The Importance of Acute Phase Proteins in the Evaluation of Critical Covid-19 Patient Prognosis

Objectives: Identifying COVID-19 patients with risk of adverse outcomes at first admission to the intensive care unit has several diagnostic challenges. The concentration of acute phase proteins synthesized by the liver increases or decreases markedly in the serum following inflammation and infection. This study aimed to investigate the predictive value of acute phase proteins in critically ill COVID-19 patients and to evaluate the efficacy of inflammatory markers in predicting mortality risk in the intensive care unit. Material-Methods: A retrospective study was conducted in critically ill COVID-19 patients treated in the intensive care unit. Overall, 123 patients with ARDS and/or multi-organ dysfunction were included in the first 24 hours of admission to intensive care unit. After 28 days, groups of survived (n=54) and dead patient (n=69) or groups of patients with (n=83) and without (n=40) invasive mechanical ventilation were formed. Serum amyloid A, C-reactive protein, albumin, and prealbumin values considered as acute phase proteins within the first 24 hours of admission to the intensive care unit were compared between groups. Result(s): Albumin and prealbumin levels significantly decreased in dead patients (p=0.011, p<0.001, respectively) and were mechanically ventilated patients (p=0.010, p=0.006, respectively). The Serum amyloid A levels in mechanically ventilated patients significantly increased (p=0.022). Conclusion(s): Low prealbumin and albumin levels and high serum amyloid A levels during admission to ICU can be used as a prognostic marker of disease severity and mortality.

Isolation and Characterization of Protein Complexes of Overexpressed Human Serum Amyloid A from Cytokines-Induced Hepg2 Cells

Serum Amyloid A (SAA) is an apolipoprotein found in the serum of many vertebrate species and is associated with the acute-phase reaction in the body with expression levels reaching up to a 1000-fold increase. The loss of its alpha-helix conformation during its expression peak is directly linked to secondary amyloidosis. Recent studies have been suggested to play a role in cholesterol and HDL metabolism, retinol transport and tumor pathogenesis. Moreover, high SAA concentration in blood have been correlated with severe symptoms or death in patients with COVID-19. However, how this protein is involved in so many diseases is uncertain or not completely understood. Therefore, the purpose of this research is to determine which protein-protein interactions with SAA occur in human cells, and to predict its biochemical role based on new discovered complexes. Two major isoforms overexpressed during an acutephase reaction, human SAA1 and SAA2, are the focus of this study. Both are primarily produced in hepatocytes. HepG2 cells were cultured and induced with interleukin-1b, interleukin-6, LPS and retinol. Protein complexes associated with SAA will be isolated through a co-Immunoprecipitation technique, resolved by SDS-PAGE, and characterized by mass spectrometry. Our hypothesis focus on those protein complexes with SAA to explain how this protein lead other undiscovered metabolic pathways involved in both cellular and survival regulation. Special thanks to The Science and Technology Competency & Education Core (Stce) for Undergraduate and Graduate Junior Research Associates Working Program from the Puerto Rico IDeA Network Biomedical Research Excellence for funding part of this research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Plasma markers of Covid-19 severity: A pilot study

SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect certain disease presentations. Our cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins by nephelometry, full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection. When compared to healthy controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, alpha-1-acid glycoprotein, alpha1-antitrypsin (AAT), ceruloplasmin, haptoglobin, and highsensitivity C-reactive protein. The concentrations of alpha1-antichymotrypsin, alpha2-macroglobulin and serum amyloid A proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose and sialylated di-antennary glycans, while the non-sialylated di-antennary glycan A2G2 significantly decreased in COVID-19 patients compared to controls. COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight involvement of different pathophysiological mechanisms and grant further investigations.

