(Please be very detailed with this assignment) and find a great article.

Public health is an evidence-based discipline. It is very important for public health professionals to recognize and rely upon primary sources to inform opinion and action. A research report is an example of a primary source, and you can tell because it will outline the materials and methods used in the study, describing how the research was done. A published primary research study is an original document that is the first account of what happened. Review articles are secondary sources. They are designed to give you an overview of the subject and often compile multiple study results from different authors, with discussion. For the purpose of this Assignment, a meta-analysis is considered secondary.

Your Instructor will provide you with two articles for this Assignment. One article will be a primary research article and the other a secondary research article, both on the same biological topic. For this Assignment, you compare and contrast the features of the articles, and identify which article is primary and which article is secondary. Then, you will find another primary research article on the same biological topic and discuss why it is primary. It is recommended that you search the Walden Library for your article. Be sure to find an article that is available in full text, and not just the abstract.

Your Instructor uses the Module 1 Assignment Rubric to grade this Assignment.

By Day 7 of Week 1

Submit your Assignment that includes the following:

• To demonstrate your understanding of primary versus secondary sources, describe the specific features of the two articles provided by your Instructor. Compare and contrast these features-what is similar and what is different between these resources and how do these features support that the resource is primary or secondary?
• Clearly identify which article is primary and which article is secondary based on these features. .
• Locate a full text primary research article on the same biological topic and provide the reference in APA format. Support that this article is primary by analyzing and explaining the features that indicate it is a primary source.
• Provide a concise reflection on how being able to distinguish primary versus secondary articles will help you as a public health professional.

3/1/2021 Rubric Detail – Blackboard Learn

https://class.waldenu.edu/webapps/bbgs-deep-links-BBLEARN/app/course/rubric?course_id=_16811594_1&rubric_id=_2237513_1 1/4

Rubric Detail
Select Grid View or List View to change the rubric’s layout.

 
Does Not
Meet
Expectations

Meets
Expectations Very Good Outstanding

Name: PUBH_6128_Module1_Assignment1_Rubric

Description: PUBH 6128D Module 1 Assignment 1 Rubric
Criteria: Adherence to Assignment Expectations (see course for details)

EXIT

Grid View List View

3/1/2021 Rubric Detail – Blackboard Learn

https://class.waldenu.edu/webapps/bbgs-deep-links-BBLEARN/app/course/rubric?course_id=_16811594_1&rubric_id=_2237513_1 2/4

 
Does Not
Meet
Expectations
Meets
Expectations Very Good Outstanding

Demonstration
of
understanding
of primary vs.
secondary
resources by
correctly
identifying and
describing the
speci�c features
of the two
articles
provided by
your Instructor,
comparing and
contrasting
what is similar
and di�erent
between these
resources and
explaining how
these features
support that the
resource is
primary or
secondary

0 (0%) – 7
(17.5%)

Missing,
unoriginal, or
did not
adequately
demonstrate
understanding
of primary vs.
secondary
sources by
correctly
identifying
and describing
the speci�c
features of the
two articles
provided by
your
Instructor,
comparing
and
contrasting
the resources
and explaining
how these
features
support that
the resource
is primary or
secondary

8 (20%) – 8
(20%)

Adequately
demonstrate
understanding
of primary vs.
secondary
sources by
correctly
identifying
and describing
the speci�c
features of the
two articles
provided by
your
Instructor,
comparing
and
contrasting
the resources
and explaining
how these
features
support that
the resource
is primary or
secondary,
but with
infrequent
and minor
issues

9 (22.5%) – 9
(22.5%)

Generally
thorough and
well organized
and supported
demonstration
of
understanding
of primary vs.
secondary
sources by
correctly
identifying and
describing the
speci�c
features of the
two articles
provided by
your
Instructor,
comparing
and
contrasting
the resources
and explaining
how these
features
support that
the resource is
primary or
secondary,
with minimal
concerns

10 (25%) – 10
(25%)

Fully
developed and
supported,
insightful, and
scholarly
demonstration
of
understanding
of primary vs.
secondary
sources by
correctly
identifying and
describing the
speci�c
features of the
two articles
provided by
your
Instructor,
comparing
and
contrasting
the resources
and explaining
how these
features
support that
the resource is
primary or
secondary

3/1/2021 Rubric Detail – Blackboard Learn

https://class.waldenu.edu/webapps/bbgs-deep-links-BBLEARN/app/course/rubric?course_id=_16811594_1&rubric_id=_2237513_1 3/4

 
Does Not
Meet
Expectations
Meets
Expectations Very Good Outstanding

Identi�cation of
a full text
primary
research article
on the same
biological topic
and supporting
that this article
is primary by
analyzing and
explaining the
features that
indicate it is a
primary source

0 (0%) – 7
(17.5%)

Missing,
unoriginal, or
did not
adequately
identify a full
text primary
research
article on the
same
biological
topic and
support that
this article is
primary by
analyzing and
explaining the
features that
indicate it is a
primary
source

8 (20%) – 8
(20%)

Adequately
identi�ed a
full text
primary
research
article on the
same
biological
topic and
supported
that this
article is
primary by
analyzing and
explaining the
features that
indicate it is a
primary
source, but
with
infrequent
and minor
issues

9 (22.5%) – 9
(22.5%)

Generally
thorough and
well organized
identi�cation
of a full text
primary
research
article on the
same
biological topic
and support
that this article
is primary by
analyzing and
explaining the
features that
indicate it is a
primary
source, with
minimal
concerns

10 (25%) – 10
(25%)

Fully
developed,
insightful,
credible, and
scholarly
identi�cation
of a full text
primary
research
article on the
same
biological topic
and support
that this article
is primary by
analyzing and
explaining the
features that
indicate it is a
primary
source

Re�ection on
how being able
to distinguish
primary versus
secondary
articles will help
you as a public
health
professional

0 (0%) – 7
(17.5%)

Missing,
unoriginal, or
does not
adequately
re�ect on how
being able to
distinguish
primary
versus
secondary
articles will
help you as a
public health
professional

8 (20%) – 8
(20%)

Adequate and
supported
re�ection on
how being
able to
distinguish
primary
versus
secondary
articles will
help you as a
public health
professional,
but with
infrequent
and minor
issues

9 (22.5%) – 9
(22.5%)

Generally
thorough, well
organized, and
supported
re�ection on
how being
able to
distinguish
primary versus
secondary
articles will
help you as a
public health
professional,
with minimal
concerns

10 (25%) – 10
(25%)

Fully
developed and
supported,
insightful,
credible
re�ection on
how being
able to
distinguish
primary versus
secondary
articles will
help you as a
public health
professional

3/1/2021 Rubric Detail – Blackboard Learn

https://class.waldenu.edu/webapps/bbgs-deep-links-BBLEARN/app/course/rubric?course_id=_16811594_1&rubric_id=_2237513_1 4/4

 
Does Not
Meet
Expectations
Meets
Expectations Very Good Outstanding

Written
Communication:
Extent to which
writing and
reference list is
professional,
appropriate,
clear, properly
formatted,
grammatically
and structurally
correct,
synthesized,
supported, and
scholarly

0 (0%) – 7
(17.5%)

Writing does
not meet basic
expectations
for a paper
(e.g. clarity,
tone,
organization,
grammar,
spelling,
punctuation,
format, source
citation,
references,
synthesis of
source
material,
insu�cient
originality,
etc.).

8 (20%) – 8
(20%)

Adequately
meets writing
expectations
for a paper,
but with
infrequent
and minor
issues

9 (22.5%) – 9
(22.5%)

Writing is
generally
sound,
structurally
and
grammatically
correct, and
properly
formatted,
with minimal
concerns.
Synthesis is
demonstrated
and ideas are
supported
without
reliance on
quoting.

10 (25%) – 10
(25%)

Writing is fully
developed,
exceptionally
well organized,
synthesized,
supported,
scholarly, and
free of writing
errors.
Concepts are
connected
throughout
paper with
appropriate
transitions
and multiple
appropriate
resources and
examples.

Total Points: 40

Name: PUBH_6128_Module1_Assignment1_Rubric
Description: PUBH 6128D Module 1 Assignment 1 Rubric
Criteria: Adherence to Assignment Expectations (see course for details)
EXIT

International Immunopharmacology 93 (2021) 10740

7

Available online 22 January 2021
1567-5769/© 2021 Elsevier B.V. All rights reserved.

Serum cytokine levels of COVID-19 patients after 7 days of treatment with
Favipiravir or Kaletra

Esmaeil Mortaz a, b, Ali Bassir c, Neda Dalil Roofchayee b, Neda K. Dezfuli b, Hamidreza Jamaati d,
Payam Tabarsi a, Afshin Moniri a, Mitra Rezaei a, Payam Mehrian d, Mohammad Varahram d,
Majid Marjani a, *, Sharon Mumby e, Ian M. Adcock e, f

a Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of
Medical Sciences, Tehran, Iran
b Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
c Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA, USA
d Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences,
Tehran, Iran
e National Heart and Lung Institute, Imperial College London and the NIHR Imperial Biomedical Research Centre, London, UK
f Priority Research Centre for Asthma and Respiratory Disease, Hunter Medical Research Institute, University of Newcastle, Newcastle, NSW, Australia

A R T I C L E I N F O

Keywords:
COVID-19
SARS-CoV-2
Cytokines storm
Flow cytometry

A B S T R A C T

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease
2019 (COVID-19) has infected 86,4 M patients and resulted in 1,86 M deaths worldwide. Severe COVID-19
patients have elevated blood levels of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)α, IL-8 and inter-
feron (IFN)γ.
Objective: To investigate the effect of antiviral treatment serum cytokines in severe COVID-19 patients.
Methods: Blood was obtained from 29 patients (aged 32–79 yr) with laboratory-confirmed COVID-19 upon
admission and 7 days after antiviral (Favipiravir or Lopinavir/Ritonavir) treatment. Patients also received
standard supportive treatment in this retrospective observational study. Chest computed tomography (CT) scans
were evaluated to investigate lung manifestations of COVID-19. Serum was also obtained and cytokines levels
were evaluated. 19 age- and gender-matched healthy controls were studied.
Results: Anti-viral therapy significantly reduced CT scan scores and the elevated serum levels of C-reactive
protein (CRP) and lactate dehydrogenase (LDH). In contrast, serum levels of IL-6, IL-8 and IFNγ were elevated at
baseline in COVID-19 subjects compared to healthy subjects with IL-6 (p = 0.006) and IL-8 (p = 0.011) levels
being further elevated after antiviral therapy. IL-1β (p = 0.01) and TNFα (p = 0.069) levels were also enhanced
after treatment but baseline levels were similar to those of healthy controls. These changes occurred irrespective
of whether patients were admitted to the intensive care unit.
Conclusion: Antiviral treatments did not suppress the inflammatory phase of COVID-19 after 7 days treatment
although CT, CRP and LDH suggest a decline in lung inflammation. There was limited evidence for a viral-
mediated cytokine storm in these COVID-19 subjects.

1. Introduction

In December 2019, an outbreak of severe acute respiratory syndrome
coronavirus 2 was first reported in Wuhan, Hubei Province, China [1].
This novel coronavirus was announced a pandemic by the World Health
Organization (WHO) on March 11th, 2020 This disease, named coro-
navirus disease 2019 (COVID-19) by the World Health Organization

(WHO), manifests clinically with fever, cough, muscle pain, fatigue, loss
of taste and smell, diarrhea and pneumonia and results in death in
susceptible subjects [2]. Approximately 20–30% of affected people may
develop the severe form of disease and require further intervention in
intensive care [3]. A high viral load and an intensified host immune
response including both innate and acquired immunity contribute to
COVID-19 pathogenesis in severe cases leading to organ damage and

* Corresponding author.
E-mail address: marjani@sbmu.ac.ir (M. Marjani).

Contents lists available at ScienceDirect

International Immunopharmacology

journal homepage: www.elsevier.com/locate/intimp

https://doi.org/10.1016/j.intimp.2021.107407
Received 10 December 2020; Received in revised form 7 January 2021; Accepted 14 January 2021

International Immunopharmacology 93 (2021) 107407

2

failure [4,5].
The hyperactive host immunity in SARS-CoV-2 infection is charac-

terized by lymphopenia, cytokine release storm (CRS), intracellular
levels of nitric oxide (NO) in red blood cells and dysfunctional immune
responses to virus-specific antigens [6]. Several studies have indicated
that increased levels of serum proinflammatory cytokines were associ-
ated with pulmonary inflammation and lung and organ failure in
COVID-19 disease [7]. Various mechanisms appear to be effective in
reducing the number of lymphocytes in the disease, the most important
being increased levels of IL-6, TNF-α and other cytokines [8,9].

