The COVID-19 Cytokine Storm; What We Know So Far.

Letter to the Editor: Influence of right ventricular structure and function on hospital outcomes in COVID-19 patients

Introduction: IL-1β and IL-6 are cytokines that have major roles in cytokine storms and endogenous pyrogens. Several studies have also displayed the effectiveness of IL-1β inhibitors in COVID-19 patients in minimizing severity and mortality. Objective: This study aims to analyze the correlation between IL-1β and body temperature with ARDS severity and mortality in COVID-19 patients. Materials and Methods: This is an analytical observational study with a prospective cohort design. A total of 54 patients have met the inclusion criteria from July to September 2020. This study mainly applied the Spearman-Rho, Mann Whitney, free sample T2 test, and Chi-Square test. Results and Discussion: The correlation between body temperature and IL-1β levels in COVID-19 patients with ARDS did not show a statistically significant difference towards mortality and ARDS severity, as shown by the p-value > 0.05 in the analysis tests of each of the variables studied. Nonetheless, the occurrence of ARDS (p = 0.022), the severity of ARDS (p = 0.001), application of mechanical ventilation (p = 0.00), secondary infection (p = 0.00), and length of stay (p = 0.042) were found to be statistically significant towards COVID-19 patients’ mortality. Conclusion: Body temperature does not correlate with the occurrence of ARDS, the severity of ARDS, mortality, and IL-1β levels. IL-1β levels and transformation in IL-1β levels also do not correlate with mortality as well as the occurrence and severity of ARDS, but the use of mechanical ventilation, secondary infection, and length of stay were correlated with mortality in COVID-19 patients.

The pandemic of COVID-19 raised many questions about the impact of comorbidities on susceptibility, severity, and outcome in these patients. Hypertension and diabetes are considered as the most prevalent comorbidities in COVID-19 patients.1, 2 It is not fully clarified whether hypertension and diabetes are independent predictors of severity and outcome in COVID-19 patients or they have synergistic negative effect in this population.3, 4 In the current issue, Sun et al5 reported that neither hypertension nor elevated blood pressure was an independent risk factor for mortality or acute respiratory distress syndrome (ARDS) and respiratory failure. Hypertension was associated only with increased risk of severe COVID-19 infection. On the other hand, diabetes alone or in combination with hypertension was independent predictor of mortality.5 The investigators divided all participants into 4 groups: (i) patients without hypertension and diabetes, (ii) patients with hypertension alone, (iii) patients with diabetes alone, and (iv) patients with concomitant diabetes and hypertension. Cardio- and cerebrovascular diseases were more prevalent in patients with hypertension and diabetes alone than in those without these conditions, and the highest prevalence was reported in patients with both conditions.5 Chronic kidney disease was more prevalent in patients with diabetes with or without hypertension, as well as in patients with diabetes and hypertension in comparison with hypertensive patients. Analyses restricted to hypertensive patients and those with neither hypertension nor diabetes showed no elevation of risk associated with hypertension, independent of whether other comorbidities were or were not included in the analysis. Even when patients were grouped according to blood pressure values and glucose levels, only glucose level, but not blood pressure, was risk factor for mortality in COVID-19 patients. Diabetes alone or in combination with hypertension increased the risk of ARDS and respiratory failure. Diabetes and elevated glucose level, but not arterial hypertension and elevated blood pressure, were independent predictors of ARDS and respiratory failure in COVID-19 patients.5 The multivariable analysis showed the diabetes alone or in combination with hypertension increased the risk of severe COVID-19 infection. Nevertheless, hypertension was also associated with a mild increase in risk of severe infection. Elevated fasting blood glucose, but not elevated blood pressure, was a risk factor for the development of severe COVID-19 infection independently of other comorbid conditions. Recent meta-analysis included 18 012 COVID-19 patients and revealed that diabetes mellitus and hypertension were moderately associated with severity and mortality for COVID-19, whereas the existence of cardiovascular disease was strongly related to both severity and mortality.6 However, the authors did not have possibility to investigate the influence of each of these comorbidities in the same model, which did not allow us to conclude whether hypertension and diabetes were independently associated with severity and outcome in COVID-19 patients.6 Systematic review that involved 15 794 COVID-19 participants showed that hypertension and diabetes were associated with admission in intensive care and mortality in unadjusted model.7 However, neither hypertension nor diabetes or their combination was not related to COVID-19 severity. Meta-analysis included 310 494 patients and analyzed the relationship between large number of variables and mortality.3 These parameters included demographics, signs, and symptoms and related morbidities, vital signs, laboratory findings, imaging studies, and underlying diseases. The authors reported that older age, hypertension, and diabetes significantly increased risk of mortality among patients with COVID-19. However, the multivariate analysis showed that only diabetes was independently associated with increased mortality.3 The main question that arises is whether hypertension and diabetes independently of sex, age, and other comorbidities are associated with severity and adverse outcome of COVID-19. In the current study, Sun et5 al tried to overcome this problem by separating patients with diabetes and hypertension from other cardiovascular and cerebrovascular diseases, chronic lung, liver, and kidney diseases, endocrine and immune system disorders, or cancer. In these circumstances, hypertension or elevated blood pressure was not recognized as an independent predictor of severity or mortality in COVID-19 patients. Recently published study that included large number of patients with type 1 and type 2 diabetes in England revealed that hypertension was not related to mortality in patients with type 1 diabetes.8 Interestingly, hypertension was weakly associated with lower COVID-19–related mortality in patients with type 2 diabetes.8 Furthermore, the use of antihypertensive drugs was related to increased mortality and the use of statins was associated with lower mortality in type 2 diabetes, whereas the relationship with type 1 diabetes was insignificant.8 The authors stated that drawing conclusions about the possible effects of antihypertensive drugs or statins on COVID-19–related mortality was not possible due to potential confounding factors. Nevertheless, these medications reduce cardiovascular and renal disease, which is