Investigation of some acute phase proteins, cytokines and hepcidin values in feline enteric corona virus antibodies and feline infectious peritonitis antigen positive cats

Feline Infectious Peritonitis (FIP)is a fatal disease caused by Feline coronaviruses. The causative agent is Feline Infectious Peritonitis Virus, a mutation of Feline Enteric Coronavirus. Feline Corona Virusinfection is very common in the cat population.In Feline Corona Virus infected cats, the development of FIP depends on the cat's immune response. FIP disease is more common in young and old cats because young and old animals have a weaker immune system. The acute phase response is a complex systemic reaction that occurs as a response to acute or chronic inflammatory processes such as infection, neoplasia or immunological disorders, tissue damage, trauma, and surgery. The study material was composed of15 cats with FIP (study group) and 10 healthy cats (control group). Serum amyloid A (SAA), haptoglobin (Hp), alpha1-acid glycoprotein (AGP), albumin, interleukin-6 (IL-6), hepcidin, alanine-amino transferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen(BUN), and creatinine levels were measured in the serum collected from both groups. There was no difference between the wet and dry FIP in albumin values (p<0.05).Haptoglobin, alpha1-acid glycoprotein, SAA, IL-6, and hepcidin values were significantly different between the two groups (P<0.001). It was also concluded that hepcidinhas a potential for use as a biomarker in Feline Infectious Peritonitis disease like other acute phase proteins.Copyright © 2023, Sima Sahinduran, Metin Koray Albay, Mehmet Karaca, Mehmet Cagri Karakurum, Reyda Kiyici

Serum amyloid A-A potential therapeutic target for hyper-inflammatory syndrome associated with COVID-19.

Serum amyloid-A (SAA) is associated with inflammatory disorders such as rheumatoid arthritis, Familial Mediterranean Fever, sarcoidosis, and vasculitis. There is accumulating evidence that SAA is a reliable biomarker for these autoinflammatory and rheumatic diseases and may contribute to their pathophysiology. Hyperinflammatory syndrome associated with COVID-19 is a complex interaction between infection and autoimmunity and elevation of SAA is strongly correlated with severity of the inflammation. In this review we highlight the involvement of SAA in these different inflammatory conditions, consider its potential role and discuss whether it could be a potential target for treatment of the hyperinflammatory state of COVID-19 with many potential advantages and fewer adverse effects. Additional studies linking SAA to the pathophysiology of COVID-19 hyper-inflammation and autoimmunity are needed to establish the causal relationship and the therapeutic potential of inhibitors of SAA activity.

Comparison of the ELISA method with the nephelometric method for determination of serum amyloid A in patients with COVID 19.

This study aimed to examine the agreement of the serum amyloid A (SAA) values determined using the ELISA test and the nephelometric automated method. This study included 80 serum samples obtained from patients with COVID-19. Samples were determined using ELISA and the nephelometric method. Wilcoxon signed ranks test showed a statistically significant difference in the calculated median values (Z = -2.432, p = 0.015). The correlation between methods was statistically significant (r = 0.603, p < 0.0001). Bland Altman analysis showed a bias of 56.6 mg/L and a relative bias of 7.4% between the methods. The results of this study indicate that further studies are needed that will examine the compliance between the ELISA and the nephelometric method for determining SAA, and the results must be carefully interpreted based on the method used.

Elevated Serum Amyloid A Levels Contribute to Increased Platelet Adhesion in COVID-19 Patients.

Coronavirus disease-19 (COVID-19) patients are prone to thrombotic complications that may increase morbidity and mortality. These complications are thought to be driven by endothelial activation and tissue damage promoted by the systemic hyperinflammation associated with COVID-19. However, the exact mechanisms contributing to these complications are still unknown. To identify additional mechanisms contributing to the aberrant clotting observed in COVID-19 patients, we analyzed platelets from COVID-19 patients compared to those from controls using mass spectrometry. We identified increased serum amyloid A (SAA) levels, an acute-phase protein, on COVID-19 patients' platelets. In addition, using an in vitro adhesion assay, we showed that healthy platelets adhered more strongly to wells coated with COVID-19 patient serum than to wells coated with control serum. Furthermore, inhibitors of integrin aIIbß3 receptors, a mediator of platelet-SAA binding, reduced platelet adhesion to recombinant SAA and to wells coated with COVID-19 patient serum. Our results suggest that SAA may contribute to the increased platelet adhesion observed in serum from COVID-19 patients. Thus, reducing SAA levels by decreasing inflammation or inhibiting SAA platelet-binding activity might be a valid approach to abrogate COVID-19-associated thrombotic complications.