An effective immune response against viral infections occurs when
cytotoxic T cells are properly activated to clear the virus and virus-
infected cells and increasing the numbers of T cells and their function
is essential for the recovery of COVID-19 patients [8]. IL-6, TNF-α and
IL-1β have been implicated as key factors in the COVID-19 cytokine
storm [1,9] and in severe COVID-19 patients may lead to multi-organ
failure or acute respiratory distress syndrome (ARDS) [3]. As such,
many therapeutic strategies involve lowering cytokine levels [10,11].
We hypothesized that anti-viral agents would reduce the cytokine storm
and improve the clinical indices of patients with severe COVID-19.

2. Materials and methods

2.1. Study design

Confirmed COVID-19 patients (n = 29; aged 32–79) were enrolled
into the study following admission to the Masih Daneshvari Hospital of
Shahid Beheshti Medical University, Tehran between 10th April-9th
May 2020. Serum samples were collected from all COVID-19 patients
upon admission to Hospital and from 19 age- and gender-matched
healthy volunteers were served as controls. Subjects were not infected
with other pathogenic microorganisms such as hepatitis B (HBV), hep-
atitis C (HCV) and (human immunodeficiency virus) HIV. Demographic,
clinical, treatment history and laboratory data were extracted from
electronic medical records.

Severe COVID-19 disease was confirmed by the presence of at least
one of the following: respiratory rate ≥30/min; blood oxygen saturation
≤93%; ratio of partial pressure of oxygen in arterial blood to the inspired
oxygen fraction (PaO2/FiO2) < 300; lung infiltrates present in >50% of
lung fields [12]. All patients provided written informed consent. This
study was approved by the institutional ethics board of Masih Danesh-
vari Hospital of Shahid Beheshti Medical University (R.SBMU.NRITLD.
REC.1399.123).

2.2. Laboratory examination of blood samples

15/29 COVID-19 patients were transferred to the intensive care unit
(ICU) because they required high-flow nasal cannula or higher-level
oxygen support measures to resolve their hypoxemia. Blood (3.5mls in
tubes without anticoagulants) was obtained at baseline and 7 days after
initiation of antiviral treatment. Serum samples were separated by
centrifugation at 2000 rpm /20 min. Blood cell counts, coagulation
profiles, serum biochemical tests (including kidney and liver function
tests including creatine kinase (CK), lactate dehydrogenase (LDH),
electrolytes, myocardial enzymes, ferritin and procalcitonin (PCT)
levels) were obtained.

Serum cytokines were measured using ELISA kits for IL-1β (R&D
Systems, Minneapolis, MN, USA), TNFα (R&D Systems), IL-8 (BD Bio-
sciences, CA, USA), IFNγ (Thermo Fisher, Waltham, Massachusetts,
United States), IL-6 (R&D Systems) according to the following Manu-
factures’ instructions. Specificity and cross reactivity for IL-8 was ≥10
ng/mL with no cross-reactivity seen (≥2pg/mL). IFN, IL-β, IL-6 (all at 50
ng/mL) exhibited no cross-reactivity.

2.3. Treatment protocol

All patients were under supportive care consisting of intravenous
fluid and supplemental oxygen. As the recommendation of the Iranian
national guideline of COVID-19 management at the time of the study
[28], all patients received Favipiravir (600 mg three times a day) or
lopinavir/ritonavir (Kaletra, 400/100 mg bid) for seven days. Dexa-
methasone was prescribed for one subject who developed moderate to
severe ARDS.

All patients underwent a chest computed axial tomography (CT) scan
on admission and 7 days after treatment. All chest CT scans were eval-
uated in a blinded fashion by one of the authors (PM). For comparing
lung involvement in CT scans before and after treatment we used a semi-
quantitative scoring system taking into account the most prevalent
patterns of COVID-19 lung disease in CT scan (consolidation, ground
glass opacity and crazy paving pattern) was used [13–15]. According to
the anatomic structure in each patient right and left lungs were divided
into the 5 lobes: Ieft upper lope (LUL), left lower lobe (LLL), right upper
lobe (RVL), right middle lobe (RML) and right lower lobe (RLL) and each
lobe was assigned a score: Score 0, 0% involvement, score 1, <5% involvement, score 2, 5% to <25% involvement, score 3, 25% to <50% involvement, score 4, 50% to <75% involvement and score 5, 75% or greater involvement. The total CT score was evaluated from all 5 lobes and sum plotted for each patient before and after treatment.

2.4. Statistical analysis

Analysis was performed using SPSS version 16.0 (SPSS, Inc. Chicago,
USA) and GraphPad Prism software (version 6; 07 Graph Pad Software,
Inc.). Non-parametric Mann-Whitney U test (Median, 95% confidence
intervals (CI) was used for the variables without normal distribution.
Parametric t-student test (mean ± SD) was used for the variables with
normal distribution. Statistical Software p-values < 0.05 were consid- ered as significant.

3. Results

3.1. Demographic and clinical characteristics of patients with COVID-19
and healthy control

Demographic and clinical characteristics of the all participants are
shown in Table 1. The mean age of the COVID-19 patients was similar
(54.2 ± 2.53 years) to that of the healthy control population (53.6 ±
1.71 years). The groups were also matched for gender although COVID-
19 patients had a greater propensity for co-morbid conditions including
hypertension, diabetes and cardiovascular disease. The serum levels of
LDH, ESR, CRP, CPK and troponin in COVID-19 patients before antiviral
therapy on admission were 570.2 ± 54.6, 42.6 ± 5.2, 30.4 ± 5.1, 154 ±
29.6 and 0.02 ± 0.0 respectively (mean ± SD). The serum levels of LDH,
ESR, CRP, CPK and troponin in COVID-19 patients after antiviral ther-
apy were 382 ± 192, 33.8 ± 23.7, 19.8 ± 17.2, 107 ± 97 and 0.02 ± 0.0,
respectively. The serum levels of LDH (p < 0.0001), ESR (p < 0.0001), CRP (p < 0.0023) and CPK (p < 0.0153), but not troponin (p = 0.9), were significantly reduced after antiviral therapy compared to before treatment (Table 1).

CT scores improved with treatment (p < 0.05) irrespective of anti- viral therapy used or whether patients were in ICU or not. Further de- tails are provided in the on-line supplement.

3.2. Serum cytokine levels at baseline and after anti-viral therapy

Serum levels of IL-6 were elevated at baseline in COVID-19 subjects
[0.1 (0.1–27.5) pg/ml, p = 0.006] compared to healthy subjects [0.1
(0.1–1.46) pg/ml] with levels being further elevated after antiviral
therapy [10.44 (0.1–31.55) pg/ml, p < 0.0001] (Fig. 1A, Table 2). Serum IL-6 levels were significantly elevated after anti-viral therapy

E. Mortaz et al.

International Immunopharmacology 93 (2021) 107407

3

compared with levels before therapy (p < 0.01) (Fig. 1A, Table 2). The levels of serum IL-8 were also elevated at baseline in patients

[0.2 (0.015–6.4) pg/ml] compared to healthy controls [0.1 (0.1–0.1)
pg/ml, p = 0.011] and were further raised after 7 days of anti-viral
therapy [0.6 (0.052–27.42) pg/ml, p < 0.0001] (Fig. 1B, Table 2). Serum IL-8 levels were significantly elevated after anti-viral therapy compared with levels before therapy (p < 0.05) (Fig. 1B, Table 2).

Furthermore, serum levels of IFN-γ were elevated at baseline in
COVID-19 subjects [4.62 (1.2–35.98) pg/ml] compared to healthy
subjects [0.1 (0.1–3.52) pg/ml, p < 0.001] with levels not being enhanced after antiviral therapy [3.41 (2.31–40.58) pg/ml] (Fig. 1C, Table 2).

In contrast, serum levels of IL-1β were not elevated at baseline in
COVID-19 subjects [0.1)0.1–5.15) pg/ml] compared to healthy subjects
[0.1 (0.1–0.19) pg/ml, p = 0.9]. However the levels of IL-1β were
elevated after 7 days of antiviral therapy in COVID-19 patients [0.3
(0.1–44.64) pg/ml, p = 0.0019) (Fig. 1D, Table 2).

Serum levels of TNF-α was similar at baseline in COVID-19 subjects
[0.1 (0.1–0.89) pg/ml] compared to healthy subjects [0.1 (0.1–0.1) pg/
ml, p = 0.9) but elevated after antiviral therapy [0.1 (0.1–25.36) pg/ml,
p = 0.0235) (Fig. 1E, Table 2).

3.3. Effect of ICU treatment on serum cytokine levels

Since some patients progressed to ICU whilst others did not, we
assessed whether there were differences at baseline and after therapy
according to ICU status. There was no significant difference in the
baseline serum IL-6 level before and after therapy in non-ICU patients
(Table 3). However, in ICU patients serum IL-6 levels were significantly
elevated between baseline [0.1 (0.1–32) pg/ml] and after treatment [17
(1–34) pg/ml, p < 0.05) (Table 3).

Similarly, there were no differences between serum IL-8 levels in ICU
patients and non-ICU patients at baseline and after anti-viral therapy
(Table 3). In addition, anti-viral therapy did not significantly affect
serum IL-8 levels in patients in ICU but there was a small but significant
increase in serum IL-8 levels in non-ICU subjects [0.1 (0.02–1.5) vs 0.45
(0.03–3.6) pg/ml, p < 0.05] (Table 3).

Serum IFNγ concentrations did not differ at baseline between pa-
tients in ICU or not in ICU and anti-viral therapy had no effect on serum
levels of IFNγ (Table 3). However, comparison of serum IFNγ levels after
7 days of anti-viral therapy showed a significantly reduced level in non-
ICU subjects [2.9 (2.3–12.36) pg/ml] compared to those in ICU subjects
[3.89 (2.7–41) pg/ml, p = 0.003) ] (Table 3).

There were no significant differences at baseline between ICU and

non-ICU patients in the serum levels of IL-1β (Table 3). Anti-viral ther-
apy significantly enhanced serum IL-1β in patients in ICU [0.1 (0.1–45)
vs 0.1 (0.1–10.2) pg/ml, p = 0.0063] but not in non-ICU subjects [0.1
(0.1–18.13 vs 0.1 (0.1–0.1) pg/ml, p = 0.482] which resulted in levels
being greater in ICU patients compared to non-ICU patients after treat-
ment (p < 0.05) (Table 3).

Serum TNFα levels before therapy were similar between subjects in
ICU and those not in ICU (Table 3). Anti-viral therapy resulted in a small,
but significant increase in serum TNFα levels in ICU patients [0.1
(0.1–27.71) vs. 0.1 (0.1–1.67) pg/ml, p < 0.05] but not in non-ICU subjects [0.1 (0.1–12.75) vs. 0.1 (0.1–0.1) pg/ml, p > 0.99] (Table 3).

3.4. Antiviral treatment on serum cytokine levels

We evaluated the effect of the two different anti-viral therapies on
serum cytokine levels. Cytokine levels varied considerably between in-
dividual patients but there were no significant differences in baseline
levels of any cytokine measured between ICU or non-ICU subjects on
Kaletra or Favipirivir treatment (Table 4A and Table 4B). In non-ICU
patients, Kaletra treatment resulted in a significant decrease in serum
IFN-γ levels (2.59 (2.3–3.83) versus 7.46 (2.48–40.07) pg/ml, p =
0.007) with no effect on the levels of the other cytokines measured
(Table 4A and Table 4B). In contrast, Faviprivir had no significant effect
on any cytokine studied. Favipriavir also had no significant effect on
cytokine levels in patients treated within the ICU whereas Kaletra
significantly enhanced the expression of IL-1β (19.6 (0.1–44.27) versus
0.1 (0.1–0.1) pg/ml, p = 0.021) in COVID-19 patients in the ICU
(Table 4A and Table 4B).