why their use can help to decrease not only non-COVID-19–related mortality, but could contribute to the reduction in COVID-19–related mortality, because these findings also revealed that a history of cardiovascular disease and impaired renal function was related to COVID-19–associated mortality.8 There are some important limitations of the current study that deserve further discussion. Therapeutic approach in hypertension and diabetes could interfere with final results. Namely, the authors provided information regarding main antihypertensive classes and reported significant differences between groups.5 Angiotensin-converting enzyme inhibitors (ACEI) and calcium channel blockers (CCB) were used more frequently in patients with hypertension and diabetes than in participants with only one of these conditions. Similar differences were found in the use of beta-blockers and diuretics between patients with both conditions and those with only hypertension or diabetes.5 ACEI were used significantly less frequently than CCB in the present population that differs from common clinical practice in Western countries, which should be considered during the analysis of obtained findings. Hypoglycemic drugs except insulin were more frequently used in patients with both conditions than in patients with isolated diabetes.5 Medications, except ACEI, were not included in multivariable analysis and therefore it is not possible to exclude them as a potential confounding factor in this analysis. Furthermore, the influence of obesity was not investigated and body mass index (BMI), as the main parameter of obesity, was not provided in this investigation. This is a very important point because previous studies showed that underweight and obese patients (BMI <20 kg/m2 and BMI >30 kg/m2) were under increased risk of mortality in type 2 diabetes.8 The investigators included only type 2 diabetic patients and excluded patients with type 1 diabetes, and therefore, obtained results do not have to be necessarily applied to type 2 diabetic patients. Underweight and obesity potentially could be a significant confounding factor in this study group. One should also notice that Asian and black races were important predictors of COVID-related mortality in patients with type 2 diabetes in previous studies.8 Considering the fact that current study is performed in the Asian population, the obtained findings should be interpreted with caution and possibly could not be fully applied worldwide. The persisting problem present also in the current study is lack of information about the percentage of patients with hypertension and diabetes prior to hospitalization for COVID-19. Patients with persistent hypertension and diabetes have significantly more pronounced endothelial damage, which is essential in pathogenesis of complications related to COVID-19. On the other hand, chronic patients have more prevalent target organ damage, which increases susceptibility for COVID-19 and risk of adverse outcome in these patients. Data regarding the impact of chronic and newly diagnosed hypertension in COVID-19 patients are scarce. Ran et al recently investigated only chronic hypertensive COVID-19 patients and reported that poor blood pressure control was independently associated with adverse outcome in these patients.9 Another study showed that stage I pre-diagnosed hypertension existed in only 37% of hospitalized COVID-19 patients, while the percentage of pre-diagnosed stage II and stage III hypertension was significantly higher among these patients (61% and 70%, respectively).10 This confirms our hypothesis that hypertension is diagnosed concomitantly with COVID-19 in significant portion of these patients. Nevertheless, the authors showed that adverse outcomes (septic shock, respiratory failure, ARDS, ICU admission) and mortality gradually increased with blood pressure elevation, despite the fact that hypertension was diagnosed and treated before hospitalization due to COVID-19.10 It is even more complicated relationship between pre-existing and new onset of diabetes among COVID-19 patients. Pre-existing diabetes increases risk of severe COVID-19. However, COVID-19 could also induce new onset of diabetes with metabolic complications and necessity for insulin therapy. Li et al11 reported that newly diagnosed diabetes was related to higher mortality than known diabetes or hyperglycemia in hospitalized patients with COVID-19. Similar findings were revealed from the Italian group of authors.12 These studies raised many questions that remained unanswered. It seems that the role of antihypertensive and anti-diabetic therapy, whenever it has been initiated—before or during COVID-19–related hospitalization, is clearer. The influence of acute and chronic therapy could also impact final results. Ran et al9 did not find any influence of chronic use of RAAS inhibitors on outcome in hypertensive COVID-19 patients. Findings from diabetic population showed that adequate blood glucose regulation was essential in this population, irrespective of when anti-diabetic therapy was initiated.13 However, differences between various antihypertensive medications remain to be determined. There is a consensus about high prevalence of hypertension and diabetes among (i) all COVID-19 patients, (ii) COVID-19 patients who were admitted in intensive care unit due to complicated course of disease, and (iii) COVID-19 patients with lethal outcome. However, there is no agreement about the role of hypertension and diabetes as independent predictors of outcome in COVID-19 patients. Current study reported independent role of diabetes, but not hypertension, on mortality in COVID-19 patients. This emphasizes the importance of glucose regulation in chronic and newly diagnosed diabetic patients. Interestingly patients with diabetes demonstrated higher risk of mortality than those with concomitant diabetes and hypertension, which is not expected due to negative influence of both conditions in COVID-19 patients. There was no difference in insulin therapy between these two groups of patients, but all antihypertensive classes (ACEI, ARB, CCB, and BB) were used significantly more frequently in patients with both conditions. The latter might be responsible for lower mortality risk in patients with hypertension and diabetes, and therefore, beneficial influence of antihypertensive agents should not be forgotten in COVID-19 patients. Even though the present study did not reveal independent effect of hypertension on severity and mortality of COVID-19, one should be careful in interpretation of obtained results and should not neglect the importance of hypertension and antihypertensive medications in these patients. Longitudinal multicenter studies with larger number of COVID-19 patients of different races and more detailed information regarding duration of hypertension and diabetes are necessary to investigate isolated and joined effect of hypertension and diabetes irrespective of other common comorbidities and therapy. No conflict of interest. Marijana Tadic wrote the review paper. Cesare Cuspidi contributed to detailed review with constructive remarks that substantially changed the review paper.