SARS-CoV-2-Induced Amyloidgenesis: Not One, but Three Hypotheses for Cerebral COVID-19 Outcomes.

The main neuropathological feature of Alzheimer's disease (AD) is extracellular amyloid deposition in senile plaques, resulting from an imbalance between the production and clearance of amyloid beta peptides. Amyloid deposition is also found around cerebral blood vessels, termed cerebral amyloid angiopathy (CAA), in 90% of AD cases. Although the relationship between these two amyloid disorders is obvious, this does not make CAA a characteristic of AD, as 40% of the non-demented population presents this derangement. AD is predominantly sporadic; therefore, many factors contribute to its genesis. Herein, the starting point for discussion is the COVID-19 pandemic that we are experiencing and how SARS-CoV-2 may be able to, both directly and indirectly, contribute to CAA, with consequences for the outcome and extent of the disease. We highlight the role of astrocytes and endothelial cells in the process of amyloidgenesis, as well as the role of other amyloidgenic proteins, such as fibrinogen and serum amyloid A protein, in addition to the neuronal amyloid precursor protein. We discuss three independent hypotheses that complement each other to explain the cerebrovascular amyloidgenesis that may underlie long-term COVID-19 and new cases of dementia.

Investigation of hemogram, oxidative stress, and some inflammatory marker levels in neonatal calves with escherichia coli and coronavirus diarrhea.

Calf diarrhea is the most common disease affecting calves in the neonatal period resulting in economic losses. Although predisposing factors play a role in the etiology of the disease, in most cases, different pathogens are involved in the development of the infection. In this study, hemogram data, glutathione and malondialdehyde levels were examined to determine lipid peroxidation and glutathione levels in E. coli- and coronavirus-infected calves. Serum amyloid A and calprotectin levels were also analyzed to determine inflammatory status. The study included a total of 45 female Montofon calves aged 0-1 week, including the E. coli group (15 calves), the coronavirus group (15 calves), and the control group (15 calves). Analysis revealed that total leukocyte, neutrophil, lymphocyte, malondialdehyde, serum amyloid A, and calprotectin levels increased in the coronavirus-infected calves compared with the E. coli group and the control group. In contrast, the levels of glutathione, one of the antioxidant markers, decreased. In conclusion, the main findings related to the determination of inflammation and oxidative status were characterized by the presence of E. coli and coronavirus diarrhea, and it is suggested that future studies may be guided by the fact that inflammatory conditions are higher in viral disease than in bacterial infection.

Human Serum Amyloid a Impaired Structural Stability of High-Density Lipoproteins (HDL) and Apolipoprotein (Apo) A-I and Exacerbated Glycation Susceptibility of ApoA-I and HDL.

Human serum amyloid A (SAA) is an exchangeable apolipoprotein (apo) in high-density lipoprotein (HDL) that influences HDL quality and functionality, particularly in the acute phase of inflammation. On the other hand, the structural and functional correlations of HDL containing SAA and apoA-I have not been reported. The current study was designed to compare the change in HDL quality with increasing SAA content in the lipid-free and lipid-bound states in reconstituted HDL (rHDL). The expressed recombinant human SAA1 (13 kDa) was purified to at least 98% and characterized in the lipid-free and lipid-bound states with apoA-I. The dimyristoyl phosphatidylcholine (DMPC) binding ability of apoA-I was impaired severely by the addition of SAA, while SAA alone could not bind with DMPC. The recombinant human SAA1 was incorporated into the rHDL (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC): cholesterol: apoA-I) with various apoA-I:SAA molar ratios from 1:0 to 1:0.5, 1:1 and 1:2. With increasing SAA1 content, the rHDL particle size was reduced from 98 Å to 93 Å, and the α-helicity of apoA-I:SAA was decreased from 73% to 40% for (1:0) and (1:2), respectively. The wavelength maximum fluorescence (WMF) of tryptophan in rHDL was red-shifted from 339 nm to 345 nm for (1:0) and (1:2) of apoA-I:SAA, respectively, indicating that the addition of SAA to rHDL destabilized the secondary structure of apoA-I. Upon denaturation by urea treatment from 0 M to 8 M, SAA showed only a 3 nm red-shift in WMF, while apoA-I showed a 16 nm red-shift in WMF, indicating that SAA is resistant to denaturation and apoA-I had higher conformational flexibility than SAA. The glycation reaction of apoA-I in the presence of fructose was accelerated up to 1.8-fold by adding SAA in a dose-dependent manner than that of apoA-I alone. In conclusion, the incorporation of SAA in rHDL impaired the structural stability of apoA-I and exacerbated glycation of HDL and apoA-I.