Serum IL-8 levels were significantly lower in non-ICU patients
treated with Kaletra (0.3 (0.1–0.5) pg/ml) compared with those treated
with Favipiravir (0.7(0.03–3.6) pg/ml) (p = 0.0444) (Table 4B. Fig. 2B).
This difference was not observed in ICU patients. There were no sig-
nificant differences in post-treatment levels of IL-6 (Fig. 2A), IFN-γ
(Fig. 2C), IL-1β (Fig. 2D) or TNFα (Fig. 2E) between patients treated with
Kaletra or Favipirivir.

Serum IL-6 levels were higher in Favipiravir-treated patients in ICU
compared to non-ICU patients (p = 0.0044) whilst IFN-γ levels were
higher in Kaltera-treated patients in the ICU compared to non-ICU pa-
tients (p = 0.007) (Table 4A and Table 4B, Fig. 2). No other effects of
anti-viral therapy was seen on serum cytokine expression.

4. Discussion

Antiviral therapy resulted in improvements in lung CT scores after 7

Table 1
Demographic data of the study population and biochemical characteristics of serum components on admission to hospital.

HC (n ¼ 19) Patients (BT) (n ¼ 29) ap value Patients (AT) (n ¼ 29) p value (BT vs AT)

Age, (yrs, mean ± SD) (range) 53.6 ± 1.705 (40–64) 54.45 ± 2.536 (32–79) 0.8025 – –
Gender
Female (n, mean ± SD) (age-range) 11 (52.55 ± 2.44) (40–64) 12 (53.73 ± 4.65) (32–79) 0.9295 – –
Male (n, mean ± SD) (age-range) 8 (54.89 ± 2.43) (40–63) 17 (54.94 ± 3.21) (34–77) 0.9196 – –
Comorbidities
Hypertension 4 – –
Chronic kidney disease – – –
COPD 3 – –
Cardiovascular disease 6 – –
Diabetes 8 – –
Malignancy 1 – –
ARDS – – –
ESR (mm/h, mean ± SD) 5.6 ± 3.12 (1–11) 42.55 ± 5.15 (5–100) <0.0001**** 33.76 ± 23.71 (4–89) <0.0001**** C-Reactive protein (mg/l, mean ± SD) 10.6 ± 3.8 (6.8–19) 30.41 ± 5.08 (0–107) <0.0001**** 19.79 ± 17.16 (0–60) 0.0023** Lactate Dehydrogenase (U/L, mean ± SD) 307 ± 49.37 (222–378) 570.2 ± 54.58 (231–1535) <0.0001**** 382 ± 192.6 (134–870) <0.0001**** Troponin (pg/ml, mean ± SD) 0.02 ± 0.0 (0.02–0.02) 0.02 ± 0.0 (0.02–0.02) 0.9 0.02 ± 0.0 (0.02–0.02) 0.9 CPK (U/L, mean ± SD) – 154 ± 29.63 (5–671) – 107 ± 96.77 (5–450) 0.0153*

Abbreviations: ARDS, acute respiratory distress syndrome; COPD, chronic obstructive pulmonary disease; ESR, erythrocyte sedimentation rate, HC, Healthy control,
CPK, Creatine phosphokinase, BT, before treatment, AT, after treatment.

a p values indicate differences between healthy and COVID-19 patients. p < 0.05 was considered statistically significant.

E. Mortaz et al.

International Immunopharmacology 93 (2021) 107407

4

days irrespective of whether subjects were in ICU or not. In addition, the
raised blood levels of ESR, CRP, LDH and CPK seen in COVID-19 patients
compared to those in healthy control subjects were also reduced by anti-
viral therapy. In contrast, serum levels of IL-6, IL-8 and IFN-γ were
elevated at baseline in COVID-19 subjects compared to healthy subjects
with IL-6 and IL-8 levels being further elevated after antiviral therapy.
IL-1β and TNFα levels were also enhanced after anti-viral treatment but
baseline levels were similar to those of healthy controls. Antiviral

therapy significantly raised IL-6, IL-1β and TNFα levels in ICU patients
and IL-8 levels in non-ICU patients.

Two major families of IFNs exist: type 1 IFNs include IFN-α, IFN-β,
and IFN-ω, which are encoded by a family of more than 20 genes and
share the type 1 cellular receptor and type II IFN (IFN-γ) which is
encoded by a single gene, has a separate cellular receptor, and is pro-
duced by T cells and NK cells. IFNγ triggers its antiviral actions in vivo
by exerting cellular effects at multiple levels [16,17]. Severe COVID-19

Fig. 1. Serum levels of cytokines at baseline in healthy and COVID-19 patients and following 7 days of anti-viral therapy in COVID-19 subjects. Serum levels of IL-6
(A), IL-8 (B), IFN-γ (C), IL-1β (D) and TNFα (E) were measured in healthy subjects (n = 19) and COVID-19 patients (n = 29) before and after antiviral therapy. Results
are presented as dot blots of individual values for each subject with the median (5–95% percentiles). *p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001.

Table 2
Serum cytokine concentrations.

Cytokine (pg/ml) HC (n = 18) BT (n = 29) AT (n = 29) p value HC vs. BT† p value (HC vs. AT)† p value (BT vs. AT)†

IL-6 0.1 (0.1–1.46) 0.1 (0.1–27.5) 10.44 (0.1–31.55) 0.0067** <0.0001**** 0.0041** IL-8 0.1 (0.1– 0.1) 0.2 (0.015–6.4) 0.6 (0.052–27.42) 0.0114* <0.0001**** 0.0228* IFN-γ 0.1 (0.1–3.52) 4.62 (1.2–35.98) 3.41 (2.31–40.58) <0.0001**** <0.0001**** 0.123 IL-1β 0.1 (0.1–0.1) 0.1 (0.1–5.15) 0.3 (0.1–44.6) 0.99 0.0104* 0.0019** TNF-α 0.1 (0.1–0.1) 0.1 (0.1–0.888) 0.1 (0.1–25.36) 0.9 0.0691 0.0235* * Values are presented as the median and 95% CI unless otherwise indicated. † Comparisons between the groups were performed using a Mann-Whitney U test.

E. Mortaz et al.

International Immunopharmacology 93 (2021) 107407

5

patients have a higher IL-6/IFNγ ratio than in patients with moderate
disease, which could be related to an enhanced cytokine storm favoring
lung damage. In addition, a suppressed T-cell immunity exists in patients
with severe COVID-19 based on decreased T-cell numbers and abnormal
IFNγ expression by T-lymphocytes [6,18,19]. Continuous high levels of
cytokines observed in COVID-19 patients may imply a key inflammatory
drive in response to infection in these subjects [20]. However, anti-IL-6
therapies have been tested in COVID-19 patients with limited success
[21].

Several cytokines in addition to IL-6 and IFNγ are elevated during the
proposed cytokine storm in COVID-19 patients and these contribute to
tissue damage in the respiratory tract and in other organs. TNFα is
important in nearly all acute inflammatory reactions, acting as an
amplifier of inflammation. IL-1β is also a highly active pro-inflammatory
cytokine, and monotherapy blocking IL-1β activity is used to treat in-
flammatory diseases including rheumatoid arthritis and inherited auto-
inflammatory syndromes such as cryopyrin-associated syndromes [22].
IL-8 is a potent pro-inflammatory cytokine playing a key role in the
recruitment and activation of neutrophils during inflammation, and,

given the frequent neutrophilia observed in patients infected with SARS-
CoV-2, it is possible that IL-8 contributes to COVID-19 pathophysiology
[23].

To our knowledge, this is the first report regarding the ineffective-
ness of antiviral medication on modulating cytokine release in COVID-
19 patients. Thwaites and colleagues analyzed 619 hospitalized and
39 milder, non-hospitalized COVID-19 patients and reported higher
levels of angiopoietin-2, CXCL10, and GM-CSF in plasma [24]. In
addition, they detected mediators of endothelial cell injury in the early
stages of disease and reported that inflammatory cytokines and markers
of lung injury were higher in patients with a poor clinical outcome [24].
Zhou and co-workers showed that treatment with IFNα2b, in presence or
absence of umifenovir, a broad-spectrum antiviral, decreased elevated
blood levels of inflammatory mediators such as IL-6 and CRP [25]. We
show a reduction in serum CRP, LDH, ESR and CPK, but not IL-6, in our
study which may reflect a dose-dependent difference in local versus
systemic effects or reflect the differences in antivirals used in our study
and these others.

The lack of IFN-γ induction seen in the current study with antiviral
treatment may due to the lymphopenia induction induced by COVID-19
virus but additional information regarding the impact upon blood total
blood lymphocyte counts are necessary to test this hypothesis. However,
the significant rise in serum IL-1β, IL-6, TNF-α and IL-8 levels observed
after 7 days of antiviral therapy treatment suggests an inability of the
anti-viral therapy to suppress systemic inflammation despite the
improvement in CT scores and other inflammatory markers associated
with COVID-19 infection. The use of corticosteroids such as dexameth-
asone is used as supportive treatment for COVID-19 patients and appears
to be highly effective [26,27]. The results from our study are not
confounded by co-treatment with dexamethasone since only one subject
was given this therapy.

Our current data suggests that antiviral therapy is able to improve
lung-associated clinical features of disease as determined by CT scans as
well as blood markers such as ESR, CRP, LDH and CPK irrespective of the

Table 3
Comparison of pre- and post-treatment cytokine levels according to ICU status.

Cytokine (pg/
ml)

ICU p value non-ICU p value p value BT (ICU-non-
ICU)

p value AT (ICU-non-
ICU)

BT AT BT AT

IL-6 0.1 (0.1–32) 17 (1–34) 0.0165* 0.1 (0.1–7.7) 1.68 (0.1–29.1) 0.1459 0.4092 0.14
IL-8 0.3 (0.01–7.2) 1.56 (0.07–28) 0.19 0.1 (0.02–1.5) 0.45 (0.03–3.6) 0.021* 0.0617 0.13
IFN-γ 5.2 (1.5–30.98) 3.89 (2.7–41) 0.4671 4.52

(1.2–40.7)
2.9 (2.3–12.36) 0.1353 0.46 0.003***

IL-1β 0.1 (0.1–10.2) 0.1 (0.1–45) 0.0063** 0.1 (0.1–0.1) 0.1 (0.1–18.13) 0.4815 >0.99 0.02*
TNF-α 0.1 (0.1–1.67) 0.1 (0.1–27.71) 0.0421* 0.1 (0.1–0.1) 0.1 (0.1–12.75) >0.99 >0.99 0.0738

Values are presented as the median (5–95% percentile).
†p value < 0.05 was determined by Mann-Whitney U test. AT: After treatment. BT: Before treatment.

Table 4A
Comparison of cytokine levels according to antiviral received.

Cytokine
(Pg/ml)

non-ICU p value non-

ICU p
value

ICU p
value

ICU p
value Kaletra (Kal) Favipiravir (Fav) Kaletra (Kal) Favipiravir (Fav)

BT (n = 7) AT (n = 7) BT (n = 7) AT (n = 7) BT (n = 7) AT (n = 7) BT(n = 8) AT(n = 8)

IL-6 1.94
(0.1–3.67)

15.7
(0.1–29.1)

0.0915 0.1
(0.1–7.7)

0.1
(0.1–16.0)

0.7308 0.1
(0.1–32)

19
(0.1–34.0)

0.0862 3.87
(0.1–23)

10.28
(0.1–24)

0.1497

IL-8 0.1
(0.02–0.3)

0.3
(0.1–0.5)

0.0892 0.1
(0.02–1.5)

0.7
(0.03–3.6)

0.0769 0.3
(0.01–7.2)

0.7
(0.07–28.0)

0.8048 0.3
(0.1–3.19)

3.5
(0.2–26.8)

0.06

IFN-γ 7.46
(2.48–40.1)

2.59
(2.3–3.83)

0.007*** 3.01
(1.2–5.2)

3.11
(2.7–12.36)

0.6991 5.2
(1.7–30.98)

3.9
(2.99–41.0)

0.8741 4.4
(1.5–29.6)

3.7
(2.7–8.9)

0.2786

IL-1β

0.1
(0.1–0.1)

0.1
(0.1–18.13)

0.4615 0.1
(0.1–0.1)

0.1
(0.1–0.1)

>0.99 0.1
(0.1–0.1)

19.6
(0.1–44.27)

0.021* 0.1
(0.1–10.2)

0.1
(0.1–45)

0.4667

TNF-α 0.1
(0.1–0.1)

0.1
(0.1–12.75)

>0.99 0.1
(0.1–0.1)
0.1
(0.1–0.1)
>0.99 0.1
(0.1–0.1)

0.1
(0.1–27.7)

0.1923 0.1
(0.1–1.67)

0.1
(0.1–23)

0.467

Table 4B
Associated p values for group comparisons.

