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Glycosylation of antibodies and the effects this has on inflammatory responses has concentrated predominately on the study of glycosylation moieties found in the Fc region of heavy chains. Light chain glycosylation and their ratios are relatively understudied. Nevertheless, variable glycosylation and ratio of {kappa} and {lambda} light chains have been associated with worse prognosis in myeloma and in tissue deposition - amyloidosis. The {kappa} & {lambda} light chains, of antibodies binding to SARS-CoV2 nucleocapsid and spike protein were analysed, using MALDI-ToF MS, in respect to their intensity, ratios, glycosylation patterns and any pattern changes correlating with COVID-19 severity. The molecular masses and signal intensity of {kappa} and {lambda} glycosylated and non-glycosylated light chains were measured for immunoglobulins isolated from plasma of sero-positive and sero-negative health care workers (HCW), and convalescent patients who had suffered from acute respiratory distress syndrome (ARDS). Overall, there was no significant changes in {kappa} to {lambda} ratio of total IgG (via protein G capture) antibodies between the groups. A non-statistically significant trend towards {lambda} light chains was found in antibodies against SARS CoV-2 Nucleocapsid and Spike proteins. However, detailed analysis of the molecular forms found a significant increase and bias towards un-glycosylated light chains and in particular un-glycosylated {kappa} light chains, in antibodies against SAR-CoV-2 spike protein, from convalescent COVID-ARDS patients. Here we have demonstrated a bias towards un-glycosylated {kappa} chains in anti-spike antibodies in those who suffered from ARDS as a result of SARS-CoV2 infection 3 months after recovery. How this relates to the immunopathology of COVID-19 requires further study.

Human ADA2 deficiency (DADA2) is an inborn error of immunity with a broad clinical phenotype which encompasses vasculopathy including livedo racemosa and lacunar strokes, as well as hemato-immunological features. Diagnosis is based on the combination of decreased serum ADA2 activity and the identification of biallelic deleterious alleles in the ADA2 gene. DADA2 carriers harbor a single pathogenic variant in ADA2 and are mostly considered healthy and asymptomatic. However, some DADA2 carriers present a phenotype compatible with DADA2. Here, we report ten patients from seven kindreds presenting with a phenotype indicative of DADA2, in whom only a single pathogenic variant (p.G47R, p.G47V, p.R169Q, p.H424N) was identified. To test whether being heterozygote for specific variants could explain the patients phenotype, we investigated the effect of the ADA2 missense variants p.G47A, p.G47R, p.G47V, p.G47W, p.R169Q, p.E328K, p.T360A, p.N370K, p.H424N and p.Y453C on ADA2 protein expression, secretion and enzymatic activity. Functional studies indicate that they exert a dominant negative effect on ADA2 enzymatic activity, dimerization and/or secretion. At the molecular level, heterozygosity for these variants mimics what is observed in DADA2. We conclude that humans with heterozygous dominant negative missense variants in ADA2 are at risk of DADA2. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/24317629v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@a7d7b9org.highwire.dtl.DTLVardef@143c268org.highwire.dtl.DTLVardef@19048daorg.highwire.dtl.DTLVardef@19f06f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

The prefusion Spike protein of SARS-CoV2 binds advanced glycation end product (AGE) glycated human serum albumin (HSA) and a higher mass, hyperglycosylated/glycated, IgG3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF MS). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct dilution and disulphide bond reduction method was development and applied to plasma samples from SARS-CoV2 seronegative (N = 30) and seropositive (N = 31) healthcare workers and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress syndrome (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass, AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for development of ARDS as a result of COVID-19 infection. Furthermore, rapid direct analysis of plasma samples by MALDI-ToF MS for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/21260186v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12bfa69org.highwire.dtl.DTLVardef@45344forg.highwire.dtl.DTLVardef@16d4f7forg.highwire.dtl.DTLVardef@17e5c34_HPS_FORMAT_FIGEXP M_FIG C_FIG