Rationale: Novel coronavirus-19 (COVID-19) has been observed to cause multi-organ failure and death. Lactic acid has been shown to predict worse outcomes in patients with multi-organ failure in various disease processes. During episodes of acute hypoxia there is increased conversion of pyruvate to lactic acid. In COVID-19 patients, the dysregulated inflammatory response, also known as the cytokine storm, has been known to cause acute respiratory distress syndrome (ARDS) causing refractory hypoxemia and worsening lactic acidosis. Various inflammatory markers have been studied to predict outcomes in COVID-19 patients. The objective of this study was to identify whether lactic acid at admission correlated with mortality in patients with acute respiratory failure due to COVID-19. Methods: Seventy-one patients admitted to our hospital with acute respiratory failure due to COVID-19 were studied. Medical records were reviewed to obtain demographics including age, gender, body mass index (BMI), outcome (survivor or non-survivor) and admission lactic acid levels. Pearson's correlation analysis was performed to evaluate the correlation between admission lactic acid levels and mortality. Independent t-test was performed to determine the impact of this parameter on mortality. p < 0.05 was deemed statistically significant. Results: Of the seventy-one (71) patients, admission lactic acid levels were obtained in sixty-four (64) patients. Mean age was 47.7 + 16.7 years, 73% were male, and mean BMI was 32.27 + 2.73 kg/m2. Twelve patients (18.75%) did not survive. The mean admission lactic acid for non-survivors was 2.05 + 1.032 mmol/L and for survivors was 1.16 + 0.85 mmol/L (p = 0.002, independent t-test) [Figure 1]. Pearson's correlation analysis showed a significant correlation between admission lactic acid levels and mortality (r = -0.372, p = 0.002). Conclusions: A lactic acid at admission has a significant correlation with mortality in COVID-19 patients with acute respiratory failure. Further studies are required to assess this correlation and of rise in lactic acid to risk of mortality in patients. In addition, more studies are also needed to determine if there is a specific value of lactic acid that would predict mortality in patients.

Coronavirus infection COVID-19 is an acute respiratory viral disease caused by a novel beta-coronavirus SARS-CoV-2. In 81 % of cases, mortality in COVID-19 patients is associated with the development of acute respiratory distress syndrome (ARDS). Another critical challenge associated with COVID-19 is the development of a cytokine storm, which is an uncontrolled release of proinflammatory mediators due to the excessive activation of immune system. Cytokine storm is another cause of high mortality because of COVID-19, as it leads to multiple organ failure, ARDS and disseminated intravascular coagulation (DIC). Thus, the management of cytokine storm and ARDS in COVID-19 patients is an urgent issue for the medical society. Recent research assessed the potential role of interleukin(IL)-17 in the pathogenesis of cytokine storm and ARDS in COVID-19 patients. Some authors also pointed to using anti-IL17 medications in the management of patients with severe COVID-19. The present article gives a literature review on the possible role of IL-17 in COVID-19 pathogenesis and our personal experience of anti-IL17 prescription for patients with severe course of COVID-19.

COVID-19 is a rapidly spreading global threat that has been declared a pandemic by WHO. COVID-19 is transmitted through droplets or direct contact and infects the respiratory tract causing pneumonia in most cases and acute respiratory distress syndrome (ARDS) in about 15% of cases. Deaths in COVID-19 patients have been linked to so-called "cytokine storms" caused by the virus. Overproduction of proinflammatory cytokines leads to worsening of ARDS and extensive tissue damage resulting in multi-organ failure and death. Suppressing cytokines during the management of COVID-19 patients can increase survival rates and reduce mortality.

