Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), more commonly known as COVID‐19, is a virus that has spread at a rapid rate beginning in 2019 causing a pandemic. Currently, we remain in ...this pandemic, and as this virus progresses it is creating mutations of itself, called variants. With these variants, it has become increasingly difficult to control COVID‐19 and keep track of new variants that continue to infect people everywhere. In Orange County, California, COVID‐19 and its variants have enabled county wide shut downs and thousands of infections and deaths. To investigate its devastating effect, five positive COVID‐19 nasal samples were collected between February 2021 to July 2021, and the RNA was extracted. After extraction, a Reverse Transcription Polymerase Chain Reaction (RT‐PCR) was done to amplify 1,727 base pairs of the S‐ Gene within the COVID‐19 genome and was sent to sequencing. These sequences were then aligned using the Basic Local Alignment Search Tool (BLAST) and compared to the original COVID‐19 S‐Gene genome to analyze possible variations. Within the five samples, 30 different mutations were detected and a phylogenetic tree was generated. Awareness of the genetic variation of SARS‐CoV‐2 provides potential future development for more effective targeted research.
Antibodies play an important part in combating SARS‐CoV‐2 infection whether generated by the infection or vaccination. However, the many epitopes generated by infection have not been fully ...investigated with only a few epitopes known and these being mostly limited to the S protein’s receptor binding domain (RBD) and the N‐terminal domain which limits vaccine and drug design 1‐4. The difference between epitopes generated by infection and vaccination has also not been studied. To address this, we employed a SARS‐CoV‐2 proteome microarray to screen for linear epitopes recognized by antibodies present in COVID‐19 patients and individuals vaccinated with the Pfizer‐BioNTech mRNA COVID‐19 Vaccine.
The proteome microarray consisted of S, N, and E proteins, as well as spotting peptides that were 15 amino acids in length with overlaps of 5‐amino acids, covering the entire SARS‐CoV‐2 proteome (MN908947.3) (Figure 1). Blood samples were incubated onto the arrays followed by an incubation of fluorescent secondary anti‐human antibodies. Fluorescent intensity data generated and normalized using the Z‐score method and then further analyzed for significance by parametric one‐way ANOVA with Dunnett's post hoc test (COVID‐19 cohort) and repeated measure ANOVAs with Dunnett's post hoc tests (vaccinated cohort).
The full‐length S protein showed a significant increase in COVID‐19 patients at around 20‐23 days after symptom onset and vaccinated individuals over all time points in both IgM and IgG antibodies (Figure 2A). Linear mapping of the IgM epitopes revealed a degree of overlap between infected and vaccinated individuals (22.2%; 6/27 total) with both having epitopes in the RBD and fusion peptide (FP) (Figure 2B). Structural mapping on 3D models of the S protein showed that all epitopes where on the surface of the protein and that COVID‐19 generated epitopes have a different pattern than those generated by vaccination (Figure 2C).
An Epitope identified in this study with future prospects is epitope S481‐495 from COIVD‐19 patients that partially overlapped the binding site of two neutralizing antibodies previously isolated from COVID‐19 patients, S2H135 and F2B‐2F61, and contacted amino acids that interact with ACE2 receptor6,7. One epitope of note from the vaccinated individuals is epitope S811‐825 which mapped adjacent to the fusion‐peptide proximal region. These epitopes may be helpful in future vaccine and antibody therapy development.