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSObjectivesC_ST_ABSAutologous blood transfusion (ABT) enhances athletes performance, is banned as doping by the World Anti-Doping Agency (WADA). Currently, there is no implemented detection method for ABT. Transfusion of ones own, long-term cryopreserved red blood cells (cryo-RBC) immediately increases circulating RBC count, hemoglobin mass, blood volume and oxygen carrying capacity, resulting in enhanced physical performance. Functional viablity of cryo-RBC are maintained for decades, but storage lesions lead to removal of damaged RBC from circulation days after transfusion, with remaining circulating cryo-RBC displaying normal half-life. MethodsThe cytosolic RBC peptidome from 22 human subjects (12 men and 10 women) was analyzed by UHPLC-MS/MS before and after ABT with cryo-RBC. As a control group and for investigation of confounders, 14 elite athletes and 5 recreational subjects were sampled multiple times, also at high altitude. ResultsHere we report alteration in the cytosolic peptidome of circulating RBC weeks after ABT, discriminating doped from non-doped human subjects. A valid discriminating multivariate model (OPLS-DA) based on <200 peptides was accomplished (R2/Q2 = 0.88/0.59, P CV-ANOVA < 0.0001, ROC AUC = 0.97). Models did not show bias for sex, high altitude or elite endurance training and racing. ConclusionIdentified peptides with low intra- and inter-individual variation, and high multivariate model weight and probability scores, create a direct method for the detection of autologous blood doping.

The asymptomatic disease stage in HIV-2 infection is approximately twice as long compared to in HIV-1 infection, still the majority of HIV-2 infected individuals progress to AIDS in the absence of antiretroviral treatment. In this study, we applied data-independent acquisition mass spectrometry analysis of blood plasma samples collected from HIV negative, and HIV-1 or HIV-2 infected individuals in Guinea-Bissau with an estimated date of HIV infection, to explore associations between plasma proteome alterations and HIV disease progression. In total, 609 proteins were quantified and mapped towards publicly available data on tissue-enhanced genes, to provide insight on the tissue-specific origin of the detected proteins. Here we identified ten proteins that could differentiate between faster and slower HIV disease progression. The analysis also suggested a larger leakage of proteins from the sigmoid colon in HIV-1 compared to HIV-2 infection. Moreover, the levels of sigmoid colon and spleen tissue proteins were associated with disease progression among all HIV infected individuals. In conclusion, these results encourage further research on the role of both target and bystander cells in HIV disease progression.

BackgroundCOVID-19 or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared throughout the World and currently affected more than 3.6 million people and caused the death of around 252,000 people. The novel strain of the coronavirus disease is transmittable at a devastating rate with a high rate of severe hospitalization even more so for the elderly population. Currently around 50,000 patients are in a seriously critical situation. Although 1.2 million patients recovered from the disease there are still more than 2.1 Million active cases. Naso-oro-pharyngeal swab samples as the first step towards detecting suspected infection of SARS-CoV-2 provides a non-invasive method for PCR testing at a high confidence rate. Furthermore, proteomics analysis of PCR positive and negative nasooropharyngeal samples provides information on the molecular level which highlights disease pathology. MethodSamples from 15 PCR positive cases and 15 PCR negative cases were analyzed with nanoLC-MS/MS to identify the differentially expressed proteins. ResultsProteomic analyses identified 207 proteins across the sample set and 17 of them were statistically significant. Protein-protein interaction analyses emphasized pathways like Neutrophil degranulation, Innate Immune System, Antimicrobial Peptides. ConclusionNeutrophil Elastase (ELANE), Azurocidin (AZU1), Myeloperoxidase (MPO), Myeloblastin (PRTN3), Cathepsin G (CTSG) and Transcobalamine-1 (TCN1) were found to be significantly altered in naso-oropharyngeal samples of SARS-CoV-2 patients. The identified proteins are linked to alteration in the innate immune system specifically via neutrophil degranulation and NETosis.