(1) Background/Aim: People infected with SARS-CoV-2 may develop COVID-19 in a wide range of clinical severity. Pulmonary fibrosis is characterized by several grades of chronic inflammation and collagen deposition in the interalveolar space. SARS-CoV-2 infection has been demonstrated to cause lung fibrosis without a currently elucidated mechanism. Some studies emphasize the role of proinflammatory cytokines. This research studies the correlation of the released cytokines with mortality or lung injury in COVID-19 patients. (2) Methods: Electronic medical record data from 40 patients diagnosed with COVID-19 in the COVID-19 Department, Galilee Medical Center, Nahariya, Israel, were collected. Epidemiological, clinical, laboratory, and imaging variables were analyzed. The cytokine levels were measured upon admission and discharge. A correlation between cytokine levels and severity and mortality or lung involvement was undertaken. (3) Results: IFN-gamma and IL-10 are the most powerful risk factors for mortality in the COVID-19 patient groups in a multivariate analysis. However, in a univariate analysis, TGF-β, CXCL-10, IFN gamma, and IL-7 affected mortality in COVID-19 patients. MMP-7 was significantly correlated with a cytokine storm and a high 4-C (severity) score in COVID-19 patients. MMP-7, TGF-β, IL-10, IL-7, TNF-α, and IL-6 were correlated with high lung involvement in COVID-19 patients. Serum concentrations of IGF-1 were significantly increased upon discharge, but MMP-7 was decreased. (4) Conclusions: Proinflammatory cytokines predict clinical severity, lung fibrosis, and mortality in COVID-19 patients. High concentrations of TGF-β, CXCL-10, IL-10, IL-6, and TNF-α are correlated to severity and lung injury. However, certain cytokines have protective effects and higher levels of these cytokines increase survival levels and lower lung damage. High levels of INF-γ, IL-7, MMP-7, and IGF-1 have protection probabilities against lung injury and severity.

Electron paramagnetic resonance spectroscopy quantitatively correlates structural damages of serum albumin with COVID-19 severity and mortality thus suggesting albumin replacement therapy as a strategy to rescue patients at risk of mortality.

Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors’ HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors' HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress. Editor's evaluation This submission is novel since it provides information on the structure changes of albumin in COVID-19. https://doi.org/10.7554/eLife.69417.sa0 Decision letter Reviews on Sciety eLife's review process Introduction COVID-19 pandemic continues as a global health crisis while the underlying SARS-CoV-2 virus defies all attempted treatment strategies. While writing this report, there have been more than 135 million confirmed cases including around 3 million deaths worldwide according to the World Health Organization Coronavirus Disease Dashboard (https://covid19.who.int/). Although 50% of cases are reported to be in the 25–64 age group, the percentage of deaths increases dramatically with age, and approximately 75% of deaths are in those aged 65 years and above (COVID-19 Hospitalization and Death by Age | CDC). People in the age groups 30–39 years, 40–49 years, and 50–64 years are 4, 10, and 30 times more likely to die from COVID-19 complications compared to the 18–29 years age group. Nevertheless, molecular and cellular factors contributing to mortality outcome in a homogeneous cohort of patients are not yet clear. Lack of diagnostic markers that predict mortality in COVID-19 patients impedes current efforts to siege the pandemic. It is thus critical to identify prognostic tests that can assess the risk of death in critically ill patients to guide clinical protocols and prioritize interventions. Furthermore, mechanistic clues for determining the underlying molecular factors contributing to the hypercoagulability, inflammation, and cytokine storm have been so far illusive. It is therefore imperative to intensify efforts focusing on understanding the molecular pathophysiology of COVID-19 infection and to identify prognostic markers to guide and prioritize clinical decisions. Human serum albumin (HSA) is the most abundant constituent of soluble proteins in the circulatory system. HSA has been suggested and used as a diagnostic and prognostic marker of numerous diseases and conditions including ischemia, rheumatoid arthritis, cancer, septic shock, among many others. In addition to its numerous physiological and pharmacological functions including the maintenance of blood/tissue osmotic balance (Singh-Zocchi et al., 1999), blood pH, metal cation transport and homeostasis (Bal et al., 2013; Stewart et al., 2003), nutrients and drug shuttling (Fujiwara and Amisaki, 2013; Wishart et al., 2018), and toxin neutralization (Ascenzi et al., 2006; Vorum and Honoré, 1996), HSA is suggested to be a major circulating antioxidant (Cha and Kim, 1996; Loban et al., 1997). HSA can remarkably bind with a diverse array of drugs and toxins thus controlling their bioavailability and pharmacologic effects (Fasano et al., 2005). It has been previously shown that more than 70% of the free radical-trapping capacity of serum was due to HSA (reviewed in Roche et al., 2008). Importantly, several reports indicated that inflammation enhances vascular permeability of various tissues to HSA apparently to confer antioxidant beneficial effects against reactive species released by activated neutrophils (Cross et al., 1994; Halliwell, 1988; Sitar et al., 2013). Although currently without direct experimental evidence, neutrophilia-mediated oxidative stress was implicated in the COVID-19 pathology and speculated to exacerbate the inflammatory immune response eventually causing multi-organ failure and death (Laforge et al., 2020). We hypothesized that COVID-19-mediated oxidative stress may be differentially reflected in HSA's