Cytokine p value (ICU vs non-
ICU)

p value (ICU vs non-ICU) p value
(Kaletra vs
Faviprivir,
AT)

Kaletra
(BT)

Favipiravir
(BT)

Kaletra
(AT)

Favipiravir
(AT)

All patients

IL-6 0.1026 0.0797 0.6818 0.0044** 0.069
IL-8 0.1515 0.3164 0.6865 0.1796 0.0444*
IFN-γ 0.8759 0.0593 0.007*** 0.2151 0.7773
IL-1β >0.99 >0.99 0.4667 0.46 0.0722
TNF-α >0.99 >0.99 0.1923 0.4667 0.4649

p < 0.05 was considered significant as determined by a Mann- Whitney U test. BT: before treatment; AT: After treatment.

E. Mortaz et al.

International Immunopharmacology 93 (2021) 107407

6

severity of the disease. This effect was independent of an action on
systemic blood cytokines that are up-regulated by viral infection. These
data suggest that the systemic inflammatory drive seen in these patients
is either no longer driven by an ongoing infection or that other driver
mechanisms exist for the enhanced cytokine expression seen in these
patients. These may reflect the host response to the initial infection that
remains despite the potential absence of ongoing viral replication. A
limitation of our study was that we did not obtain PCR analysis for SAR-
CoV-2 presence in all patients after anti-viral therapy. Moreover, the
small sample size in this study is another limitation of the study and
larger studies should be conducted to confirm the results.

The local effect of therapy on the CT imaging and on serum CRP and
LDH measures of COVID-19 infection without reducing serum cytokine
levels supports the concept of a local virus-induced lung inflammation.
Furthermore, it suggests a systemic inflammatory response that is pro-
longed even though local lung inflammation is decreased. In this
retrospective single-centre observational study, antiviral therapy has a
significant local effect in the lung but appears unable to suppress the
systemic cytokine storm and control the down-stream inflammatory
cascade. Further studies are needed to confirm this data.

5. Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Dr. Masih
Daneshvari Hospital, and all patients gave signed informed consent.

6. Consent for publication

All authors have read the manuscript and consent to publication in
the Journal.

Funding

This study was supported by internal funding.

Declaration of Competing Interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Fig. 2. Serum levels of cytokines in patients treated with Favipiravir or Kaletra. Serum levels of IL-6 (A), IL-8 (B), IFN-γ (C), IL-1β (D) and TNFα (E) were measured in
non-ICU and ICU patients after 7 days of antiviral treatment. Results are presented as dot blots of individual values for each subject with the median (5–95%
percentiles) also indicated. *p < 0.05; ns, not significant.

E. Mortaz et al.

International Immunopharmacology 93 (2021) 107407
7

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.intimp.2021.107407.

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Life Sciences 267 (2021) 11892

3

Available online 23 December 2020
0024-3205/© 2020 Elsevier Inc. All rights reserved.

Review article

Targeting inflammatory cytokine storm to fight against COVID-19
associated severe complications

Rishabh Hirawat, Mohd Aslam Saifi, Chandraiah Godugu *

Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, Telangana, India

A R T I C L E I N F O

Keywords:
COVID-19
SARS-COV-2
Acute respiratory distress syndrome
Cytokine storm
Interleukins
TNF-α
KV1.3

A B S T R A C T

Such testing and trying time probably never seen before in the human history. The novel coronavirus disease
abbreviated as COVID-19 is the ongoing health crisis which entered into human life in late December 2019. The
ease of transmission between humans and the undetectability in early stage makes COVID-19 frightening and
unprecedented. The disease is characterised by pneumonia progressing to breathing difficulty, acute respiratory
distress syndrome (ARDS) and multi-organ failure. Clinical studies suggest excessive release of inflammatory
mediators leads to cytokine storm, a phenomenon which appears to be potentially life-threatening in COVID-19.
Across the globe, when the world authorities are grappling to contain the virus, our review provides a glimpse on
structure, pathophysiology of the virus and further sheds light on various clinical complications associated with
the disease in order to open up/raise new horizons to explore various possible theoretical targets for COVID-19.
The review also portrays a question and debates: Can targeting cytokine storm can be a feasible approach to
combat COVID-19?

1. Introduction

Coronavirus disease 2019 (COVID-19) caused by severe acquired
respiratory syndrome coronavirus 2 (SARS-CoV-2) is an ongoing global
health crisis. The first case of COVID-19 was announced in Wuhan,
Hubei Province, China in late December 2019 [1] and since then it has
shaken the whole world. As of 9th December2020, almost 68 million
people have been confirmed to be infected, and around 1.6 million
people have already succumbed to this deadly viral disease. The previ-
ous virus attacks such as SARS CoV infection of 2003 killed fewer than
800 people [2], whereas middle east respiratory syndrome coronavirus
(MERS-CoV) infection of 2012 killed less than 700 people [3]. However,
previous virus attacks appear mild as compared to what the world has
witnessed so far this year due to high transmissibility of SARS-CoV-2.
World Health Organization (WHO) brought the outbreak to global
attention and declared it pandemic on March 11, 2020 (https://www.
euro.who.int/en/health-topics/health-emergencies/coronavirus-co
vid-19/novel-coronavirus-2019-ncov).

Coronavirus (CoV) is a member of family Coronaviridae, order
Nidovirales. It is an enveloped positive-sense single-stranded RNA
(ssRNA) virus. Genotypically and serologically they are divided into four

genera namely α, β, γ and δ- coronaviruses. Human coronavirus in-
fections are caused by α- and β-CoVs [4]. The SARS-CoV-2 viruses were
isolated in bronchoalveolar lavage fluid (BALF) samples of the patients
and upon sequence analysis, it was confirmed to be the member of
β-coronaviruses [5]. Genome wide studies suggest SARS-CoV-2 shares
around 79% and 50% sequence similarity with SARS-CoV and MERS-
CoV, respectively [5–7]. However, the amino acid sequence of SARS-
CoV-2 differs from other coronaviruses specifically in the regions of
1ab polyprotein and surface glycoprotein [8]. Respiratory droplets and
contact transmission are considered to be the major transmission routes
[9]. Studies suggest that SARS-CoV-2 have been detected in the urine
and stools of laboratory confirmed patients as well [10]. Recently, WHO
has also acknowledged the possibility of airborne transmission (https
://www.who.int/news-room/commentaries/detail/transmissio
n-of-sars-cov-2-implications-for-infection-prevention-precautions). The
ease of transmission between humans and the mildness or undetect-
ability of its symptoms makes COVID-19 frightening and unprecedented.
Patients infected have reported no or little flu like symptoms including
dry cough, fever, runny nose, throat pain and anosmia. In severe forms
of the disease, marked inflammation and pneumonia have been identi-
fied, progressing to breathing difficulty, acute respiratory distress

* Corresponding author at: Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad,
Telangana 500037, India.

E-mail address: chandragodugu@gmail.com (C. Godugu).

Contents lists available at ScienceDirect

Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

https://doi.org/10.1016/j.lfs.2020.118923
Received 17 October 2020; Received in revised form 9 December 2020; Accepted 15 December 2020

Life Sciences 267 (2021) 118923

2

syndrome (ARDS) and multi-organ failure. Older as well as comorbid
patients suffering from diabetes, hypertension and lung diseases are
more susceptible to COVID-19 complications [6,11]. A study suggests
that smokers are 1.4 times more prone to COVID-19 infection and
approximately possess 2.4 times more chances of getting admitted to
intensive care unit (ICU) or die as compared to non-smokers [12].
Clinical evidences indicate that there is an involvement of wide variety
of cytokines and the situation is worsened by the excessive release of
pro-inflammatory cytokines including Interleukin (IL)-1, IL-6, IL-12,
Interferon (IFN) γ and Tumour Necrosis Factor (TNF) α which mainly
targets the lung tissue [11]. The dysregulated release of signalling me-
diators is broadly known as “cytokine storm” and is sometimes, also used
interchangeably, with cytokine release syndrome (CRS). Although, the
concept of cytokine storm is not new; it has garnered the attention pri-
marily due to its striking role in COVID-19. It is a race against time with
containment the only option since there are no specific therapeutics and
vaccines available for the prophylaxis and treatment of this infection.
However, a better understanding of pathophysiology is highly recom-
mended for the development of vaccine and suitable therapeutic agents
against the virus. In order to provide a summary to public health au-
thorities and potential readers across the planet, we provide a detailed
review summarising the structure, pathogenesis, immunopathological
progression, pathological complications and the potential agents avail-
able which can be explored to control the cytokine storm observed in
COVID-19.

2. Structure and pathogenesis of SARS-COV-2

SARS-CoV-2 is an enveloped, pleomorphic or spherical shaped pos-
itive sense RNA virus having varying genome length of around 30 kb
with a 5′- cap structure and 3′- poly A tail with an arrangement as 5′-
replicase open reading frame (ORF) 1ab-spike(S)-envelope(E)-mem-
brane(M)-N-3′ [13,14]. Population genetics analysis of 103 SARS-CoV-2
genomes indicate that these viruses have evolved into two major line-
ages namely the more prevalent CT haplotype or the L lineage and the
TC haplotype also called the S lineage [15]. Two-third of the genomic
RNA is used as a template to directly translate two large polyproteins
namely pp1a and pp1ab which encode for non-structural proteins
(nsps). Remaining one-third of the genome encodes for four structural
proteins namely Spike (S), Envelope (E), Membrane (M) and Nucleo-
capsid (N) [16]. The genes encoding S, E, M and N proteins are around
3822, 228, 669 and 1260 nucleotides in length [13]. The replication and
transcription of genome of SARS-CoV-2 occurs via RNA dependent RNA
polymerase (RdRp). Structurally, RdRp resembles that of right-hand
comprising fingers, thumb and palm subdomain. The cryo-electron
microscopic structure of the SARS-CoV-2 reveals that the structure of
RdRp consists of nsp7, nsp8 and nsp12 subunits with over two turns of
RNA template-product duplex. The nsp12 is composed of three domains:
N-terminal nidovirus RdRp associated nucleotidyltransferase (NiRAN)
domain, an interface domain and a C-terminal RdRp domain. The sub-
unit nsp7 binds to the thumb whereas two copies of nsp8 which adopt
different structure in RdRp binds to the finger and thumb subdomains
[17]. The densely glycosylated S proteins, similar to SARS-CoV S pro-
tein, is a clove shaped, trimeric class I fusion protein on the virus surface
consists of a large ectodomain, a single-pass transmembrane anchor and
a short intracellular tail. The ectodomain segment consists of a receptor-
binding subunit S1 and a membrane-fusion subunit S2. The S1 compo-
nent having a receptor binding domain shows conformational move-
ments and can hide or expose the determinants for receptor binding. In
each spike, three S1 heads attach at the top of a trimeric S2 stalk. The C-
terminal domain of S1 binds to the host receptor surface whereas the N-
terminal of S1 is in contact with the S2 [18]. The S2 protein has showed
much higher sequence similarity (around 93%) with bat-SL-CoVZC45
and bat-SL-CoVZXC21 in comparison to S1 domain which had only
around 68% identity with these bats derived viruses [7]. The E-protein
localised in the ER-Golgi Intermediate Compartment (ERGIC) is

involved in protein-protein interaction and is a determinant of SARS-
CoV-2 virulence. E-protein PDZ-binding motif activates p38 MAPK
which is involved in over expression of inflammatory cytokines [19].
The M proteins are the most abundant structural proteins which define
the shape of the virion and act as an organiser of coronavirus activity
[20]. A simplified pictorial representation of virus and its spike protein
is shown in Fig. 1.