Systematic review and meta-analysis of serum amyloid A prognostic value in patients with COVID-19

INTRODUCTION: This study was designed to assess the levels of human serum amyloid A (SAA) among COVID-19 patients. MATERIAL AND METHODS: A systematic review and meta-analysis were conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis. A comprehensive literature search was performed (PubMed, Web of Science, Scopus, and Cochrane network), and studies comparing SSA levels in: (A) with non-severe vs severe COVID-19;(B) severe vs critical COVID-19 condition;(C) survived vs died due to COVID-19 in-hospital treatment period - were included. Random-effects meta-analyses were performed to obtain pooled estimates. RESULTS: Thirty studies met the criteria and were included in the meta-analysis. Pooled analysis showed that SAA levels were statistically significantly lover in non-severe group 58.7 ± 53.9 mg/L compared to 154.5 ± 169.6 mg/L for patients with severe condition (MD = -120.29;95% CI: -135.35 to -105.22;p < 0.001). SAA levels among patients with critical condition were 89.5 ± 90.4 mg/L compared to 195.3 ± 206.2 mg/L (MD = -56.66;95% CI: -101.81 to -11.51;p = 0.01). SAA levels in patient who survived were 108.7 ± 157.3 mg/L, and 206.8 ± 58.8 mg/L for patients who not survived (MD = -85.04;95% CI: -145.78 to -24.29;p = 0.006). CONCLUSIONS: In conclusion, this updated meta-analysis suggests that SAA concentrations are positively correlated with the severity of the COVID-19. Therefore, SAA can be considered a biomarker for predicting the severity and prognosis of COVID-19. Measurement of this parameter might assist clinicians in monitoring and evaluating the severity and prognosis of COVID-19. Copyright © 2022 Via Medica.

Validation of the Performance of A1HPV6, a Triage Blood Test for the Early Diagnosis and Prognosis of SARS-CoV-2 Infection.

BACKGROUND AND AIMS: Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. METHODS: In a prospective observational study of hospitalized patients with nonsevere SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7482 controls representing the general population. The primary diagnostic endpoint was the area under the receiver operating characteristic curve in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-and sex-adjusted risk of A1HPV6 to predict patients with WHO-stage > 4 (W > 4) severity. We assessed the kinetics of the A1HPV6 components in a nonhuman primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. RESULTS: The area under the receiver operating characteristic curve for A1HPV6 was 0.99 (95% CI 0.97-0.99) in the validation subset, which was not significantly different from that in the construction subset, 0.99 (0.99-0.99; P = .80), like for sensitivity 92% (85-96) vs 94% (88-97; P = .29). A1HPV6 was associated with W > 4, with a significant odds ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P < .01) at D2 and normalized at D4; HP levels increased at D2 and peaked at D4. In patients, A1 concentration was very low at D2 vs controls (P < .01) and increased at D14 (P < .01) but was still lower than controls; HP increased at D2 and remained elevated at D14. CONCLUSION: These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, HP, and alpha-2-macroglobulin were observed in the NHP model. ClinicalTrials.gov number, NCT01927133.

Role of Polypeptide Inflammatory Biomarkers in the Diagnosis and Monitoring of COVID-19.