The Fc{gamma} receptors (Fc{gamma}Rs) act as modulators of the immune system and have previously been shown to play a role in immune disorders such as systemic lupus erythematosus and immune thrombocytopenic purpura. Thus far, their role in primary immunodeficiencies (PID), including common variable immunodeficiency disorders (CVID), has not been studied. In this paper we explored whether there is an association between the following single nucleotide polymorphisms (SNPs) and CVID: FCGR2A H131R (rs1801274), FCGR2B I232T (rs1050501), and FCGR3A F158V (rs396991). We compared the genotypes of a cohort of 83 patients with PID, including 56 with CVID, against controls. We found a significant difference between our mixed PID cohort and controls at the FCGR2A H131R SNP (X2 =7.884, p=0.019). There was not a significant difference at either of the other SNPs studied. Further, we examined the effect of FCGR SNPs on the incidence of the most common CVID complications within our cohort: anaemias, organ-specific autoimmunity, bronchiectasis, splenomegaly, granulomata, and cytopenias. We found no significant association between SNPs and the development of these complications. In summary, we have shown that there is a link between the FCGR2A H131R SNP and the development of a PID.

Long-COVID (LC) is a serious clinical condition characterised by debilitating fatigue together with a wide array of symptoms that significantly reduce the quality of life of patients. Currently no holistic or even symptom specific treatment options are available, likely due to both a lack of insight into the disease processes that drive LC symptoms and an extreme heterogeneity in patients profiles. We characterised patients and post infection controls, with respect to their immunological profiles with a non-exhaustive panel of biomarkers rationalised based on their potential role in driving symptoms. We observed that the patients symptoms could be grouped into 4 clusters suggesting possible stratification. Systemic inflammation persisted and did not normalize over time in LC. This was not related to persistent SARS-CoV-2 infection, as the presence of circulating N-protein was detected similarly in both patients and controls. No obvious deviation in B-cells and monocytes profiles were observed with minor changes for NK-cells (CD62L+/CD16+/HLA-DR+). Major changes affected CD4+T-cells (and to a lesser extent CD8+T-cells) with respect to exhaustion (PD1+/LAG3+/CD44+), regulation (Treg) and differentiation (naive/memory-CD62L+). Several candidate biomarkers (cytokines, microRNAs, phosphate metabolism) were present more frequently in LC at high levels and provided information on underlying disease processes. While frequencies of candidate autoantibody+ participants were not different, levels of some antibodies were higher in LC. Yet none of these candidates stood out as a universal biomarker for LC, with the exception of CRP (73% cases), and loss of Treg (50%). However, we confirmed that several overlapping underlying aetiologies may be involved in this complex disease. Specific groups of biomarkers also associated with the 4 cluster of patients. Although to be taken with caution due to small numbers, 3 biomarkers discriminated controls from patients (Treg/CD4+PD1+/CD4+CD161+), others were associated with symptoms recovery (low IL10/IL12/IL4 and high miR766) or deterioration (high CD4+CD38+/ CD8+naiveCD62L+/low IL2) over 12 months. This study provides rational for developing targeted therapeutic strategies as well as biomarkers to stratify LC patients most likely to respond.

