EQ-Visual Analogue Scale (EQ-VAS) on 0-100% scales. Their associations with 20 symptoms, socio-demographic and clinical information were explored in median regression models, stratified by RRMS and PMS. RESULTS: We included 611 participants with RRMS and 244 with PMS. In RRMS, gait (-6.5%) and balance problems (-5.1%) had the largest EQ-5D-index reductions, and were also important at the population level (frequencies 45% and 52%). Fatigue, depression, and spasticity (frequencies 74.1%, 31%, 38%) also contributed to the population disease burden. In PMS, spasticity, paralysis, and bowel problems had the largest impact on EQ-5D-index, both at the individual and population levels. The largest impact on EQ-VAS at population level was associated in RRMS with balance problems, depression, dizziness, and spasticity, while in PMS with weakness, pain, and paralysis. CONCLUSIONS: While HRQoL at population level is most affected by balance problems, spasticity, and depression in RRMS, the biggest HRQoL losses in PMS are caused by spasticity, paralysis, weakness, and pain. Many symptoms with the largest effects in individuals substantially contribute to the population disease burden.