The COVID-19 (coronavirus disease 2019) pandemic that took over the world in December 2019 has had everlasting devastating impacts on the lives of people globally. It manifests a huge symptom spectrum ranging from asymptomatic to critically ill patients with an unpredictable outcome. Timely diagnosis and assessment of disease severity is imperative for effective treatment. Possibilities exist that by the time symptoms appear the viral load might increase beyond control. However, it is advisable to get adequately diagnosed as soon as the first symptom appears. There is an immediate requirement of reliable biomarkers of COVID-19 manifesting an early onset for effective clinical management, stratification of high risk patients and ensuring ideal resource allocation. In this review, we attempt to explore and describe important polypeptide inflammatory biomarkers, namely C-reactive protein, Procalcitonin, Ferritin, Lactate Dehydrogenase, Serum amyloid A, Interleukin-6, Tumor necrosis factor-alpha and LIGHT used in the detection and management of COVID-19. Viral pathogenesis and the role of these inflammatory biomarkers is highlighted, based on the evidences available till date. An integrative data monitoring along with their correlation with the natural disease progression is of utmost importance in the management of COVID-19. So further research and in-depth analysis of these biomarkers is warranted in the present scenario.

Serum amyloid A, ferritin and carcinoembryonic antigen as biomarkers of severity in patients with COVID-19.

In view of the rapid spread of COVID-19 and the high mortality rate of severe cases, reliable risk stratifying indicators of prognosis are necessary to decrease morbidity and mortality. The aim of the present study was to evaluate the value of serum amyloid A (SAA) and carcinoembryonic antigen (CEA) as prognostic biomarkers in comparison to other predictors, including C-reactive protein (CRP) and ferritin levels. This study included 124 patients diagnosed with COVID-19, and they were assigned to one of two groups: Mild and severe, based on the severity of the infection. Radiological and laboratory investigations were performed, including evaluation of CRP, ferritin, D-Dimer, SAA and CEA levels. Significantly higher levels of CRP, ferritin, D-Dimer, SAA and CEA were observed in severe cases. SAA was significantly correlated with CRP (r=0.422, P<0.001), ferritin (r=0.574, P<0.001), CEA (r=0.514, P<0.001) and computed tomography severity score (CT-SS; r=0.691, P<0.001). CEA was correlated with CRP (r=0.441, P<0.001), ferritin (r=0.349, P<0.001) and CT-SS (r=0.374, P<0.001). Receiver operator characteristic (ROC) curves for performance of SAA, CEA, ferritin, CRP and SAA showed the highest AUC value of 0.928, with a specificity of 93.1%, and a sensitivity of 98.5% at a cut-off of 16 mg/l. The multi-ROC curve for SAA and ferritin showed 100% specificity, 100% sensitivity and 100% efficiency, with an AUC of 1.000. Thus, combining SAA and ferritin may have guiding significance for predicting COVID-19 severity. SAA alone showed the highest prognostic significance. Both SAA and CEA were positively correlated with the CT-SS. Early monitoring of these laboratory markers may thus provide significant input for halting disease progression and reducing mortality rates.

HDL in COVID-19 Patients: Evidence from an Italian Cross-Sectional Study.

A number of studies have highlighted important alterations of the lipid profile in COVID-19 patients. Besides the well-known atheroprotective function, HDL displays anti-inflammatory, anti-oxidative, and anti-infectious properties. The aim of this retrospective study was to assess the HDL anti-inflammatory and antioxidant features, by evaluation of HDL-associated Serum amyloid A (SAA) enrichment and HDL-paraoxonase 1 (PON-1) activity, in a cohort of COVID-19 patients hospitalized at the Cardiorespiratory COVID-19 Unit of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico of Milan. COVID-19 patients reached very low levels of HDL-c (mean ± SD: 27.1 ± 9.7 mg/dL) with a marked rise in TG (mean ± SD: 165.9 ± 62.5 mg/dL). Compared to matched-controls, SAA levels were significantly raised in COVID-19 patients at admission. There were no significant differences in the SAA amount between 83 alive and 22 dead patients for all-cause in-hospital mortality. Similar findings were reached in the case of PON-1 activity, with no differences between alive and dead patients for all-cause in-hospital mortality. In conclusion, although not related to the prediction of in-hospital mortality, reduction in HDL-c and the enrichment of SAA in HDL are a mirror of SARS-CoV-2 positivity even at the very early stages of the infection.