Initial symptoms of COVID-19 infection depend on viral replication, while hyperinflammation is a hallmark of critical illness and may drive severe pneumonia and death. Among the mechanisms potentially involved in the hyperinflammatory state, we focused on the unfolded protein response, because the IRE1-XBP1 branch can be activated as result of the endoplasmic reticulum stress produced by the overwhelming synthesis of viral components and synergizes with Toll-like receptor signaling to induce cytokine expression. Viral RNA may trigger the IRE1-XBP1 branch via TLR7/8 activation and like TLR2 and TLR4 may underpin cytokine expression trough XBP1 splicing (sXBP1). The expression of IL1B, IL6, and TNF mRNA in bronchoalveolar aspirates (BAAs) were higher in COVID-19 patients under mechanical ventilation and intubation who showed sXBP1. The scrutiny of monocytic/macrophagic markers during active infection showed a reduction of those involved in antigen presentation and survival, as well as the IFN stimulated gene MX1. These changes reverted after infection tests turned negative. In contrast, the expression of the mRNA of the serine protease TMPRSS2 involved in S protein priming showed a high expression during active infection. TLR8 mRNA showed an overwhelming expression as compared to TLR7 mRNA, which suggests the presence of monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a positive-sense, single-stranded RNA (+ssRNA) like SARS-CoV-2 RNA, induced sXBP1 and the expression of IL-1{beta}, IL-6, and TNF at mRNA and protein levels. These responses were blunted by the IRE1 ribonuclease inhibitor MKC8866. Given the analogies between the results observed in BAAs and the effects induced by +ssRNA in MDDCs, IRE1 ribonuclease inhibition might be a druggable target in severe COVID-19 disease. O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/22269752v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@13b04b3org.highwire.dtl.DTLVardef@1b1af7corg.highwire.dtl.DTLVardef@780104org.highwire.dtl.DTLVardef@8ad0ba_HPS_FORMAT_FIGEXP M_FIG C_FIG Author summaryCOVID-19 pandemics put an unprecedented pressure on health systems. The need of new therapies urged research on the mechanisms triggered by the interaction of SARS-CoV-2 virus with host cells and the ensuing pathophysiology driving pneumonia and multiorgan failure. Hyperinflammation soon appeared as a mechanism involved in mortality that could even proceed after viral infection comes to an end. Hyperinflammation is supported by an inappropriate production of cytokines, and this explains the use of the term cytokine storm to refer to this phase of the disease. Given that insight into the molecular mechanisms driving cytokine storm should focus on the interaction of viral components with immune cells, experiments addressing the effect of viral components on its cognate receptors were carried out. It was observed that viral RNA induces a cytokine pattern like the one observed in bronchoalveolar aspirates of COVID-19 patients with critical disease. Overall, the study revealed that both cell organelle overload and receptors involved in the recognition of viral RNA may team up to induce proinflammatory cytokines. This mechanism can be exploited to develop new treatments for COVID-19 disease.

Multisystem inflammatory syndrome in children (MIS-C or PIMS) is a rare but serious complication after an infection with SARS-CoV-2. A possible involvement of pathogenetically relevant autoantibodies has been discussed. Recently neutralizing autoantibodies against anti-inflammatory receptor antagonists progranulin (PGRN) and IL-1-receptor antagonist (IL-1-Ra) were discovered in adult patients with critical COVID-19. Plasma of an index case with severe PIMS/MIS-C was analyzed for autoantibodies against IL-1-Ra and PGRN. The study was extended by a case series of 12 additional patients. In addition to ELISA for of antibodies, IL-1-Ra plasma levels were determined and IL-1-Ra was analyzed by Western-blot and isoelectric focusing. Functional activity of the autoantibodies was examined in vitro with IL-1{beta} reporter assays. Antibodies against IL-1-Ra could be detected in 10 of 13 (76.9%) patients with PIMS/MIS-C, but not in controls. In contrast to critical COVID-19 in adults, no IL-1-Ra antibodies of the IgM class were detected in PIMS/MIS-C. IL-1-Ra-antibodies exclusively belonged to IgG1. No antibodies directed against PGRN were detected. Western blots and ELISA showed a concomitant reduction of free IL-1-Ra plasma levels in the presence of IL-1-Ra-antibodies. The antibodies inhibited IL-1-Ra function in IL-1{beta} reporter cell assays. Notably, an additional, hyperphosphorylated, transiently occurring atypical isoform of IL-1-Ra was observed in all IL-1-Ra autoantibody-positive patients. To conclude, IL-1-Ra autoantibodies were observed in high frequency in children with PIMS/MIS-C. They may represent a diagnostic and pathophysiologically relevant marker for PIMS/MIS-C. Their generation is likely to be triggered by an atypical, hyperphosphorylated isoform of IL-1-Ra.