1 Ju, B. et al. Nature 584, 115‐119.
2 Robbiani, D. F. et al. Nature 584, 437‐442.
3 Seydoux, E. et al. bioRxiv.
4 Wu, Y. et al. Science 368, 1274‐1278.
5 Piccoli, L. et al. Cell 183, 1024‐1042 e1021.
6 Casalino, L. et al. ACS Cent Sci 6, 1722‐1734.
7 Wang, Q. et al. Cell 181, 894‐904 e899.
Covid‐19 vaccination and social barrier efforts worldwide have deescalated the death and morbidity toll due to Sars CoV‐2 infection in most countries. However, intramuscular vaccination leads to a ...variable antibody response insufficient to provide airway mucosal immunity that prevents contagion and reinfection in asymptomatic carriers. Nasal vaccines could provide this missing piece and are currently being tested with encouraging results, but large‐scale availability and efficacy in humans are unknown. Moreover, previous exposure to the virus would render nasal spray vaccines less effective. Past literature on upper airway infections has shown that water steam inhalation therapy (WSIT) may modulate airway mucosa immunity responses. Airway mucosal immunity in COVID‐19 infection is a critical aspect to consider with WSIT. Based on a small but slowly growing body of evidence, we propose that WSIT may be a relevant home remedy worth revisiting to help halt mass contagion. Here, we review current and past studies on WSIT use in upper respiratory infections (URI). Previous studies reported mixed results on the efficacy of steam therapy on viral respiratory diseases. Safety concerns with steam therapy include possible nosocomial infections associated and burns. There is a paucity of studies with high methodological quality exploring the outcomes of this ancient therapy. Upper airway blood flow increases substantially after 15‐min of steam inhalation with temperatures of 42 ℃. Still, methods to measure bronchial blood flow are lacking, and this may be an essential variable for the analysis of how steam may impact mucosal immune responses. Studies with better methodologies demonstrate that nebulized steam at 80°C for 20 min reduces neutrophils compared to controls in COPD patients indicating an anti‐inflammatory effect. The best evidence for using WSIT for upper airway viral disease shows that 60% of asymptomatic carriers increased viral shedding immediately post steam inhalation, and Rt‐PCR testing confirmed viral negativity at 10‐days post‐treatment. Subjects used steam for a total of 20 minutes per hour with steam temperatures ranging from 55 to 65℃. Efficacy rates decline with research methodologies that use WSIT for less than 20 minutes. In summary, more recent studies using WSIT for URI show encouraging results and may help to disentangle former controversies. Lessons emerging from previous studies indicate that exposure of WSIT less than 20 minutes may be ineffective. Adopting WSIT as an inexpensive public health measure may lead to secondary prevention of airway viral infections, especially in new viral strands that have the potential to become pandemic.
Introduction
Coronavirus disease 2019 (COVID‐19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV2). The portal of entry for the virus is peptidase ACE2, a major key player of ...the renin‐angiotensin‐aldosterone system (RAAS), resulting in severe lung injury. Although COVID‐19 mainly manifests as an acute respiratory distress syndrome (ARDS), there is increasing evidence of neurological symptoms in patients infected by COVID‐19. Yet, there is limited understanding of how COVID‐19 impacts the central nervous system (CNS). We speculate that such neurological symptoms maybe a consequence of a dysfunction of the blood‐brain barrier (BBB) with our central hypothesis that the neurological effects of SARS CoV‐2 are driven by chronic hypoxic stress‐impaired ACE2 at the BBB.
Methods
An in‐vitro human induced pluripotent stem cells (hiPSCs) BBB model was used in the study. Such model was exposed to various levels of hypoxia (1,5 and 10%) for up to 24 hours. In addition, normoxic cells were treated with Angiotensin II (AngII) or Angiotensin 1‐7 (degradation by product of AngII by ACE2). Changes in the barrier function was assessed using TEER, permeability to fluorescein and tight junction staining. Changes in ACE2 and MasR expression was assessed by immunofluorescence, whereas ACE2 shedding and HIF‐1 alpha expression was assessed by ELISA.
Results
Mild (10%) hypoxia was sufficient to induce the loss of barrier function. Secretion of ACE2 under hypoxia followed a biphasic pattern, with highest levels at 5% and 10%. Ang II and Ang1‐7 had little effect on the barrier function under normoxic condition. The hypoxic exposure induced shedding of the membrane bound ACE2 and molecular mechanism of hypoxic exposure in regulation of ACE2 occurs in a HIF1α‐ dependent manner.
Discussion
Our preliminary data suggest that our human model of the BBB responds to hypoxia and express critical components of RAAS. Both ACE2 and MasR negatively respond to mild hypoxia followed by a decreased barrier function with no changes in tight junction complex. Such loss was correlated with increased ACE2 shedding in HIF1α‐ dependent manner. We are currently investigating role of Ang 1‐7 in rescuing barrier function under hypoxic stress.