Proteomic Profiles in Patients with Thrombosis Due to COVID-19 Are Distinct from Non-COVID-19 Thrombosis

BACKGROUND. COVID-19 is a prothrombotic disease, characterized by endotheliopathy, hypercoagulability, and thromboembolic complications. We hypothesized that the pathogenesis of thromboembolism associated with COVID-19 might differ from thromboembolism in patients without COVID-19. In this study, we sought to evaluate the proteomic signatures of plasma from patients with venous thromboembolism with and without COVID-19. METHODS. Between December 17, 2020 and February 25, 2021 blood was collected from 48 hospitalized patients. Of these 24 had a confirmed diagnosis of COVID-19 infection (COVID+) and radiologic confirmation of arterial or venous thromboembolism (TE+);17 had COVID-19 infection with absence of arterial thrombosis clinically and absence of venous thromboembolism on lower extremity Doppler ultrasound or chest CT angiography (COVID+/TE-), while 7 were arterial or venous thromboembolism in the absence of COVID-19 (COVID-/TE+). Blood was collected in sodium citrate tubes and centrifuged at 4000 rpm for 20 minutes, with resulting plasma supernatant used for protein profiling performed at Eve Technologies (Calgary, Alberta, Canada). Institutional Review Board approval was obtained for this study. Statistical analysis was performed using GraphPad Prism (v9.1, GraphPad Software, San Diego, CA) and R (v4, R Core Team). P values <0.05 were considered statistically significant. A heatmap was generated using Heatmapper (heatmapper.ca) to represent the concentrations of proteins. RESULTS. The median age was 63 years;overall 25 (52%) were men (13 [54%] among COVID+/TE+, 11 [65%] among COVID+/TE-, and 1 [14%] among COVID-/TE+). In COVID-19 patients who developed thromboembolic events, several proteins associated with inflammation, complement activation, and hemostasis were present at higher levels than in non-COVID-19 patients who developed thromboembolic events (Fig. 1). These included complement factors C2 and C5a, pentraxin-3 (PTX-3), lipocalin-2 (LCN2), resistin (RETN), platelet endothelial cell adhesion molecule-1 (Pecam1), serum amyloid A (SAA), and tissue factor (TF). The heatmap indicates relative protein levels detected in each subject (columns) for proteins (rows) that had statistically significant differences between groups (Fig. 2). Heatmap revealed relatively lower levels of all proteins in patients with thromboembolism without COVID-19 and relatively higher levels of proteins in patients with COVID-19, and especially in ICU patients with COVID-19 and thromboembolism. CONCLUSIONS. Thromboembolic complications in patients with COVID-19 are associated with increased levels of various proteins involved in complement activation and immunothrombotic cascades, compared to thrombotic events in the absence of COVID-19. Activation of the classical complement pathway as evidenced by a relative increase in complement factor C2 may lead to increased TF activation, reflecting more substantial endothelial damage in COVID-19 patients. Higher levels of Pecam1, SAA, LCN2, and RETN all point to increased endotheliopathy, inflammation, and tissue damage in COVID-19 compared to non-COVID-19 thrombosis. These findings may offer insights into novel therapeutic strategies to treat immunothrombotic complications of COVID-19. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

A novel method for multiplex protein biomarker analysis of human serum using quantitative MALDI mass spectrometry.

In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4-5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established.