BackgroundHereditary angioedema (HAE) is a rare and potentially life-threatening disease. Noninvasive and disease-specific biomarkers are needed for the early diagnosis and clinical management of HAE. ObjectiveWe sought to apply untargeted proteomics profiling and targeted proteomics validation to identify pathogenic mechanisms and candidate biomarkers of HAE. MethodsData-independent acquisition (DIA)-based proteomics profiling was performed in urine samples of HAE patients and healthy controls. Bioinformatics analysis was used for functional annotation and pathway enrichment of differentially expressed proteins. Furthermore, promising biomarker candidates were validated in another independent clinical cohort using parallel reaction monitoring (PRM) targeted proteomics quantification. ResultsDifferent urinary proteomics profiles were identified among type 1 HAE, type 2 HAE and healthy controls. A total of 401 differentially expressed proteins were identified between type 1 HAE and healthy controls. Bioinformatics analysis showed that several biological processes and pathways were significantly enriched in HAE, including complement and coagulation cascades, cell adhesion molecules, immune response, proteolysis, and bradykinin catabolic process. Moreover, a promising biomarker panel (C1-INH, KNG1 and EGF) were validated in another independent clinical cohort. The area under the curve (AUC) value of this biomarker panel reached 0.910 for HAE diagnosis (sensitivity: 91.7, specificity: 88.9, P <0.001). ConclusionsThis study describes the first application of a DIA-PRM workflow to identify noninvasive and disease-specific biomarkers in HAE patients. These findings will contribute to the pathogenesis research and biomarker discovery of HAE. Key MessagesO_LIDifferent urinary proteomics profiles were identified among type 1 HAE, type 2 HAE and healthy controls. C_LIO_LISeveral biological processes and pathways were significantly enriched in HAE, including complement and coagulation cascades, cell adhesion molecules, immune response, proteolysis, and bradykinin catabolic process. C_LIO_LIA urinary biomarker panel (C1-INH, KNG1, and EGF) could be a promising noninvasive diagnostic tool for HAE. C_LI

The COVID-19 pandemic is an unprecedented global challenge. Highly variable in its presentation, spread and clinical outcome, novel point-of-care diagnostic classifiers are urgently required. Here, we describe a set of COVID-19 clinical classifiers discovered using a newly designed low-cost high-throughput mass spectrometry-based platform. Introducing a new sample preparation pipeline coupled with short-gradient high-flow liquid chromatography and mass spectrometry, our methodology facilitates clinical implementation and increases sample throughput and quantification precision. Providing a rapid assessment of serum or plasma samples at scale, we report 27 biomarkers that distinguish mild and severe forms of COVID-19, of which some may have potential as therapeutic targets. These proteins highlight the role of complement factors, the coagulation system, inflammation modulators as well as pro-inflammatory signalling upstream and downstream of Interleukin 6. Application of novel methodologies hence transforms proteomics from a research tool into a rapid-response, clinically actionable technology adaptable to infectious outbreaks. Highlights- A completely redesigned clinical proteomics platform increases throughput and precision while reducing costs. - 27 biomarkers are differentially expressed between WHO severity grades for COVID-19. - The study highlights potential therapeutic targets that include complement factors, the coagulation system, inflammation modulators as well as pro-inflammatory signalling both upstream and downstream of interleukin 6.