The novel coronavirus SARS‐Cov‐2 or COVID‐19 became a global pandemic and currently few medically approved curative treatments exist. SARS‐Cov‐2 acts similarly to SARS‐CoV‐1 from where it may have ...evolved. The COVID‐19 virus can survive ~3 hours in air and < 72 hours on distinct surfaces. COVID‐19 mutates by introducing sequence errors in the host’s RNA genome or by modifying proteins and enzymes. Vaccination, including booster shots, social distancing and isolation are the most generally practiced guidelines in the global management of COVID‐19. Globally the most frequently utilized pharmacologic treatments include ivermectin, hydroxychloroquine, glucocorticoids, the anti‐viral agent remdesivir, monoclonal antibodies, and convalescent plasma in combination with an antimicrobial agent such as azithromycin to minimize secondary microbial infection and nutritional supplementation (vitamins C, D3, and zinc) to enhance cellular immune responses and are included in the routine protocols of some emergency rooms. COVID‐19 viral transmission occurs via respiratory microdroplets, by inhaling COVID‐19 laden airborne particles and contact with contaminated surfaces on which these droplets have been deposited. SARS‐CoV‐2 targets ACE2 receptors in the upper and lower respiratory tracts in addition to the heart, brain, and gastrointestinal tract, and may cause thromboses in the liver, heart, and kidney.
Significant risk factors for the severity of COVID‐19 infection include advanced age and pre‐existing comorbid conditions obesity, hypertension, cardiovascular disease, diabetes, and compromised immune status, which all correlate with greater mortality. Outcome‐effecting factors include viral load, viral mutations, and pre‐existing conditions. Since its origination, genomic studies of the virus have identified numerous variants which have become regionally prevalent in different countries. Infective factors, comorbidities and viral load strongly affect outcomes; patients infected with the greatest viral load showed a higher mortality. It is opined that the number of deaths attributed to COVID‐19 may be inaccurate due to errors in diagnosing and reporting, since other similar illnesses may exhibit similar symptoms.
Future research should focus on prevention practices, comorbidities, genetic prevalence, reliable systematic and consistency in country‐by‐country testing and reporting procedures, further scrutiny regarding the efficacy of current vaccines and protocols, and the pursuit for innovative therapies for Coronaviruses and variants including biophotonics and exploration of emerging bioenergetic, nutritional, pharmacological, immunotherapeutic and vaccination‐preventive applications for eradication of COVID‐19.
Refs: 1. Cheng, RZ. (2020a). Med Drug Disc, 5, 100028; 2. Shankar, AH, & Prasad, AS. (1998). ACJN, 68(2), 447S‐463S; 3. Petrilli, CM, Jones, SA, et al. (2020); medRxiv; 4.van Doremalen, N, Bushmaker, T et al. (2020). NEJM, 382(16), 1564‐1567
Abstract only
Introduction
Recent research suggests that endothelial activation plays a role in COVID‐19 pathogenesis by promoting a pro‐coagulative and pro‐inflammatory state. However, the mechanism ...by which the endothelium is activated in COVID‐19 is unclear.
Objective
To investigate the mechanism by which COVID‐19 activates the pulmonary endothelium.
Hypothesis
The pulmonary endothelium generates reactive oxygen species (ROS) upon exposure to the “inflammatory load” of the systemic circulation.
Methods
COVID‐19 was recreated
in vitro
and
ex vivo
, by exposing human lung endothelial cells (EC) or donor human lung slices (human precision‐cut lung slices or huPCLS) to medium supplemented with serum from COVID‐19 affected subjects. Sera were acquired from patients with COVID‐19 infection admitted to the Intensive Care Unit of the Hospital at the University of Pennsylvania. ROS (fluorescent dye, CellROX) and intercellular adhesion molecule (ICAM‐1) levels were assessed by fluorescence labeling and imaging.
Results
Both EC activation (as monitored by ROS production) and pro‐inflammatory phenotype (as assessed by ICAM‐1), were significantly higher with COVID‐19 as compared to normal subjects.