The virus may gain entry through various routes such as droplets
released in the environment by the infected patient via coughing,
sneezing, saliva or through faecal-oral route [9,10]. Upon exposure, S
protein of virus binds to the Angiotensin converting enzyme-2 (ACE-2)
receptor [6] unlike MERS-CoV which binds to dipeptidyl peptidase 4
(DPP 4) [3]. The receptor is expressed on the type II pneumocytes in the
respiratory tract as well as on vital organs including kidney, liver, heart
and intestine. [21]. The entry of virus into the lung epithelium occurs by
cleavage of S protein into S1 domain (responsible for receptor binding)
and S2 domain (helps in membrane fusion) similar to influenza or by
endocytosis [18]. Studies demonstrate that 3CL like protease (3CLpro),
papain like protease (PLpro) [16] and RdRp are the major enzymes
responsible for proteolysis, replication and production of new virions
[17]. Upon membrane fusion to the host surface, viral RNA genome is
released into the cytoplasm and hijacks the host’s translational ma-
chinery. The RNA is uncoated to allow translation of two polyproteins,
the newly formed envelop glycoproteins are inserted into the Rough
endoplasmic reticulum (RER) or Golgi membranes. Viral particles
germinate in ERGIC and which fuse with plasma membrane to release
the virus out of the cell to transfect other host cells [16]. Fig. 2 gives a
bird eye view of the pathogenesis of SARS-CoV-2 virus.

3. The cytokine storm

The pathophysiology of SARS-CoV-2 involves a particular phenom-
enon associated with aberrant release of several mediators such as in-
terleukins, interferons, chemokines etc., as a result of hyperactive
immune response. Pathological findings of patients admitted to ICU
reported increased levels of cytokines including IL-2, IL-7, Macrophage-
Colony Stimulating Factor (M-CSF), Granulocyte CSF (G-CSF), inter-
feron γ -induced protein (IP-10), Monocyte Chemoattractant Protein-1
(MCP-1), Macrophage Inflammatory Protein 1-α (MIP1-α) and TNF-α.
This vicious cycle of cytokines overproduction and excessive release is
termed as “cytokine storm” or “hypercytokinemia” [11,22]. Although,
these mediators are primarily involved in physiological signalling and
are part of strictly regulated immune system for surveillance and
clearance of foreign objects from the body; the unfettered release of
these agents, in response to infection, may result in exaggerated immune
response which negatively affects the host cells. In context of SARS-CoV-
2 infection, cytokine storm has caught the attention of research scien-
tists, health workers and general public due to its possible close asso-
ciation with pathogenesis of the virus. The release of aforementioned
mediators, notably TNF-α show a good correlation with severity of
SARS-CoV-2 based on the results of clinical studies. Adding to this fact,
the monoclonal antibodies such as Tocilizumab and Sarilumab
(although, intended for rheumatoid arthritis) have shown their effec-
tiveness in SARS-CoV-2 and have entered the clinical trials highlighting
the striking role of TNF-α in disease.

4. Immunopathological basis of cytokine storm in SARS-CoV-2

Once SARS-CoV-2 encounters the host cells, the immune system of
host gets activated and triggers adaptive as well as innate immune re-
sponses [23]. The early activation of innate immune response is an
incumbent reaction of the body to eliminate the infection. Cellular
component of innate immunity such as natural killer (NK) cells, mac-
rophages, mast cells work in concert with soluble components including
cytokines, chemokines, complement system etc. to provide a first line of
defence mechanism to the body. The details of immune response

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3

activation are beyond the scope of this review and the interested readers
are requested to refer certain excellent reviews on the topic [24,25].
However, the activation of cytokine storm as a result of hyperactive
immune response leads to progressive inflammation in the organs.
Although, our knowledge of SARS-CoV-2 pathogenesis is quite limited;
the in vitro as well as clinical studies have shown a definite role of
cytokine storm in SARS-CoV-2. Within hours of infection with virus, the
body starts producing and releasing various inflammatory cytokines
followed by a delayed production of IFN. The sudden spurt in the levels
of proinflammatory mediators dominates the relatively slower defence
mechanism of the body. The released cytokines attract a number of in-
flammatory cells such as neutrophils, mast cells etc. further fuelling the
inflammatory response. Inflammation begins when the virus replicates
within the local macrophages resulting in apoptosis. Antigen presenting
cells (APCs) engulf the foreign antigen and digests them into small

peptide fragments further releasing pro-inflammatory cytokines, mainly
IL-1β, IL-12, IL-6, TNF-α and chemokines including MCP-1 and IP-10 to
attract other immune cells to the site of infection [11,26]. Macrophages
bind to Toll like receptors present on CD4+ T-cells and activate them.
Activation promotes the secretion of IL-2 which acts in an autocrine
manner and binds to specific receptor on itself causing proliferation and
differentiation, which in turn generates the CD4+ T-effector and mem-
ory cells. The effector T-cells promote the production of T-helper cells
which stimulate the release of IL-4, IL-5 and IL-6, which is manifested as
inflammation [27]. Patients infected with COVID-19 have presented
high amounts of IL-1β, IFNγ, IP-10, and MCP-1, leading to activated T-
helper-1 (Th1) cell responses. However, evidences suggest infected pa-
tients also showed increased secretion of T-helper-2 (Th2) cytokines
namely IL-4 and IL-10 that suppress inflammation, which is opposite to
what was encountered in SARS-CoV infection [28]. CD8+ T-effector

Fig. 1. A simplified structure of SARS COV-2 virus and spike protein. SARS-CoV-2 is an enveloped, pleomorphic or spherical shaped positive sense RNA virus. The
genome of the virus encodes for four structural proteins namely Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N). S protein is clove shaped and consists
of a receptor-binding subunit S1 and a membrane-fusion subunit S2.

Fig. 2. Pathogenesis of SARS-COV-2 virus. SARS-CoV-2 infects the host lungs epithelium by binding to ACE-2 receptor and releases its genome into host cell. The
viral genome hijacks host cellular machinery. Viral particles germinate in ERGIC and fuses with plasma membrane to release more copy of viruses.

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4

cells secrete cytotoxic substances like granzyme and perforin in order to
kill the virus infected cells resulting in local tissue damage [29]. Acti-
vated forms of B-lymphocytes undergo differentiation to generate
memory B-cells and plasma cells in order to protect the body from
invading exogenous antigens. Plasma cells liberate IL-5 and IL-6 to
protect the host cells by neutralization and lysing the infected cells. The
cytokine storm syndrome has been associated with infectious diseases
including influenza, SARS, COVID 19 and non-infectious diseases like
multiple sclerosis and pancreatitis [30]. Hence, immune response in-
duction may not always be good as it is used to remove the infected cells
by committing suicide of host cells to clear the virus. Pattern recognition
receptors (PRRs) such as retinoic acid – inducible gene I protein (RIG) or
melanoma differentiation associated protein 5 (MDA 5) trigger complex
signalling cascades involving MYD88 leading to production of type I
Interferons (IFNs) and activation of transcription factor nuclear factor –
κB (NF-κB) [31]. Type I IFN signals through downstream molecules such
a signal transducer and activator of transcription (STAT) proteins to
stimulate the production of antiviral proteins. Hence, establishing the
antiviral response to limit the virus replication in neighbouring cells
[32]. Healthy serum IL-6 levels are 4 pg/ml but this level rises to several
folds in case of novel coronavirus infected patients [11]. High levels of
IL-6 bind to its receptor CD126 (also called IL-6R) and forms a complex.
IL-6-IL-6R complex further binds to CD130 (also called gp130) and
forms a hexameric structure. This hexameric structure having two
molecules each of IL-6, CD126 and CD130 formed activates and phos-
phorylates JAK followed by phosphorylation of tyrosine residues on
gp130. Furthermore, STAT1, STAT3 and SH2 domain containing protein
tyrosine phosphatase (SHP2) recruitment occur on gp130. STAT1 and
STAT3 are phosphorylated by JAKs and translocate into the nucleus
causing transcription of target genes [33]. On the other hand, SHP2 acts
by activating the Ras-MAP pathway which causes inflammation [34]. It
can also be expected that cAMP hydrolysing enzymes i.e.; phosphodi-
esterase which is expressed in monocytes, lymphocytes and neutrophils
might also contribute to inflammation upon stimulation by cytokines
such as TNFα, IL-1β, IL-6 and matrix metalloproteases (MMPs) [35,36].
Retrospective longitudinal studies of recovered patients when compared
to died patients demonstrated early expression of IFNα, IFNγ, Chemo-
kine C–C motif ligand 2 (CCL2) and proteins that are encoded by IFN-
stimulated genes in all patients but only the patients who survived had
expression profiles, which indicates development of adaptive immunity.
Also the newly recruited immune cells recognise the viral pathogen and
release more anti-inflammatory cytokines such as IL-10 to recruit more
immune cells thus amplifying the cytokine response [37]. It has been
well understood that when there is an over-exuberant release of cyto-
kines and severe inflammation, the tissue repair process is unable to
keep up resulting in a major damage to the lung tissue. In severe cases
these inflammatory cytokines may enter the systemic circulation
resulting into shock like condition and ultimately causing multi organ
failure. Table 1 summarises the physiological role of various cytokines
and evidences which suggests their possible involvement in COVID-19.

5. Complications associated with the cytokine storm

Clinical findings suggest that patients infected with SARS-CoV-2
initially appears stable and the situation deteriorates rapidly with hyp-
oxia finally, inching towards ARDS. The interval from appearance of
symptoms to the development of ARDS takes approximately 8–12 days
[60]. Comorbid patients are more severely and rapidly affected as
compared to patient without any underlying disease [11,59]. Chest scan
of patients confers ground glass opacities and consolidation are the
noteworthy features in the lungs of patients infected by SARS-CoV-2 and
the lesions predominantly appear in the peripheral and sub pleural re-
gion [5,11]. The patients showed characteristic reverse halo sign and
pulmonary nodules with halo sign which is not been reported in SARS
and MERS patients [61,62]. Clinicians have suggested that deformation
of bronchus due to fibrosis and strip like lesions may cause irreversible

Table 1
Physiological role of various cytokines and their evidences reported in COVID-
19.

SL.
No.

Cytokines Physiological Role of
cytokines

Evidences related
to COVID-19

References

1. IL-1β Pyrogenic, pro-
inflammatory,
proliferation and
differentiation

COVID-19
patients with
severe symptoms
have elevated
levels of IL-1β

[38,39]

2. IL-2 Proliferation of T cells,
generation of effector
and memory T cells. It
increases glucose
metabolism to promote
the proliferation and
activation of T-cells, B-
cells and NK cells.

A direct
relationship
between elevated
IL-2 levels and
disease severity.

[11,40,41]

3. IL-6 Differentiation into
plasma cells, IgG
production

IL-6 levels
elevated in
patients with
COVID-19 and
related to poor
prognosis.
Additionally,
they were found
to be markedly
higher in patients
who died from
COVID-19 than in
those who
recovered.

[38,42,43]

4. IL-4 Proliferation of B and
cytotoxic T cells,
enhances MHC class II
expression.

Various studies of
COVID-19
patients have
detected elevated
IL-4 levels
associated with
severe
respiratory
symptoms.

[11,38,44]

5. IL-10 Inhibits the production
of pro-inflammatory
cytokines such as TNF-
α, IL-1β, IL-6. Also
prevents dendritic cell
maturation by blocking
IL-12

IL-10 levels were
found to be
higher in patients
with COVID-19 as
compared to
those with SARS-
CoV or MERS
It has also been
indicated that IL-
10 may be over-
expressed in anti-
SARS-CoV-2
immunity.

[11,38,45]

6. IL-7 Plays vital role in
lymphocyte
differentiation to
promote the
development of B and T
cells.

IL-7 levels are
elevated in
patients with
COVID-19.