structure and functions and employed electron paramagnetic resonance (EPR) spin labeling spectroscopy to explore HSA's structural changes in correlation with severity and mortality of critically ill COVID-19 patients. Spin-labeled fatty acids (SLFAs) are established probes to explore structural and functional changes in albumin by EPR spectroscopy (Ge et al., 1990; Haeri et al., 2019). This approach relies on the well-studied ability of albumin to strongly and exclusively bind with fatty acids in blood. Albumin has at least seven different specific binding sites for long-chain fatty acids located in different domains within the protein (Bhattacharya et al., 2000; Curry et al., 1999; Simard et al., 2006). Effectively, structural and functional changes in HSA may be assessed through the detection of parallel changes in mobility and binding affinity of SLFAs, in addition to the distribution of the spin labels on the albumin molecule (Haeri et al., 2019). EPR spectra of spin labels bound to different domains of the protein provide information on the local fatty acids/protein interactions, which may probe changes in the overall structure of the protein under unfolding or damaging conditions (Figure 1A; Bhattacharya et al., 2000). Here, we compare changes that occur to the mobility, binding affinity, and distribution of the HSA-bound SLFA in whole blood and plasma from COVID-19 patients in critical care unit relative to those observed in normal healthy individuals. Figure 1 Download asset Open asset Probing structual changes of serum albumin through spin labeling EPR spectroscopy. (A) HSA crystal structure containing seven copies of stearic acid. (B) Representative EPR spectra of free and HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood from the same COVID-19 recovered patient. Chemical structures of the two spin-labeled fatty acids are given on the right side of the figure. EPR, electron paramagnetic resonance; HSA, human serum albumin. Results Demographic, clinical, and laboratory hematologic characteristics of COVID-19 patients Table 1 lists demographic data, comorbidities, ongoing medications, and administered anti-COVID-19 medications applied to treat current study participants that were divided into survivors (Sev-R) and deceased (Sev-D). No clinical or demographic characteristic showed statistically significant difference between Sev-R and Sev-D groups when analyzed by Pearson's Chi-square test. In Table 2, we show and statistically compare laboratory results of survivors versus non-survivor COVID-19 groups. Although when comparing all parameters in the two COVID-19 groups, we observed changes following the same reported trends in the literature, means' comparisons by Tukey test reported non-significant changes in all parameters except for a significant decrease in albumin level (p<0.05) and a strong trend observed for C-reactive protein (CRP) (greater levels in Sev-D group, p=0.06). Nevertheless, non-survivors' blood carried the frequently observed hallmarks of increased CRP, D-dimer, IL-6, ferritin, and the liver enzymes ALT and AST (reviewed in: Singh et al., 2021; Velavan and Meyer, 2020). However, it is conceivable that the clinical severe category and the same ICU status of patients in the two groups in addition to relatively small sample sizes underlie the observed lack of robust statistical differences between these parameters. Table 1 Demographic and clinical characteristics of the studied subjects. Sev-RSev-DTukey 95% CIpn1623Age (mean ± SD)60.7±9.567.8±13.24.1–17.80.09Male56.25%63.16%0.677†sO2 (mean ± SD)82.1±18.776.1±18.6–20.5 to 8.40.40Hypertension12.5%42.1%0.053†Diabetes25%75%0.08†Cardiovascular disease0%15.8%0.10†Cancer0%10.5%0.18†Bronchial asthma6.25%10.5%0.65†ACE inhibitors0%7.14%0.47†ARBs9.09%7.14%0.85†calcium channel blocker14.28%7.14%0.60†Beta blockers0%7.14%0.47†Diuretics0%7.14%0.47†Sulphonylurea14.28%21.43%0.69†Other oral hypoglycemic0%21.43%0.18†Insulin42.85%21.43%0.30†Anticoagulant57.14%50.0%0.76†Steroids71.42%64.28%0.74†Hydroxychloro-quine14.28%7.14%0.60†IL-6 receptor antibody28.57%21.42%0.72†Proton-pump inhibitor28.57%42.85%0.52†Azithromycin14.28%28.57%0.47†Cephalosporin42.85%21.43%0.30†Carbapenem42.85%42.85%1.0†Oxazolidinone42.85%28.57%0.51†Fluoro-quinolone42.85%21.43%0.30†Nitrofuran14.28%0%0.15†Remdesivir14.28%28.57%0.47†Ivermectin0%28.57%0.11† sO2, blood oxygen saturation level; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blocker; IL-6, interleukin-6. † p values obtained through Pearson's χ2 test. Table 2 Laboratory parameters of the current study patients. WBC, white blood cell; INR, international normalized ratio; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; ALT, alanine transaminase; AST, aspartate transaminase. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Sev-R (mean ± SD)Sev-D (mean ± SD)Tukey 95% CIpWBCs (×103 /ml)10.6±4.013.9±8.0–1.47 to 8.060.17Platelets (×106 /ml)260±75.5213.7±115.8–116.7 to 24.20.19INR1.29±0.561.23±0.23–0.38 to 0.260.69CRP (mg/L)51.19±54.4103.77±86.3–2.35 to 107.50.06D-dimer (mg/ml)1.47±1.93.17±3.56–0.55 to 3.960.13IL-6 (pg/ml)314.1±527325.3±619–591 to 6140.97Ferritin922.6±5651078±578–281 to 5940.47Albumin (g/ml)31.47±7.9526.97±5.1–8.7 to –0.260.038Hemoglobin (g/dl)12.26±2.012.16±2.0–1.53 to –1.330.97ALT (U/L)33.64±24.1546.5±37.2–10.37 to 36.13 ALT, alanine transaminase; AST, aspartate transaminase; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; INR, international normalized ratio; WBC, white blood cell. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Neutrophils are a major source of reactive oxygen species It has been recently proposed that the high neutrophil-to-lymphocyte ratio (NLR) observed in critically ill COVID-19 patients may tip the redox homeostasis due to increased reactive oxygen species (ROS) production (Laforge et al., 2020). Our hypothesis implicates elevated oxidative stress as a major cause of HSA damage in severe COVID-19 patients. As a result, we started by following the dependence of clinical outcomes and mortality on ROS levels in blood cells. First, we used flow cytometry to assess percentages of neutrophils, lymphocytes, and platelets in all patients as described in Materials and methods (Figure 2A). Furthermore, we used the ROS-sensitive DCF dye to probe intracellular ROS levels in various cell populations in whole blood from all groups. Figure 2 shows that while lymphocyte counts decrease, a parallel dramatic increase in neutrophil counts (% total) was observable when going from Control (40.78±14.0, n=9) to Sev-R (64.0±20.0, n=10) to Sev-D (76.4± 6.8, n=11) groups (overall ANOVA p=3.9×10–5). Similar trend was clearly seen in the heat map depicting parameters for all patients analyzed by flow cytometry (Figure 2B). It is also clear from Figure 2B&C that changes in DCF-positive neutrophils follow similar trend observed for neutrophil counts. To confirm this relation, we compared neutrophil counts with DCF-positive neutrophil counts and found that the two parameters were strongly correlated (Pearson's r=0.8, p=3×10–7; Figure 2C). Moreover, both parameters individually showed statistically significant increases in both of the studied COVID-19 groups when compared with the control group (Figure 2D&E). These results suggest that neutrophils are major sources of elevated oxidative stress in critically ill patients. Note that the observed trends in platelets, lymphocyte, neutrophils, and NLR are similar to reported values (Sun et al., 2020; Yang et al., 2020). Figure 2 Download asset Open asset Hematologic cellular counts and neutrophil-ROS levels reflect severity and mortality in COVID-19 patients. (A) Representative flow cytometric diagrams comparing morphologic, hematologic, and ROS levels in control (representative of n=9; upper row), Sev-R (representative of n=10; middle row), and Sev-D (representative of n=11; lower row) groups. (B) Heat diagram comparing lymphocyte, neutrophils, platelets, and DCF-positive neutrophil counts as the percentage of total cell counts in all of the studied subjects. Yellow areas are either group separators or missing data due to insufficient sample size or processing errors. (C) A diagram showing statistically positive correlation between neutrophil count and count of neutrophils stained positive for DCF dye in all groups (black dots denote controls; blue are Sev-R; and red represent Sev-D patients). (D) When neutrophil counts were compared for all groups, both Sev-R and Sev-D groups showed statistically significant neutrophilia relative to control groups. However, only a weak trend has been observed when comparing the two groups with COVID-19. (E) DCF staining revealed increased levels of ROS in Sev-R and Sev-D groups relative to control neutrophils. Sev-D showed a trend of increased ROS level relative to Sev-R group. Multiple comparisons were carried out using ANOVA followed by Tukey test and p values are given. ROS, reactive oxygen species. Figure 2—source data 1 Raw source data for Figure 2B-E. https://cdn.elifesciences.org/articles/69417/elife-69417-fig2-data1-v2.xlsx Download elife-69417-fig2-data1-v2.xlsx Hydrogen peroxide levels in plasma correlate with mortality Next, we reasoned that elevated oxidative stress in both groups with critical COVID-19 infection would be echoed in plasma levels of hydrogen peroxide. Hydrogen peroxide is the most stable ROS and is highly stable under prolonged storage at low temperatures. We used a highly specific catalase-based assay that we developed and verified in our laboratory to quantify [H2O2] in plasma samples of all groups. The assay relies on high-resolution detection and quantification of released oxygen due to hydrogen peroxide decompostion by catalase (Figure 3A&B). We constructed a calibration curve to confirm the catalase-mediated H2O2 to O2 stoichiometric conversion (Figure 3B). A linear relation was obtained with zero intercept and slope of 0.47±0.03 which closely matches the theoretically expected value of 0.5 (95% confidence interval [CI]: 0.37‒0.56, p=5.6×10–4, Pearson's r=0.994). Indeed, we detected striking differences between groups even with relatively small sample sizes (Mean ± SD, Control, n=11: 2.95±0.77, Sev-R, n=16: 7.21±2.4, Sev-D, n=23: 9.67±2.0; overall ANOVA p=2.6×10–11; Figure 3C). The differences between groups have reached statistical significance (Sev-R vs. Control, 95% CI: 2.37‒6.13, p=4.9×10–6; Sev-D vs. Control, 95% CI: 4.95‒8.48, p=0.0; Sev-D vs. Sev-R, 95% CI: 0.90‒4.03, p=0.001). It appears from these results that a measure of oxidative stress, that is, [H2O2] in plasma, is doubled in survivors and tripled in deceased COVID-19 patients relative to controls' plasma average levels. Figure 3 Download asset Open asset Hydrogen peroxide levels in plasma and neutrophils reflect mortality in COVID-19 patients. Catalase was used to specifically and quantitatively determine levels of hydrogen peroxide in identical plasma volumes collected from control (n=11), Sev-R (n=16), and Sev-D (n=23) groups. (A) Oxygen levels are monitored and recorded while 50 μl batches of plasma from control, Sev-R, and Sev-D subjects are sequentially infused into tightly air-controlled O2k chamber containing catalase (315 units/ml) in deoxygenated buffer. In addition to the initial rise due to residual oxygen in the added plasma samples, the decomposition of hydrogen peroxide in these samples produces oxygen quantitatively. (B) To verify the assay we measured the released oxygen upon adding an increasing volume of standard hydrogen peroxide solution in PBS buffer with 0.2, 0.8, 1.2, and 1.6 μM final concentrations; inset. Linear fitting of the plotted [O2] versus [H2O2] relation yielded a slope=0.47±0.03 (Pearson's r=0.994, p=5.6×10–4), which is very close to the theoretically expected value of 0.5 as the catalase-mediated decomposition of one mole of H2O2 produces ½-mole O2. (C) Plasma contents of H2O2 in plasma