Conclusions
The endothelium is activated with COVID‐19 via ROS production; thus, the ROS produced drive a pro‐inflammatory phenotype by inducing the expression of ICAM‐1, a pivotal marker of endothelium inflammation. As ROS mediates EC activation and inflammation during COVID‐19, ROS blockade could be a therapeutic target in maintaining vascular health.
The adaptive immune response is under circadian control, yet, the benefit of this rhythmicity for the organism is unknown. Furthermore, it is not understood why adaptive immune reactions continue to ...exhibit circadian changes over long periods of time. Using a combination of experimental and mathematical modelling approaches, we show here that dendritic cells (DCs) migrate from the skin to the draining lymph node (LN) in a time‐of‐day‐dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Greater numbers of infiltrating DCs induce rhythmic expression of TNF‐α in the draining LN, which enhances ICAM‐1 expression in high endothelial venules (HEVs), resulting in lymphocyte infiltration and LN expansion. Icam1 is controlled by binding of the core circadian clock transcription factor BMAL1 to its promoter region, and rhythmic Icam1 expression is lost in mice lacking endothelial cell BMAL1. LN cellularity continues to be different for weeks after the initial time‐of‐day‐dependent challenge, which determines the immune response to vaccinations directed against Hepatitis A virus as well as SARS‐CoV‐2, as evidenced by rhythmic T cell reactions, germinal center formation and antibody production. Our results provide the mechanistic understanding of the time‐of‐day dependent development and maintenance of an adaptive immune response, demonstrating its dependency upon the timing of the initial challenge and the interactions of rhythmicity in multiple parameters. This provides a strategy for using time‐of‐day to optimize vaccination regimes.
Endothelial cells (ECs) are in the inner layer of blood vessels, and it controls the transportation of nutrition and essential molecules from the extracellular matrix to the intracellular space ...making a homeostasis environment. Vascular endothelium can be disrupted with viruses, which are among the main reasons for epidemic and pandemic outbreaks, and it leads to health issues such as cardiovascular‐related diseases. Wide range of viruses can potentially target the endothelium including Dengue virus estimated to infect 390 million people per year. Zika virus leads to pregnancy complications such as preterm birth and miscarriage. Tick‐borne encephalic damaged the central nervous system and, Hantavirus has a mortality rate of 38% through hantavirus pulmonary syndrome. Reviewing these several diseases, including SARS‐CoV‐2, has given an in‐depth look at what the endothelial cells are going through when these viruses are infecting the host bodies. Following the virus infection, ECs are going through activation mode, which is accompanied with glycocalyx degradation, plasma leakage, phosphorylation of P120, and cadherin, increased proinflammatory responses and, disruption of barrier integrity.
The SARS‐CoV‐2 pandemic has taken a global toll; With a total of 362 million cases, there are 5.63 million deaths occurring. With the usage of immunofluorescence and vascular permeability assay, we monitor how Human Umbilical Vascular Endothelial Cells (HUVEC) and Human Lung Microvascular Endothelial Cells (HLMVEC) are impacted when treated with UV‐inactivated & heat‐inactivated SARS‐CoV‐2, and tumor necrosis factor (TNF)‐α. TNF‐α is an inflammatory cytokine prominent during inflammation and its known to increase the permeability of endothelium. Like the TNF‐α, both cells treated with UV‐inactivated and heat‐inactivated SARS‐CoV2 demonstrated boosted permeability. With the considerable effect of SARS‐CoV2 on the permeability of ECs, it is essential to understand the mechanisms and pathways behind the boosted leak, including the impact of the virus on the expression of adhesion molecules like platelet endothelial cell adhesion molecule (PECAM‐1). One can see in the images of HUVEC and HLMVEC that SARS‐CoV‐2 diminish the localization of PECAM‐1. On a non‐treated negative control, cells are spaced evenly and are close to another since their junctions are not targeted. Once the cells are treated with either heat‐inactivated or UV‐inactivated SARS‐CoV‐2, PECAM‐1 localization is disrupted, which can be seen from the space between cells since the PECAM‐1 has been altered and is no longer holding the cells together. From those exact images, one can detect the enlarged and irregular shapes of nucleuses and change in morphology of these cells since the SARS‐CoV‐2 is not only altering the PECAM‐1 complex but it can also modify various proteins and mRNAs. By combining information of various viruses and knowledge from our studies, we can understand the mechanisms of ECs and take measurements to protect ECs from viruses. In future experiments, we will detect if the PECAM‐1 expression is lowered when treated with SARS‐CoV‐2 and TNF‐α or if PECAM‐1 is internalized in the cell. Moreover, we can do mRNA and immunoblotting analysis to elaborate the changes in protein pathways as a consequence of COVID‐19.