[11,38,46]

7. IL-12 Major role in the
development of Th1
and Th17 cells. It
activates NK cells.

Elevated serum
IL-12 levels have
been reported in
patients infected
with SARS-CoV-2

[11,38,44,47]

8. IL-13 Regulates immune
responses mediated by
Th2-type cytokines.

Researchers have
found no
difference in
serum IL-13
levels between
those requiring
ICU admission
and those who
did not. In
contrast, a study
has reported
direct

[11,48]

(continued on next page)

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Life Sciences 267 (2021) 118923

5

damage to the lung and deteriorate the respiratory functions of the pa-
tients [63]. On the other hand, the cytokines entering the circulating
blood cause relaxation of the smooth muscles lining the blood vessels
and increase the permeability by contracting the epithelial cells. The
increased permeability of blood vessels may also promote the accumu-
lation of plasma in the extra cellular spaces decreasing the blood volume
which may instigate hypovolemic shock [64]. In response to the cyto-
kines release, the host defence mechanism activates neutrophils gener-
ating reactive oxygen species (ROS) and proteases insulting the gaseous
exchange by destructing the alveolar lining figuring hypoxia like con-
dition in the patients [65]. Almost 80% of the patients suffering from
novel coronavirus have reported elevated body temperature. Data sug-
gest that patients suffering from SARS-CoV-2 infection demonstrate
elevation in the heart rate due to stimulation of chemoreceptor by the
prevalent hypoxic condition triggering sympathetic nervous system. The
exact mechanism of cardiovascular injury in SARS-CoV-2 infection has
not been well understood. However, it has been suggested that due to
expression of ACE2 on cardiomyocytes, binding of SARS-CoV-2 can
occur promoting cardiac insufficiency and poor cardiac outcomes [66].
Further, infected patients have also reported elevated expression of
markers of myocardial injury such as Troponin T, creatine kinase
isoenzyme and N-terminal of the prohormone brain natriuretic peptide
(NT-proBNP) [67]. The excessive release of cytokines can cause poten-
tial changes in the ECG arrhythmic markers such as QTc, QTd, TPe/QTc.
Elevated cytokines mainly IL-6, IL-1 may result in the electrophysio-
logical changes in cardiomyocytes, which may cause electrical remod-
elling of the heart prolonging QT interval and ventricular arrhythmia
[68]. Patients infected with SARS-CoV-2 virus exhibit elevated C-

Table 1 (continued )

SL.
No.
Cytokines Physiological Role of
cytokines
Evidences related
to COVID-19
References

relationship
between IL-13
levels and the
viral load of
SARS-CoV-2.

9. IL-17 It plays a major role in
tissue damage,
physiological stress,
and infection. There
functions vary
according to the tissue.

Elevated IL-17
levels have been
reported in
patients with
SARS-CoV-2 and
have been
associated with
disease severity.
Paradoxically a
study, reported
normal IL-17
levels in COVID-
19 patients.

[11,49,50]

10. M-CSF Regulates the growth,
proliferation, and
differentiation of
hematopoietic cells,
including monocytes,
macrophages, and
osteoclasts.
Mechanically they act
via type III tyrosine-
kinase receptors.

The elevated
levels of M-CSF in
patients with
COVID-19 may
be associated the
hyper-expression
of itself and other
cytokines
resulting into
lung damage.

[11,38,51]

11. G-CSF Required for the
proliferation and
maturation of
polymorphonuclear
granulocyte cells
(PMNs). PMNs act by
the release of lysosomal
enzymes and other
signalling molecules

Studies suggest
elevated G-CSF
levels in patients
with COVID-19
and the level rises
even more in ICU
patients.

[11,52]

12. GM-CSF Maintains the immune
homeostasis in lungs
and gut. It stimulates
the proliferation and
activation of
monocytes,
macrophages,
eosinophils,
neutrophils, dendritic
cells, and microglial
cells.

Serum GM-CSF
levels have been
detected to rise in
SARS-CoV-2
infection in
comparison to
healthy
individuals.

[11,53]

13. IP-10 It binds to chemokine
receptor 3 (CXCR3) and
regulates immune
system responses by
activating and
recruiting leukocytes.

Studies have
reported
elevation in IP-10
levels in patients
infected with
COVID-19 and
the levels raises
more in those
who required ICU
admission. Hence
it can be
concluded that
IP-10 over-
expression has
significant role in
lung damage and
disease severity
Findings report
high levels of IP-
10 in the most
severe cases of
COVID-19 which
can be related to
disease
progression and
mortality.

[11,54]

14. MCP-1 [11,55,56]

Table 1 (continued )
SL.
No.
Cytokines Physiological Role of
cytokines
Evidences related
to COVID-19
References

It regulates the
migration and
infiltration of
monocytes, memory T
cells, and NK cells.

Elevated levels of
MCP-1 in the
broncho-alveolar
lavage fluid of
COVID-19
patients.
Levels have also
been detected to
rise in lung tissue
of patients
infected with
SARS-CoV-2.

15. TNF-α It is mediated by IL-1β
and IL-6. TNF-α is
involved in the
regulation of
inflammatory processes
and infectious diseases.

A study of 522
patients with
COVID-19
reported an
inverse
relationship
between TNF-α
levels and T-cell
counts.
In contrast,
another study
reported normal
TNF-α level in
patients with
COVID-19.

[56–58]

16. IFN-γ It is associated with
macrophage activation,
signal transduction,
anti-bacterial and
antiviral immunity.

Serum IFN-γ
levels are found
to be higher in
patients with
COVID-19 than in
healthy
individuals.
Elevated levels
may be a result
from the
activation of Th1
and Th2 cells.

[11,59]

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6

Reactive Protein, creatine kinase and D-dimer suggesting cardiovascular
injury [60]. The exact mechanism of liver injury in COVID-19 infected
patients is not known. However, from prevalent knowledge it can be
ascertained that, since the biomarkers of cholangiocyte injury namely
γ-Glutamyl transferase (GGT), alkaline phosphatase are found to be
elevated, there might be a direct binding of SARS-CoV-2 virus to the
cholangiocyte expressing ACE-2 receptors [69,70]. Moreover, under
hypoxia like conditions due to respiratory distress depletion of adeno-
sine triphosphate, lipid accumulation, glycogen consumption of hepa-
tocyte can inhibit cell survival mechanism inducing death of
hepatocytes. In addition, lack of oxygen supply generates oxidative
stress contributing to increased ROS and lipid peroxidation products
which can promote the transcription of pro-inflammatory cytokines
mediated liver damage [70,71]. The prevalence of kidney involvement
in novel coronavirus disease is not so high. It can be argued that ARDS
may promote to renal medullary causing hypoxia, insulting tubular cells
and increasing serum creatinine levels [72]. Fig. 3 gives an overview of
complications associated with different organs in COVID-19 disease.

6. Potential targets and agents to control cytokine storm

Strong inflammatory cytokine and chemokine response upon viral
infection paint a frightening spectre. As the world authorities are
grappling to contain the COVID-19 virus, here are a few targets which
can be explored to control and manage the over-exuberant release of
cytokines. A brief overview of different potential targets has been shown
in the Fig. 4.

6.1. IL-6 inhibitors

In COVID 19 patients, pathogenic Th1 cells and inflammatory
monocytes with high expression of IL-6 exist [11]. They are also
responsible for the pathogenesis of autoimmune disorders like

Rheumatoid Arthritis. A recent study of patient suffering from COVID-19
reported IL-6 as one of the most robust prognostic markers of survival
and is associated with severity and predictive outcome when linked with
ventilation and end organ damage [73]. The biopsy samples of autopsy
from COVID-19 patients demonstrate pathogenic T cells as well as in-
flammatory monocytes may enter the pulmonary circulation in large
numbers and lead to inflammation [74]. The inflammation occurs via
two major signalling pathways namely the mitogen-activated protein
kinase (MAPK) pathway [75] and the JAK/STAT pathway [76]. Early
detection of increased serum IL-6 levels and its attenuation using suit-
able approaches can be one of the potential paths to manage SARS-CoV-
2 associated complications. A recent study of Tocilizumab (humanized
anti-monoclonal antibody) treated COVID-19 patients along with Lopi-
navir, Methylprednisolone and oxygen therapy demonstrated improved
respiratory functions and CRP levels within 3–5 days. In addition, pla-
que like lesions which appeared before the treatment were significantly
reduced. Surprisingly, no adverse drug reactions were reported during
the treatment with Tocilizumab at the selected dose [77]. It has also
been reported that treatment with Tocilizumab reduced QT prolonga-
tion which might be associated with comorbid cases in COVID-19 pa-
tients [78]. Though, Tocilizumab has been proved as a promising
therapeutic agent in reducing the mortality of severe COVID-19 patients;
it should be administered carefully because early administration can
increase viral load leading to adverse effects. In addition, the drug is
contraindicated in pregnant or lactating women, patients with active
pulmonary tuberculosis, patients with mental disorders, those having
neutrophil count <0.5 × 109/L, platelet count <50 × 109/L or have undergone organ transplant [77].

6.2. IL-1 inhibitors

IL-1β blockade has been found to be effective in the treatment of
various inflammatory diseases such as Still’s disease, rheumatoid

Fig. 3. Release of inflammatory cytokines. An overview of excessive release of inflammatory cytokines and their negative impact on the host cell.

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Life Sciences 267 (2021) 118923

7

arthritis, osteoarthritis, gout, multiple sclerosis, macrophage activation
syndrome and hyper IgD syndrome [79]. IL-1α is released from cells
undergoing hypoxic damage, lost membrane integrity and bind to
Interleukin-1 receptor type 1 (IL-1RI) receptor and further triggers a
cascade of chemokines and inflammatory cytokines. It also promotes the
leukocyte infiltration to the site of inflammation. Mechanistically, IL-
1binding to its receptor induces phosphorylation dependent pathway
such as p38 MAP kinase, and NF-κB pathway which further induces
expression of various inflammatory genes. Therefore, blocking IL-1 can
limit the production of inflammatory factors such as TNFα, IL-6 and G-
CSF [80] and may also prove to be a powerful tool in reversing the
damage in SARS-CoV-2 patients. One possible strategy to inhibit in-
flammatory cytokines could be the use of IL-1 soluble receptors so that
the available free IL-1 binds to its specific soluble IL-1 receptors (sIL-1R)
and neutralizes the cytokine action. Another possible intervention could
be inhibiting the production or release of IL-1β by using IL-1 receptor
antagonist (IL-1Ra) such as Anakinra. Several in-vitro and animal
studies have shown beneficial effects of Anakinra in disorders with
enhanced cytokine levels such as osteoarthritis. In case of osteoarthritis,
IL-1RI receptor are increased on the human chondriocytes and synovial
fibroblasts due to the biological activation of IL-1β, major cytokine
involved in osteoarthritis [79]. The safety and efficacy of Anakinra are
well established. It was approved in 2001 for treatment of RA and later
for hyper-IgD syndrome and multiple myeloma [79] A multi-centre
study involving 472 patients suffering from RA treated with placebo
as well as IL-1RI antagonist at 30 mg/kg, 75 mg/kg and 150 mg/kg doses
for 24 weeks demonstrated statistically significant results by decreasing
the rate of joint damage, inhibiting expression of metalloproteinase, IL-1
induced matrix erosion and reducing CRP value as well as proteoglycan
synthesis [81]. In addition, the use of monoclonal antibodies against IL-
1 or against IL-1RI can be a good strategy to provide relief to the SARS-
CoV-2 suffering patients. Canakinumab, a humanized immunoglobulin
monoclonal antibody that binds selectively to the human IL-1β receptor

has been found to be more promising than other IL-1β inhibitors because
of its selective affinity for IL-1β receptors and long half-life of around 21
to 28 days [82]. In such a testing time, therapeutic agents targeting IL-1
signalling can be a ray of hope for patients suffering from SARS-CoV-2
infection.

6.3. Corticosteroids

Corticosteroids are one of the first line treatments available for the
inflammatory disorders. Corticosteroids were found to be primary
means of immunomodulation during 2003 SARS outbreak [83]. His-
torical data suggests treatment with systemic glucocorticoids in SARS
and Influenza infected patients were associated with decreased mortal-
ity rates [84]. In SARS, treatment with high dosage of Methylprednis-
olone (up to 1000 mg/kg) was associated with survival benefits, ARDS
reduction and early discharge from hospital [85]. A comparison of 2100
Dexamethasone treated patients who received a dose of 6 mg/kg for 10
days against 4300 people who received standard care showed impres-
sive results. Mortality rates were reduced by 20% among patients with
O2 therapy. In case of SARS-CoV-2, treatment with Dexamethasone
showed statistically significant reduction of hospital stay and earlier
chances of discharge from hospital (https://www.sciencemag.org/
news/2020/06/cheap-steroid-first-drug-shown-reduce-death-covid-19
-patients). Dexamethasone has been reported to reduce the expression of
a number of cytokines in various cell lines and animal models. Expres-
sion of IL-22 has been reported to be impaired in-vitro in human pe-
ripheral blood mononuclear cells (PBMC) as well as in rat sepsis model.
It causes a robust reduction in the expression of IL-22 and IL-8. IL-5 gene
expression assessed in unfractioned PBMC obtained from normal human
volunteers stimulated by various agents [86]. On the contrary, the
administration of glucocorticoids in MERS was associated with adverse
outcomes [87]. The use of corticosteroids also brings certain concerns
such as decreased immunity and increased chances of infections.