significantly increased in the order Sev-D>Sev-R>Cont using ANOVA followed by Tukey test applied on n=11, 16, and 23 for control, survivors, and non-survivors, respectively. (D) Fluorescence imaging was used to assess levels of ROS in freshly isolated neutrophils using DCF (2,7-Dichlorodihydrofluorescein diacetate, green) staining in all groups. Hoechst binds strongly to adenine–thymine-rich regions in DNA thus mapping nuclei through emitting blue fluorescence. Merged DCF and Hoechst images are shown in the third column. Images were acquired using Cytation 5 Cell Imaging Multi-Mode Reader (Agilent) and analyzed using Gen5 Software package 3.08. Scale bar: 100 µm. Figure 3—source data 1 Raw polarographic data for released oxygen (A), calibration curve (B), and calculated plasma hydrogen peroxide levels in all groups (C). https://cdn.elifesciences.org/articles/69417/elife-69417-fig3-data1-v2.xlsx Download elife-69417-fig3-data1-v2.xlsx To confirm this finding, we performed DCF fluorescence imaging on freshly isolated neutrophils of representative group of individuals from each group (Figure 3D). We simultaneously stained neutrophils' nuclei with Hoechst 33342 (blue stain) to follow nuclear morphologic changes and DNA diffusion in all groups. Although requiring more detailed studies, a closer look at the acquired images of Hoechst-stained neutrophils from a survivor patient showed significantly reduced average neutrophil size DNA vs. Sev-R DNA two samples with more more nuclei and However, neutrophils from a non-survivor and in a DNA than normal and times that of Sev-R neutrophils vs. both control and Sev-R of the DCF fluorescence images indicated that the neutrophils populations of appears to be and that are highly of mean DCF fluorescence cell confirmed results obtained by flow cytometry and catalase assay increased levels of ROS in the order ± Control Sev-R Sev-D two samples for all parameters reflecting albumin changes are of COVID-19 mortality It has been previously shown that non-survivor COVID-19 patients relative to survivors et al., 2020). We started by albumin levels in the studied cohort of subjects to confirm follow similar We found that in plasma in the order (Mean ± SD, Control, Sev-R, n=16: Sev-D, n=23: overall ANOVA Figure We detected statistically significant decrease in in plasma of Sev-R and Sev-D groups (Sev-R vs. Control, 95% CI: to Sev-D vs. Control, 95% CI: to Sev-D vs. Sev-D, 95% CI: to The of HSA in serum is approximately we and groups found that COVID-19 mortality with et al., 2020). However, high of in numerous and the of this protein its diagnostic and As a result, we parameters to HSA protein in whole blood and plasma of all groups as of this critical protein functions. Figure Download asset Open asset EPR of binding strong dependence of binding on mortality in COVID-19 patients. (A) Albumin level in plasma of control Sev-R (n=16), and Sev-D groups showed that both survivors and COVID-19 patients statistically significant between representative spectra showing changes in that are to mobility and of HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood of a control (black a Sev-R (blue and a Sev-D patients. parameters including order rotational correlation and the ratio between strongly bound to bound spin labels as in Figure 1 and described in Materials and methods comparisons by ANOVA followed by Tukey tests were used for means' comparisons and revealed decrease in the binding and packing of the local the spin calculated parameters with p values are given in the into acids binding are followed by with which into the EPR Representative EPR of 16-DSA in whole blood of control and Sev-D are showing the of 16-DSA and 5-DSA bound to HSA by in whole blood. are shown as the percentage of the of the middle samples spin and 3 and measured at and of the EPR the of the spin EPR, electron paramagnetic resonance; HSA, human serum albumin. Figure data 1 and EPR parameters used for statistical Download reported that changes in HSA may be to reflect critical functional changes in albumin and diagnostic and prognostic values of these changes in (Haeri et al., et al., 2006). It has also been found that long-chain fatty acids binding the binding of in the two major drug binding sites of HSA et al., We employed EPR spectroscopy to probe binding and protein in all groups as detailed in the Materials and methods of the 5-DSA and 16-DSA EPR spectra revealed changes in between control and COVID-19 groups (Figure and Figure Figure 4, 16-DSA in whole the in Figure and used to the protein packing order is to effects as interactions, on the spin probe that is in one of the HSA fatty acids binding the S/W ratio Figure to strongly bound populations of 16-DSA spin probe may reflect changes in protein that can Furthermore, the rotational correlation which is a measure of the spin probe rotational mobility is also Figure 5 Download asset Open asset of HSA transport function of HSA is assessed through the of fatty by following the rise in both strongly (A) and (B) bound SLFA with blood from representative subjects for each These results the fatty by HSA of critically ill patients relative to To the function of HSA, increasing volumes of were added to identical of all groups and the EPR spectra were acquired (C) to follow (D) and strongly (E) bound populations of of the fatty populations are through and increased of free fatty This is remarkably in critically ill patients reflecting and of fatty acids from HSA in those patients. HSA, human serum spin-labeled fatty acid. EPR parameters for all groups are in 1 including ANOVA and Tukey test p values with the of subjects 5-DSA and 16-DSA were used to probe the of local in sites the SLFA is in the protein mobility, and closer to the mobility due to with and and Indeed, 5-DSA reflected significantly values relative to 16-DSA both in plasma and in whole blood (Figure However, of the spin probe and both in plasma and