A case report of refractory Kawasaki disease

Introduction: Objectives: Methods: Results: A four-months old infant was referred for fever for two days and dyspnea. He had bilateral conjunctival hyperemia;bilateral cervical lymphadenopathy;truncal exanthem;perineal erythema;dry cough;normal cardiac, thoracic, and abdominal examination. Nasopharyngeal swab was negative for SARS-CoV-2. His routine analysis showed: Hb 9,9 g/dl;leukocytes 12000/mm3 (neutrophils 60%;lymphocytes 13%);platelets 430000/mm3;CRP 19 mg/dl (normal values < 0,5 mg/dl);ESR 94;Serum Amyloid A 1430 mg/l;ferritin 883 ng/ml. His chest X-ray showed an interstitial pattern. His first echocardiographic evaluation only showed a minimal pericardial effusion, and normal coronary arteries. The patient received two consecutive intravenous infusions of immunoglobulins (2 g/kg), associated with 2 mg/kg/day methylprednisolone, with consequent apyrexia and reduction of CRP (4,5 mg/dl). He also started 3 mg/kg/day aspirin. After two days from the second infusion, fever reappeared together with coronary dilatation: diameter was 2,4 mm for LCA (z-score 3,16);2,4 mm for LAD (z-score 4,49);2,5 mm for RCA (z-score 4,13). Exams showed CRP 6,57 mg/dl;leukocytes 62860/mm3 (73% neutrophils);platelets 1532000/mm3. The child then received Intravenous pulses of 30 mg/kg/day methylprednisolone in three consecutive days, and started 4 mg/kg/day anakinra. The pulses were followed by apyrexia, with CRP 6,54 mg/dl. Four days later CRP was 8,96 mg/dl, so anakinra was interrupted, and 5 mg/kg infliximab was started. Echocardiogram showed rapidly evolving giant coronary aneurysms: diameter was 6 mm for LCA (z-score 15,79);6,5 mm for LAD (z-score 16,41);5 mm for RCA (z-score 11,72). Due to the rapid evolution of aneurysms and an elevated z-score (> 10), 1 mg/kg/dose enoxaparin was started, according to the Kawasaki disease guidelines of the American Heart Association (AHA) (Circulation, 2017). The patient also started ECG, troponin and BNP monitoring. Three days later, the coronary arteries showed further dilatation. Seven days after infliximab infusion, RCA developed a rosary bead - like pattern. There was a gradual improvement of general clinical conditions with reduction of inflammatory markers (negative CRP, ESR 22, leukocytes 28580/mm3, platelets 800000/mm3 after ten days from infliximab infusion), so steroid tapering was started. Because of an increased thromboembolic risk (due to thrombocytosis and to the accelerated blood flow in stenotic coronary traits), clopidogrel 0,5 mg/kg/day was added, according to AHA guidelines. Thirty days after symptom onset, also LDA developed a rosary bead - like pattern. CRP was 1,27 mg/dl and ESR was 37. The patient was then transferred to Pediatric Cardiac Surgery, started 5 mg/kg/day cyclosporin and, after further coronary dilatation (LDA 11 mm;RCA 9 mm), 3 mg/kg/day propranolol and warfarin. A CT angiogram confirmed the aneurysms, and excluded any aortic dilatations. Epiaortic vessels were spared. An increase of cyclosporin dose to 8 mg/kg/day was followed by stabilization of coronary aneurysms. Conclusion: The extreme refractariety of coronary disease, despite aggressive therapy and biologics, is rather clear. The rapid evolution could be explained, though only in part, by a few risk factors such as age below 12 months and elevated CRP. These factors are well known, even though they are not mentioned in guidelines. ed.

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. METHODS: We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. RESULTS: We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. CONCLUSIONS: Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Hypothesis: AA amyloidosis is a factor causing systemic complications after coronavirus disease.

The severe course of COVID-19 causes systemic chronic inflammation and thrombosis in a wide variety of organs and tissues. The nature of these inflammations remains a mystery, although they are known to occur against the background of a high level of cytokine production. The high level of cytokines provokes overproduction of the Serum amyloid A (SAA) protein. Moreover, the number of studies has shown that the severe COVID-19 causes SAA overproduction. The authors of these works regard a high level of SAA exclusively as a biomarker of COVID-19. However, it should be borne in mind that overproduction of SAA can cause systemic AA amyloidosis. SAA forms cytotoxic amyloid deposits in various organs and induces inflammation and thrombosis. The consequences of COVID-19 infection are surprisingly similar to the clinical picture that is observed in AA amyloidosis. Here I present the hypothesis that AA amyloidosis is a factor causing systemic complications after coronavirus disease.