Parkinsonism, the clinical term for a disorder with prominent bradykinesia and variable associated extrapyramidal signs and symptoms, is accompanied by degeneration of the nigrostriatal dopaminergic ...system, with neuronal loss and reactive gliosis in the substantia nigra found at autopsy. Parkinsonism is pathologically heterogeneous, with the most common pathologic substrates related to abnormalities in the presynaptic protein α-synuclein or the microtubule binding protein tau. In idiopathic Parkinson's disease (PD), α-synuclein accumulates in neuronal perikarya (Lewy bodies) and neuronal processes (Lewy neurites). The disease process is multifocal and involves select central nervous system neurons and peripheral autonomic nervous system neurons. The particular set of neurons affected determines nonmotor clinical presentations. Multiple system atrophy (MSA) is the other major α-synucleinopathy. It is also associated with autonomic dysfunction and in some cases with cerebellar signs. The hallmark histopathologic feature of MSA is accumulation of α-synuclein within glial cytoplasmic inclusions (GCI). The most common of the Parkinsonian tauopathies is progressive supranuclear palsy (PSP), which is clinically associated with severe postural instability leading to early falls. The tau pathology of PSP also affects both neurons and glia. Given the population frequency of PD, α-synuclein pathology similar to that in PD, but not accompanied by neuronal loss, is relatively common (10% of people over 65 years of age) in neurologically normal individuals, leading to proposed staging schemes for PD progression. Although MSA-like and PSP-like pathology can be detected in neurologically normal individuals, such cases are too infrequent to permit assessment of patterns of disease progression.
Autopsies of deceased with a confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can provide important insights into the novel disease and its course. Furthermore, ...autopsies are essential for the correct statistical recording of the coronavirus disease 2019 (COVID-19) deaths. In the northern German Federal State of Hamburg, all deaths of Hamburg citizens with ante- or postmortem PCR-confirmed SARS-CoV-2 infection have been autopsied since the outbreak of the pandemic in Germany. Our evaluation provides a systematic overview of the first 80 consecutive full autopsies. A proposal for the categorisation of deaths with SARS-CoV-2 infection is presented (category 1: definite COVID-19 death; category 2: probable COVID-19 death; category 3: possible COVID-19 death with an equal alternative cause of death; category 4: SARS-CoV-2 detection with cause of death not associated to COVID-19). In six cases, SARS-CoV-2 infection was diagnosed postmortem by a positive PCR test in a nasopharyngeal or lung tissue swab. In the other 74 cases, SARS-CoV-2 infection had already been known antemortem. The deceased were aged between 52 and 96 years (average 79.2 years, median 82.4 years). In the study cohort, 34 deceased were female (38%) and 46 male (62%). Overall, 38% of the deceased were overweight or obese. All deceased, except for two women, in whom no significant pre-existing conditions were found autoptically, had relevant comorbidities (in descending order of frequency): (1) diseases of the cardiovascular system, (2) lung diseases, (3) central nervous system diseases, (4) kidney diseases, and (5) diabetes mellitus. A total of 76 cases (95%) were classified as COVID-19 deaths, corresponding to categories 1–3. Four deaths (5%) were defined as non-COVID-19 deaths with virus-independent causes of death. In eight cases, pneumonia was combined with a fulminant pulmonary artery embolism. Peripheral pulmonary artery embolisms were found in nine other cases. Overall, deep vein thrombosis has been found in 40% of the cases. This study provides the largest overview of autopsies of SARS-CoV-2-infected patients presented so far.
PDF