whole blood samples, the order has been lower in Sev-R which was in Sev-D patients relative to the control group (Figure In whole similar results that showed more statistically robust differences have been of as described in Materials and methods showed rotational mobility of 16-DSA is significantly faster when bound with HSA from COVID-19 patients relative to that from control subjects in plasma or whole blood. similar trends have been observed for the S/W which of the strongly and bound of 16-DSA in different fatty acids these results that COVID-19 pathology is with structural changes in the HSA protein that the of of this critical into in whole blood of COVID-19 patients We followed the spin labels through analysis of the EPR by the et al., Figure experimental 16-DSA and in whole blood of control subjects remarkably faster when compared with both Sev-R and Sev-D group, and of the EPR of COVID-19 patients by the of the spin within the It is clear from these data that the HSA of COVID-19 patients is relative to control However, the of the HSA of both COVID-19 groups was not significantly different in of transport function of HSA in critically ill COVID-19 patients in severe patients relative to (Figure with the observed significant changes in the are to functional damage and 1990; et al., et al., et al., et al., In this changes in the are to functional changes in the solution of albumin due to of paramagnetic in

The coronavirus disease 2019 (COVID-19) pandemic constitutes a global health emergency. Currently, there are no completely effective therapeutic medications for the management of this outbreak. The cytokine storm is a hyperinflammatory medical condition due to excessive and uncontrolled release of pro-inflammatory cytokines in patients suffering from severe COVID-19, leading to the development of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) and even mortality. Understanding the pathophysiology of COVID-19 can be helpful for the treatment of patients. Evidence suggests that the levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1 and IL-6 are dramatically different between mild and severe patients, so they may be important contributors to the cytokine storm. Several serum markers can be predictors for the cytokine storm. This review discusses the cytokines involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, focusing on interferons (IFNs) and ILs, and whether they can be used in COVID-19 treatment. Moreover, we highlight several microRNAs that are involved in these cytokines and their role in the cytokine storm caused by COVID-19.

Background: SARS-CoV-2, a new strain of the coronavirus, caused a global outbreak of fatal acute pneumonia. Globally, WHO has recorded 709,511 deaths from COVID-19, and the number is increasing. This study aimed to determine the risk factors for mortality in COVID-19 patients. Subjects and Method: A systematic review was conducted by searching for articles from ScienceDirect, PubMed, SpringerLink, Scopus, and Google Scholar databases. The inclusion criteria were open access, English-language, and full-text articles published in journals between 20019 and 2020. The keywords were (Coronavirus Disease 2019 AND clinical characteristics AND epidemiological characteristics AND comorbidities) OR (COVID-19 AND clinical characteristics AND epidemiological characteristics AND comorbidities). A total of eight articles was reviewed to answer the research question. The data were analyzed by PRISMA flow chart. Results: Based on the reports from China and Korea, a total of 1,314 (100%) COVID-19 patients who died was aged ≥60 years with comorbidity, in which 845 (64%) were male patients. Before the death of patients, the increase D-dimer level of ≥1 μg/ mL and Sequential Organ Failure Assessment (SOFA) score of ≥4 were reported. It indicated the occurrence of multi-organ failure and Acute Respiratory Distress Syndrome (ARDS). Most of the comorbidities were hypertension, diabetes mellitus, and cardiovascular diseases. Conclusion: Risk factors for mortality in COVID-19 patients include age at ≥60 years, male, and presence of comorbidity. The clinical features are D-dimer levels ≥1 μg / mL, high SOFA score (≥4), and ARDS. Comprehensive efforts are needed to identify risk factors early and conduct effective treatment timely to reduce the mortality of COVID-19 patients. Keywords: SARS-CoV-2, COVID-19, risk factors, mortality, comorbidity Correspondence: Ratna Yustinawati. Master of Public Health Program, Faculty of Public Health, Universitas Indonesia, Depok, West Java, Indonesia. Email: ratnayustinawati@gmail.com. Mobile: +628179324304. DOI: https://doi.org/10.26911/the7thicph.01.26

Does baricitinib reduce mortality and disease progression in SARS-CoV-2 virus infected patients? A systematic review and meta analysis

Delving deeper in the eye of the hurricane: Immunopathogenesis & molecular characterization of cytokine storm in COVID-19, association with disease severity & the therapeutic regimens.

COVID-19 disease manifests itself in a wide range of signs and symptoms, beginning with mild symptoms, such as fever, cough, and dyspnea, progressing to acute respiratory distress syndrome (ARDS) and death in some cases. The cytokine storm, or an excess of cytokines released locally, is assumed to be the primary cause of ARDS and mortality in COVID-19 patients. To enhance the survival rate of COVID-19 patients, early management of the cytokine storm with immunomodulators is crucial. Although the effectiveness of some immunosuppressants, such as corticosteroids and tocilizumab, has been studied in clinical trials, the administration of these drugs should be exercised cautiously. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid from Cannabis sativa extracts with anti-inflammatory properties. This review is intended to discuss the possible utility of CBD for the management of COVID-19 patients, particularly those with ARDS.