Fig. 4. Multi-organ complications associated with COVID-19. The figure illustrates the evidences and markers which are reported to be elevated in various major
organs namely heart, lung, liver and kidneys.

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8

Though, recovery using corticosteroids has been quite promising; the
immunosuppressive effects may aggravate the viral load and increase
the risk of secondary infections [84]. Timing of treatment and blood
levels should be taken into consideration while treating with cortico-
steroids as it may have both stimulatory as well as inhibitory effects on
immune system [84,88]. Hence it can be strived upon that corticosteroid
therapy has been found to reduce the mortality rate in various infections
and can be taken ahead in case of COVID-19. Nevertheless, the use of
corticosteroids in COVID-19 warrants the critical evaluation of their risk
vs benefit ratio for a safe and efficacious therapeutic regimen.

6.4. Peroxisome proliferator–activated receptor-γ (PPAR-γ) agonist

PPAR-γ is a nuclear receptor which endogenously regulates meta-
bolism, inflammation and immunity. It is expressed in airway epithe-
lium, bronchial mucosa, smooth muscle cells and endothelial cells. T
cells, B cells, dendritic cells, neutrophils, macrophages also express
PPAR-γ during differentiation or upon activation. It plays a major role by
modulating inflammation by interaction with NF-κB which is one of the
main regulator of immune response and inflammatory cascade [89]. The
activation and functioning of PPAR-γ takes place by heterodimerization
of PPAR-γ with nuclear receptor Retinoid X Receptor (RXR) [90]. Upon
ligand binding PPAR-RXR complex migrates into the nucleus and binds
to peroxisome proliferator hormone response element (PPRE) causing
target gene expression [91,92]. PPAR-γ has been shown to downregulate
the production of IL-17 and IL-2 by CD4+ cells contributing to decreased
proliferation and differentiation of Th1 and Th17 [89]. Xiong et al.
demonstrated decreased expression of IL-6 and TNFα in Human
Mesangial cells (HMCLs) upon treatment with PPAR-γ agonist [92].
Studies have suggested ovalbumin-induced murine model of asthma
presented decreased eosinophilic inflammation and airway hyper-
responsiveness upon PPAR-γ agonist treatment. Anti-inflammatory
cytokine IL-10 expression increased in the lung tissues upon treatment
with PPAR-γ agonist [93]. Reddy et al. demonstrated that PPAR-γ sup-
pressed the expression of endothelial pro-inflammatory mediators such
as cytokines and adhesion molecules facilitating migration of neutro-
phils into alveolar spaces in LPS mediated sepsis model in mice. PPAR-γ
agonists not only down regulate the inflammatory response induced by
cytokine storm but also promote their survival in mouse model of H1N1
influenza virus [94]. Sirtuin 1 activated PPAR-γ in gouty arthritis mouse
model contributes to increased mRNA expression of PPAR-γ, inhibition
of infiltration of inflammatory cells, decreased production of pro-
inflammatory cytokines such as IL-1, IL-6 and chemokines [95]. The
significant inhibition of wide variety of proinflammatory cytokines by
PPAR agonists make them a better choice for the control of inflamma-
tory conditions. Hence, repositioning PPAR-γ agonist such as Rosigli-
tazone, Pioglitazone can be one of the potential strategies to counteract
cytokine storm and multi-organ failure involved in observed in SARS-
CoV-2 patients.

6.5. JAK-STAT inhibitors

JAK-STAT pathway plays a major role in the progression and path-
ogenesis of Psoriatic Arthritis (PsA). A dysfunctional response from the
innate and adaptive immune systems in arthritis results in over-
expression of multiple inflammatory cytokines. Tofacitinib is a small
molecule having a short half-life of 3 h which acts either as a kinase
inhibitor of JAK or by competitive binding to the docking site with
STAT. It has been reported that Tofacitinib significantly inhibited the
secretion of IL-6, IL-8 and MCP-1 from PsA explant cultures. The long
term safety and efficacy profile in patients receiving Tofacitinib as
monotherapy or with non-biologic disease modifying anti-rheumatic
drug is well demonstrated [96]. It is also used in the treatment of In-
flammatory Bowel Disease (IBD) where JAK-STAT mediated release of
cytokines such as IL-2, IL-4, IL-7, IL-9 is of significance [97]. Baricitinib,
a selective inhibitor of JAK1/2, showed significant decrease in the

expression of CD80/86 on LPS stimulated human monocyte-derived
dendritic cells which inhibits the differentiation of naïve CD4+ T cells
into Th1 and Th17 cells. It shows significant reduction in the production
of chemokine MCP-1 as well as reduced expression of pSTAT3 which
further inhibits the production of IL-17 and IL-22 in collagen induced
arthritis in BALB/c mice [98]. On the other hand, Methotrexate (MTX) is
a JAK/STAT inhibitor and a disease modifying anti-rheumatic drug
(DMARD) licenced for treatment of Rheumatoid Arthritis (RA) in 1988
by Food and Drug Administration [99]. It has been established that MTX
directly interferes with the binding of IL-1β to its receptor and inhibits
the inflammatory activity of IL-1β. Moreover, there is an inverse rela-
tionship between long intergenic (noncoding) RNA–p21 (lincRNA-p21)
and NF-κB activity in RA. Depressed levels of lincRNA-p21 leads to
increased activity of NF-κB which then contributes to the release of
cytokines. MTX increases the levels of lincRNA-p21 leading to decreased
expression of NF-κB [100]. Additionally, it has also been demonstrated
that MTX inhibits the expression of TNFα induced NF-κB activation by
degradation of IκBα. The results have been found to be consistent in-vivo
when mice were treated with MTX upon collagen induced arthritis
[101]. As the JAK-STAT pathway is one of the important pathways for
the cytokine storm observed in COVID-19, it can be deduced that the
targeting of JAK-STAT pathway could be a successful approach to con-
trol inflammatory response in COVID-19.

6.6. PDE-4 inhibitors

PDE-4 inhibitors have attracted a considerable attention because of
their potential use in pulmonary inflammatory diseases, contact
dermatitis and psoriatic arthritis [35]. PDEs act by converting intra-
cellular secondary messenger cyclic adenosine 3′,5′-monophosphate
(cAMP) to Adenosine Monophosphate (AMP) thereby reducing Protein
Kinase A activity which contributes to the production of pro-
inflammatory mediators like TNFα, IL-22 and IL-17 in endothelial
cells, inflammatory cells and keratinocytes [35,36]. Apremilast is a se-
lective PDE-4 inhibitor approved by the US Food and Drug Adminis-
tration for the treatment of moderate to severe plaque psoriasis as well
as for psoriatic arthritis, which is a chronic, systemic inflammatory
disease characterised by release of IL-23 and IL-12 [102]. Apremilast
exhibits a significant reduction of cytokines mainly IL-17, IL-22 and
TNFα involved in the pathogenesis of psoriasis [103]. In vivo studies in
immune-deficient mice xenotransplanted with normal human skin and
triggered with psoriatic Natural Killer (NK) cells when treated with
Apremilast led to absence of lymphocyte infiltration and decreased
proliferation index. Immunohistochemistry presented decreased TNFα,
intracellular adhesion molecule-1 and human leukocyte antigen
expression [104]. Analysis of clinical trial data of patients suffering from
moderate to severe psoriasis upon treatment with 20 mg of Apremilast
for 29 days presented a reduction in the number of T cells and dendritic
cells in dermis and epidermis [105]. Similarly, another drug, Roflumi-
last belonging to same class is used in the treatment of Acute Lung Injury
(ALI). ALI is characterised by recruitment of neutrophils, their activation
followed by increased levels of TNFα, IL-6 and IL-8 in the early phase of
ALI [103]. The overabundant release of cytokines destroys the endo-
thelial layer of the lungs causing increased vascular permeability. In
vivo data suggest that Roflumilast when administered to saline lavage-
induced ALI demonstrated inhibition of endothelial barrier function,
marked reduction in the concentration of IL-6, IL-8 and TNFα [106].
Interestingly, in another study, Roflumilast has been shown to act by
inhibiting inflammatory inhibitors via NF-κB, p38 mitogen activated
protein kinase and c-Jun NH2-terminal kinase inhibition in murine
macrophage cell line RAW264.7 cells [107]. These studies suggesting a
potential inhibition of induced cytokine signalling by PDE-4 inhibitors
opens an opportunity to explore their effects in the SARS-CoV-2.

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6.7. Anti-TNF-α therapy

TNF-α is one of the most important mediators of inflammatory pro-
cess which is produced by a variety of cells including macrophages and
T-cells [108]. Stimulation of TNF receptor leads to activation of NF-κB
which then translocate into the nucleus and upregulates various tran-
scriptional genes. Dysregulated TNF-α signalling has been shown to
trigger cytokine storm which leads to cell death whereas optimal levels
of TNF-α is required for tissue repairing upon acute injury [109]. Hence
TNF-α act as an agent in inducing inflammatory cytokine storm at one
hand and providing protection during tissue injury on the other side.
Exaggerated TNF-α release occurs in various diseases including Rheu-
matoid Arthritis (RA), Crohn’s Disease, PsA, spondyloarthritis and in our
context COVID-19 has been reported [110]. Anti-TNFα therapy using
Etanercept, Adalimumab, Infliximab, Golimumab, Certolizumab has
been found to be successful in the treatment of various inflammatory
diseases. TNF-α-targeting aptamer and its PEG-derivate with antago-
nistic functions have been shown to improve oxygen saturation,
decrease fluid leakage and neutrophil infiltration in the alveolar spaces.
Pro-inflammatory cytokines and chemokines in the tissue were reported
to be suppressed [111]. Blockade of TNF-α in B27 transgenic rats, an
experimental model of colitis prevented the progression of the disease
[112]. In vivo findings suggest that treatment with a combination of
TNF-α and IFN-γ neutralizing antibodies has provided protection against
SARS-CoV-2 infection associated complications in mouse model [113].
Hence, it is not overstating to suggest the TNF-α therapy in management
of cytokine storm conditions prevalent in COVID-19 patients. However,
this remains entirely speculative and their use demands a better un-
derstanding of SARS-CoV-2 which could be achieved in future.

6.8. Oxidised-1-palmitoyl-2-arachidonoyl-phosphaticylcholic (OxPAPC)
inhibitors

In lungs, phospholipids are found in lung surfactants which form a
layer at the alveolar air-water interface and reduce surface tension.
Surfactant is composed of phospholipids including unsaturated phos-
phatidylcholine. Administration of oxidised phospholipids induced
increased levels of IL-6 in the lungs and triggered acute lung injury
[114]. The inherent property to sense pathogen-associated molecular
patterns (PAMPs) is found in humans which plays a major role in the
early identification and host defence against pathogenic microorganisms
[115]. Toll like receptors (TLRs) are one of the family of PAMP re-
ceptors. OxPAPC which stimulates TLR leads to intracellular signalling
and production of TNF-α, IL-1 and other pro-inflammatory pathway
causing capillary break and endothelial damage [116]. Eritoran, struc-
turally analogous to lipid A portion of LPS, antagonizes LPS-mediated
TLR signalling. Eritoran when administered to mice infected with
influenza virus demonstrated decreased ALT, AST levels, blunted pro-
and anti-inflammatory gene expression, decreased expression of IL-6
and IL-10 and normal lung architecture. In contrast, another study re-
ported no improvement in lung histology, lung congestion, edema and
haemorrhage after Eritoran treatment when compared to vehicle treated
group. In the same study, plasma ALT, AST levels, gut permeability, liver
inflammation were reduced and liver presented fewer and smaller areas
of necrosis with decreased expression of NF-κB [117]. Another study
demonstrated the prevention of uncontrolled inflammatory response
along with inhibition of pro-inflammatory cytokine production and
sepsis by Eritoran in endotoxin induced retinochoroidal damage [118].
Hence, we suggest that Eritoran can be a potential regimen in SARS-
CoV-2 infected patients which can prevent multi-organ damage and
decrease the mortality rate and benefit the patients.