ATPase inhibitory factor 1 (IF1) is a 9.5 kDa protein that binds to mitochondrial and plasma membrane ATP synthase and selectively inhibits ATP hydrolysis. Recently, IF1 was identified in systemic circulation in humans. IF1 appeared as an independent determinant of HDL-cholesterol with lower levels in coronary heart disease (CHD) patients. Moreover, IF1 was also found to negatively associate with mortality in these patients, supporting the notion that circulating IF1 could be a promising biomarker of cardiovascular disease. However, in previous studies, IF1 was quantified by a non-standardized competitive enzyme-linked immunosorbent assay (ELISA). Herein, we have validated a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) enabling the accurate quantification of IF1 in human plasma. Plasma IF1 was trypsin-digested through an optimized procedure before LC-MS/MS analysis. The method was successfully validated over 4 independent experiments into the range of 100-1,500 ng/mL. Intra- and inter-assay variation coefficients had never exceeded 14.2% and accuracy ranged between 95% and 102% for the selected EAGGAFGK peptide marker. Subsequently, the results of the LC-MS/MS method were compared with those obtained using ELISA in 204 individuals from the GENES study. We found that IF1 plasma levels obtained using both techniques were strongly correlated (r =0.89, p <0.0001), while the Bland-Altman plot did not indicate any major statistically significant differences. To clinically validate LC-MS/MS, we confirmed the positive correlation between IF1 plasma levels and HDL-cholesterol (r =0.38, p <0.0001). Besides, we found lower IF1 plasma levels in CHD patients compared to controls (431{+/-}132 ng/mL and 555{+/-}173 ng/mL, respectively; p <0.0001). Hence, it can be concluded that the presented LC-MS/MS method provides a highly specific strategy for IF1 quantification in human plasma and could be proposed as a reference technique. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/20097816v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@19e95f7org.highwire.dtl.DTLVardef@87bf40org.highwire.dtl.DTLVardef@1d0a33org.highwire.dtl.DTLVardef@88d4cc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIATPase inhibitory factor 1 (IF1) is a biomarker of coronary heart disease in humans. C_LIO_LIThus far, IF1 plasma concentrations have been determined by non-standardized competitive ELISA. C_LIO_LIHere, we have developed and validated an LC-MS/MS assay for the accurate IF1 quantification in human plasma. C_LIO_LIOur LC-MS/MS assay is rapid and incurs reasonable costs, thus making it amenable for clinical practice. C_LI

BackgroundThe human urine peptidome reflects physiological and pathological states, making it a valuable resource for biomarker discovery. However, endogenous peptides often exist as cascades of truncated variants, complicating comparative analyses. To address this, we developed a "peptide cluster" approach, grouping overlapping peptides into representative clusters for robust statistical evaluation. MethodsUrine samples from 55 healthy volunteers (23 males, 32 females) were analyzed via LC-MS/MS. Identified peptides were assembled into clusters based on sequence overlap, with the longest peptide designated as the "precursor" and truncated variants as "truncated" ResultsWe identified 30,471 endogenous peptides, assembled into 13,163 peptide clusters--the largest urinary peptidome dataset to date. Gender-specific differences were observed in 26 clusters, while 57 clusters correlated significantly with age. Notably, male-enriched clusters included hepcidin-25 and progranulin-derived peptides, whereas female-enriched clusters were linked to immunoglobulin gamma-1. Age-associated clusters highlighted collagen degradation patterns, consistent with extracellular matrix remodeling. ConclusionOur peptide clustering approach facilitated a comprehensive characterization of the endogenous peptidome, capturing the diversity of naturally occurring truncated peptide forms. The resulting age- and sex-specific peptide clusters serve as a valuable reference framework for future investigations into disease-associated biomarkers.

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of Von Willebrand Factor, platelet factor 4,serum amyloid A, -2antiplasmin E-selectin, and platelet endothelial cell adhesion molecule-1, in the soluble part of the blood. It was noteworthy that the mean level of -2-antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We also determined that by individually adding E-selectin and PECAM-1 to healthy blood, these molecules may indeed be involved in protein-protein interactions with plasma proteins (contributing to microclot formation) and platelet hyperactivation. This investigation was performed as a laboratory model investigation and the final exposure concentration of these molecules was chosen to mimic concentrations found in Long COVID. We conclude that presence of microclotting, together with relatively high levels of six inflammatory molecules known to be key drivers of endothelial and clotting pathology, points to thrombotic endotheliitis as a key pathological process in Long COVID. This has implications for the choice of appropriate therapeutic options in Long COVID. SENTENCE SUMMARYThe presence of fibrinaloid microclots and multiple inflammatory molecules in the soluble part of blood points to thrombotic endotheliitis as a key pathological process in Long COVID.