6.9. Xanthine oxidase inhibitors

Xanthine oxidase (XO) is one of the major enzymes involved in the
production of free radicals and ROS during inflammation and hypoxia

like condition [119]. Under normal physiological conditions it is found
in the form of Xanthine dehydrogenase, while under hypoxia like con-
ditions, it gets converted to XO. XO oxidises purine substrates such as
xanthine and hypoxanthine, to produce uric acid and ROS [120]. The
expression of XO has been suggested to be upregulated in the lung tis-
sues by various inflammatory stimuli such as endotoxin, hypoxia, and
cytokines. Allopurinol, structural analogue of purines and pyrimidines,
inhibits XO leading to the inhibition of uric acid production in gout.
Febuxostat (FBX) is a non-purine inhibitor of XO and is advantageous
over Allopurinol as it can suppress both oxidised as well as reduced
forms of XO [120,121]. FBX has been reported to inhibit number of
pathways such as MAPK, NF-κB, receptors for Advanced Glycation end
product/Phosphatidyl inositol-1,4,5-bisphosphate-3-kinase (RAGE/
PI3K/Akt) in various disease models including acute lung injury, Ul-
cerative Colitis, myocardial ischaemia [122]. FBX has been shown to
reduce elevated lactate dehydrogenase (LDH), nitrogen oxides (NOx)
levels, serum C-reactive protein (CRP), TNF-α levels and edema and
alveolar thickening in LPS induced lung injury in rats [123]. Treatment
with FBX resulted in improvement in the cardiac functions, improved
haemodynamic and ventricular functions with characteristic decrease in
the expression of inflammatory biomarkers have been reported in
myocardial ischaemic reperfusion (IR) injured rats [124]. Similarly, FBX
attenuated acetic acid induced Ulcerative Colitis in mice at a dose level
of 20 mg/kg by inhibiting NF-κB [125]. A comparison of chest scans of
patients upon 14 days treatment demonstrated 47% reduction in lung
involvement when treated with FBX, whereas Hydroxychloroquine
(HCQ) treated patients reported 58.3% reduction in lung involvement
[126]. However, FBX could be a superior choice for COVID-19 patients
as it has lower side-effects in comparison to HCQ where QT prolongation
is one of the major concerns.

6.10. Histone Deacetylase (HDAC) inhibitors

Histones play a vital role in the regulation of gene expression by
acetylation or deacetylation. Acetylation of histones leads to relaxation
of chromatin following binding of transcription factors promoting
transcription. In contrast, deacetylation of histone condenses chromatin
and suppress gene transcription [127]. In mouse model of cecal ligation
and puncture (CLP) induced lethal sepsis, Tubastin A, a selective HDAC
class IIb and a specific HDAC 6 inhibitor has been reported to improve
survival by decreasing the expression of pro-inflammatory cytokines and
biomarkers such as TNF-α and IL-6 [128]. Suberoylanilide hydroxamic
acid (SAHA) also known as Vorinostat is an HDAC 6 inhibitor approved
by the U.S. Food and Drug Administration (FDA) for the treatment of
Cutaneous T-Cell Lymphoma (CTCL) [129]. SAHA binds to the zinc
containing pocket in the HDAC 6 and cause their reversible inhibition.
Studies suggest that micromolar concentration of SAHA have anti-
tumour effects, whereas nanomolar concentration decreases the secre-
tion of cytokines [130]. It has been proposed that SAHA may suppress
TLR4-MyD88-dependent pathway by acetylating STAT-1, which further
reduces the nuclear translocation of NF-κB [131,132]. Since limited and
elusive information exists regarding the actual mechanisms that are
altered by different HDAC inhibitors but in the current scenario it ap-
pears attractive agent to be explored which may surmount cytokine
storm in COVID-19.

6.11. Pyruvate dehydrogenase kinase inhibitors

Relating cytokine cycle with metabolism, pyruvate dehydrogenase is
a key enzyme that regulates the glucose, lipid, lactate and ATP levels.
Pyruvate dehydrogenase complex plays a major role in oxidative
decarboxylation of pyruvate and links glycolysis, fatty acid synthesis
and citric acid cycle. Pyruvate dehydrogenase kinase phosphorylates the
α subunit of pyruvate dehydrogenase leading to its inactivation causing
decreased metabolism and lack of energy [133]. The use of pyruvate
dehydrogenase kinase inhibitors can be one of the strategies in order to

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prevent multi-organ failure due to cytokine storm. It has been reported
through in-vivo studies that mice when infected with H1N1 virus at sub-
lethal dose presents restoration of pyruvate dehydrogenase activity
upon treatment with Diisopropylamine dichloroacetate (DADA). Results
showed suppressed induction of pro-inflammatory cytokines, increased
survival rate of mice and histopathology shows reversal of pathological
treatment at 50 mg/kg twice daily for 14 days [134]. Therefore, these
inhibitors could be explored in suitable preclinical models and also plan
to evaluate in suitable clinical conditions to evaluate their efficacy
against cytokine storm.

6.12. KV 1.3 channel blockers

Immune cells express a variety of ion channels and transporters that
allow movement of ions across the plasma membrane and the membrane
of intracellular organelles. In T cells, the interaction of the T cell re-
ceptors with its antigens induce an increase in the extracellular Ca2+

concentration, regulating numerous downstream signalling pathways
that control clonal expansion, differentiation and cytokine production
[135]. Upon activation, the number of Kv1.3 channels of effector
memory (TEM) cells increase while that of KCa3.1 channels remain
constant, emphasizing the role of Kv1.3 channels in TEM lymphocytes
[136]. TEM cells rapidly produce and release inflammatory and cytotoxic
mediators such as IFN-γ, IL-4 and perforin [137]. It has been reported

that blocking of Kv1.3 channels in TEM cells reduced the influx of Ca
2+,

resulting in TEM cells specific immunomodulatory effects without
compromising naïve and central memory lymphocytes effector functions
such as protection against pathogens [138]. Similarly, in COVID-19,
activation of T cells leads to generation of effector and memory T
cells, which further directs the release the cytokines and chemokines
[28,139]. Hence, selectively targeting TEM by blocking Kv1.3 channels to
control the release of cytokines in COVID-19 can be a novel approach
which requires further exploration. There are few selective Kv1.3 in-
hibitors which were proven to be safe upon clinical use, therefore, these
inhibitors can be repurposed for the management of cytokine storm.

6.13. IL-12/23 axis

IL-12 and IL-23 are major players in activating adaptive immunity.
Activation of dendritic cells and macrophages generates different cyto-
kines such as IL-12, IL-23, IL-1β, transforming growth factor-β (TGF-β),
and interferon γ (IFN-γ) [140]. IL-12 helps in differentiating a naive T
cells into the Th1 subtype which further secretes TNF-α, IL-2, and IFN-γ
[141]. On the other hand, IL-23 can activate the secretion of IL-17A, IL-
17F, and IL-22. Moreover, IL-23 stabilises Th17 and suppresses T regu-
latory (Treg) cell differentiation from a naive T cell [142]. IL-17A and IL-
17F enhance the expression of various inflammatory cytokines, che-
mokines and adhesion molecules. IL-17A induces stromal cells to

Fig. 5. Various potential targets and agents to control cytokine storm in COVID-19.

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11

produce extracellular matrix-degrading proteases which can cleave
components of the extracellular matrix and promote tissue degradation.
Inhibition of Treg differentiation further potentiates the inflammatory
cascades [143]. Ustekinumab is a human IgG1 kappa (κ) monoclonal
antibody that blocks the IL-12/23 p40 subunit and prevents the p40
subunit of IL-12 and IL-23 from interacting with their receptors. Pre-
vention from binding subsequently neutralizes IL-12 and IL-23 mediated
cell activation and cytokines generation [141]. Targeting this pro-
inflammatory cytokine pathway can be an area of intense therapeutic
exploration in case of novel coronavirus disease where cytokine storm
syndrome has been identified as a major culprit to promote the damage.
Fig. 5 summarises different potential targets for the management of
cytokine storm in COVID-19.

7. Contradictions and unanswered questions

The involvement of cytokine storm in the pathogenesis of SARS-CoV-
2 is well evident from clinical studies. However, the concept of cytokine
storm is quite a complex phenomenon and a critical analysis is war-
ranted before reaching any consensus. The first and foremost question is
how relevant is the cytokine storm in SARS-CoV-2? Although, the clin-
ical studies have shown upregulated levels of cytokines in SARS-CoV-2
patients; the levels do not show a strong, well-established correlation
with severity of the infection. For exampleIL-6 is one of the most
important mediators involved in pathogenesis of SARS-CoV-2 and is also
considered as a potential biomarker of COVID-19 progression [144].
However, the induced expression of IL-6 in SARS-CoV-2 has been found
to be erratic and heterogenous [145]. Also evidences from studies sup-
port the notion that timing is of the utmost importance in targeting in-
flammatory cytokines in COVID-19 [73]. A relevant question is that out
of more than 20 cytokines/chemokines elevated in COVID-19, which
one should be the prime focus at the earliest to prevent the complica-
tions in clinical conditions. Further, there are many limiting factors
involved in analysis of cytokine expression such as time of sampling,
methodology used, calibration of instrument, lab to lab variation, stage
of infection and comorbid conditions etc. which are needed to be
considered before evaluation. Another point of concern is that these
mediators are mostly involved in pleiotropic effects covering a wide
range of physiological functions. Hence, it is quite difficult to establish a
direct correlation between their high levels and the severity of patho-
genesis. In fact, the induced expression of these cytokines may not al-
ways translate to the pathological conditions, further limiting our
inference. Furthermore, most of the cytokines have target cell specific
functions and the effects of cytokines may vary with cell to cells. In
addition, the effects of cytokines are defined by their cross talk with
other cytokines/signalling molecules, hence the direct inhibition of a
particular cytokine may not be a feasible approach apparently. Adding
the complexity to the subject and limiting the efficacy of direct inacti-
vation strategies.

In addition, the use of different immunosuppressants such as corti-
costeroids also brings their own complications with their use. As the
hyperimmune response observed in patients is a consequence of acti-
vation/hyperactivation of body’s protective mechanism with an aim to
neutralise the infections; the suppression of immunity might also result
in negative outcomes such as secondary infections and weakened ability
of the body to eliminate SARS-CoV-2 infection.

As our knowledge of pathogenesis of SARS-CoV-2 infection is still
limited; most of the agents tried in combating the infection are majorly
speculative based on the similarities with other disease conditions such
as influenza, rheumatoid arthritis etc. In this case, the likelihood of the
success entirely depends on the similarity with other disease conditions.
For example, Tocilizumab, which has shown effectiveness in rheumatoid
arthritis is under clinical trials for SARS-CoV-2 due to resemblance on
inflammatory basis. However, with increase in our knowledge about
mechanisms involved and pathogenesis of virus, it is possible that these
strategies could be fruitful approaches to control cytokine storm.

8. Concluding remarks

The SARS-CoV-2 has been a great burden for the global health, public
and economy sectors which has affected almost every aspect of our life.
Our limited knowledge about this novel virus is one of the major chal-
lenges for the discovery of safe and effective vaccine. Hence, there is an
urgent need to explore the different pathways used by virus. One of the
major events observed in the SARS-CoV-2 infection is the cytokine
storm. The incidences of multiple complications associated with cyto-
kine storm highlight its importance in SARS-CoV-2 infection and further
present it as a possible major target. The different targeting strategies as
discussed here are needed to be explored by keeping in mind the prin-
ciples of viral pathogenesis and the physiological roles of inflammatory
mediators. Further, as most of the studies are preliminary in nature;
there is a need to replicate and reproduce the results of potential studies
before declaring a valid target. Nevertheless, cytokine storm is a com-
plex phenomenon associated with SARS-CoV-2 and its exploration may
endow us with potential lead molecules for tackling one of the deadliest
viruses, SARS-CoV-2. Moreover, the cytokine storm may also show its
involvement with other future viral infections, hence, targeting cytokine
storm may become a lifesaving strategy in different conditions.

CRediT authorship contribution statement

RH and MAS wrote the manuscript.
CG corrected and revised the manuscript.

Declaration of competing interest

None.

Acknowledgment

The authors would like to thank Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Government of India and Director,
NIPER Hyderabad for the support.

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