COVID-19 syndrome does not occur in all who are infected with SARS-CoV-2, and symptoms vary. The anti-SARS CoV-2 Spike immune responses is confounded by the Spike proteins ability to bind Ig{gamma}3 heavy chains. This appears to be via sialic acid glycans found on the O-Linked glycosylation moieties of this heavy chain extended neck domain. Furthermore glycosylation of light chains, particularly Kappa ({kappa}), is an associated feature of antibodies binding to SARS-CoV-2 antigens nucleocapsid and Spike protein. COVID-19 recovered patients had increased IgG1 and IgM levels and un-glycosylated {kappa} light chains; possibly In order to counter this immune system subjugation of IgG3. These molecular finding, together with our previous finding that Spike protein binds glycated human serum albumin (HSA), may explain the micro-vascular inflammatory clots that are a causative feature of COVID-19 acute respiratory syndrome (ARDS). The postulated molecular sequelae are that SARS-CoV-2 virion, entering the blood circulation, being coated with IgG3 and glycated HSA forms a colloid and deposits into micro-focal clots which are also inflammatory. It is not that all IgG3 and albumin is being bound by the virus; this depends on the affinity the SARS-CoV2 virion has for binding an individuals IgG3 and albumin due to glycosylation and glycation status. The degree of glycosylation and terminal sialyation of an individuals antibodies is both a genetic and age-maturity dependant feature of the immune system. The degree of HSA glycation is also age related feature particularly related to type 2 diabetes. Thereby establishing the molecular basis of the association of severe COVID-19 disease syndrome and deaths with diabetes, metabolic disorders, and old age. Furthermore, already having cardiovascular disease, with hardened arteries, SARS-CoV2-glycated HSA-IgG3 deposition is going to exacerbate an already compromised circulatory physiology. The binding of IgG3 might also drives a shift in the immune repertoire response to SAR-CoV-2 anti-spike antibodies of increased IgG1 and prolonged IgM levels. This may be associated with Long Covid. In summary, SARS-CoV-2 Spike protein binding of IgG3, via sialic acid glycan residues, along with increased glycosylated {kappa}-light chains and glycated-HSA may form a focal amyloid-like precipitate within blood vessels which in turn leads to the inflammatory micro-thrombosis characteristic of COVID-19 immuno-pathology.

BackgroundTreatment of severely injured patients represents a major challenge, in part due to the unpredictable risk of major adverse events, including death. Preemptive personalized treatment aimed at preventing these events is a key objective of patient management; however, the currently available scoring systems provide only moderate guidance. Molecular biomarkers from proteomics/peptidomics studies hold promise for improving the current situation, ultimately enabling precision medicine based on individual molecular profiles. MethodsTo test the hypothesis that proteomics biomarkers could predict patient outcomes in severely injured patients, we initiated a pilot study involving consecutive urine sampling (on days 0, 2, 5, 10, and 14) and subsequent peptidome analysis using capillary electrophoresis coupled to mass spectrometry (CE-MS) of 14 severely injured patients and two additional ICU patients. The urine peptidomes of these patients were compared to the urine peptidomes of age- and sex-matched controls. Previously established urinary peptide-based classifiers, CKD274, AKI204, and CoV50, were applied to the obtained peptidome data, and the association of the scores with a combined endpoint (death and/or kidney failure and/or respiratory insufficiency) was investigated. ResultsCE-MS peptidome analysis identified 281 peptides that were significantly altered in severely injured patients. Consistent upregulation was observed for peptides from A1AT, FETUA, and MYG, while peptides derived from CD99, PIGR and UROM were consistently reduced. Most of the significant peptides were from different collagens, and the majority were reduced in abundance. Two of the predefined peptidomic classifiers, CKD273 and AKI204, showed significant associations with the combined endpoint, which was not observed for the routine scores generally applied in the clinics. ConclusionsThis prospective pilot study confirmed the hypothesis that urinary peptides provide information on patient outcomes and may guide personalized interventions based on individual molecular changes. The results obtained allow the planning of a well-powered prospective trial investigating the value of urinary peptides in this context in more detail.

Mevalonate kinase (MVK) deficiency, a rare autosomal recessive disease, significantly impacts metabolism and immunity, leading to mevalonic aciduria in severe cases and hyper-IgD syndrome (HIDS) in partial deficiency. These conditions arise due to disruptions in the mevalonate pathway, which is essential metabolic pathway responsible for the synthesis of non-sterol isoprenoids and other molecules. The resulting metabolic blockade triggers autoinflammatory responses, primarily due to deficient isoprenoid intermediates such as geranylgeranyl pyrophosphate (GGPP). This first reported pilot study evaluates the safety and efficacy of dietary geranylgeraniol supplementation (GG) in three patients with HIDS. Over three months, GG supplementation showed no liver toxicity and did not alter lipid profiles. Although GG did not rise the plasma levels of GGPP, the plasma proteomics showed significant changes induced by GG. Proteomic analysis further revealed that GG supplementation can reverse some of the features of HIDS-specific plasma protein signature, highlighting its potential to modulate inflammation and protein prenylation pathways. These findings suggest that GG supplementation could be a promising metabolic intervention to mitigate inflammation in HIDS, warranting further, more targeted investigation in larger clinical trials.

BackgroundAntinuclear antibodies (ANA) and extractable nuclear antigens (ENA) are crucial biomarkers for the diagnosis of autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), Sjogrens syndrome, systemic sclerosis, and polymyositis. In the present study, we assessed the most frequent ANA patterns associated with the most detectable ENA antigen (Ag) that could be used as a diagnostic and efficient prognostic marker of AID. Materials and MethodsThe primary objective of this study was to investigate the association between immunofluorescence (IF) ANA and ENA in patients with AIDs. This was a retrospective cross-sectional study. The study was performed at the Immunology Department of the Armed Forces Institute of Pathology, Rawalpindi. Retrospective data from 76 patients were tested for ANA and ENA from June 2020 to Nov 2020. ResultsA total of 76 patients comprising 14 (18.4%) males and 62(81.6%) females were tested for AIDs. The most frequent pattern among AID patients was coarse speckled, followed by the peripheral ANA pattern. The most frequent ENA Ags were Sjogrens syndrome A (SSA) and B (SSB). SSA was significantly associated with coarse speckled and peripheral ANA patterns, whereas SSB was associated with coarse speckled ANA patterns. These associations are relevant for accurate diagnosis of autoimmune diseases. ConclusionSSA was associated with coarse speckled and peripheral ANA patterns, whereas SSB was associated with coarse speckled ANA patterns. The ANA patterns were significantly associated with ENA antigens.

Coronaviruses are sharing several protein regions notable the spike protein (S) on their enveloped membrane surface, with the S1 subunit recognizing and binding to the cellular receptor, while the S2 subunit mediates viral and cellular membrane fusion. This similarity opens the question whether infection with one coronavirus will confer resistance to other coronaviruses? Investigating patient serum samples after SARS-CoV-2 infection in cross-reactivity studies of immunogenic peptides from Middle East respiratory syndrome coronavirus (MERS-CoV), we were able to detect the production of antibodies also recognizing MERS virus antigens. The cross-reactive peptide comes from the heptad repeat 2 (HR2) domain of the MERS virus spike protein. Indeed, the peptide of the HR2 domain of the MERS spike protein, previously proven to induce antibodies against MERS-CoV is sharing 74% homology with the corresponding sequence of SARS-CoV-19 virus. Sera samples of 47 convalescent SARS-CoV-2 patients, validated by RT-PCR-negative testes 30 days post-infection, and samples of 40 sera of control patients (not infected with SARS-CoV-2 previously) were used to establish eventual cross-bind reactivity with the MERS peptide antigen. Significantly stronger binding (p< 0.0001) was observed for IgG antibodies in convalescent SARS-CoV-2 patients compared to the control group. If used as an antigen, the peptide of the HR2 domain of the MERS spike protein allows discrimination between post-Covid populations from non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection has the ability to recognize and probably decrease infectiveness of MERS-CoV infections as well as other coronaviruses. The high homology of the spike protein domain suggests in addition that the opposite effect can also be true: coronaviral infections producing cross-reactive antibodies affective against SARS-CoV-19. The collected data prove in addition that despite the core HR2 region being hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections.