Elevated MUC1 and MUC5AC mucin protein levels in airway mucus of critical ill COVID-19 patients

Wenju Lu, Xiaoqing Liu, Tao Wang, Fei Liu, Airu Zhu, and Yongping Lin 

Patients with coronavirus disease 2019 (COVID-19) exhibit a spectrum of respiratory symptoms like cough and dyspnea.13 Airway mucus is an adhesive viscoelastic gel composed mostly of high-molecular-weight mucous glycoproteins and water, which is important in maintaining lung function and health, pathological mucus hypersecretion may cause airway obstruction and lead to respiratory distress. Mucin (MUC) glycoproteins are the major macromolecular components of mucus, which are classified into two major types: the gel-forming secreted MUC5AC and the membrane-tethered MUC1.4 Here, with an attempt to understand the lung changes, we sought to provide a delineation of the components of airway mucus from patients with COVID-19.

To clean airway obstruction, respiratory tract mucus was aspirated and collected via bronchoscopy from COVID-19 patients with a critical illness, and optical coherence tomography (OCT) was applied via bronchoscopy to obtain cross-sectional images of the bronchiole. For healthy control, sputum was induced by inhaled hypertonic (3%) saline solution delivered with an ultrasonic nebulizer. After collection, sputum was processed as previously described for components analysis.5 Medical history, and clinical and laboratory data of the participants were extracted from electronic medical records. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (No. 2020-65). The requirement for informed consent was waived because the study was observational and the family members were in quarantine.

MUC5AC (sc-21701; Santa Cruz, Shanghai, China) and MUC1 (sc-6827; Santa Cruz) in airway mucus were measured using enzyme-linked immunosorbent assay, and MUC1-cytoplasmic tail (CT, clone EP1024Y; Abcam, Shanghai) levels were measured by Western blot analysis as described previously.5 The levels of MUC5AC and MUC1 were normalized to their average signal reading of a healthy control group.

A total of 16 patients with COVID-19 were included in this study, the clinical characteristics of the recruited subjects were shown in Table S1. There was no significant deviation in the distribution of age, or sex between the cohorts of case and control subjects. All the patients with COVID-19 were admitted to the intensive care unit because of low oxygenation index (199 ± 23 mm Hg), and 79% of them received mechanical ventilation. Blood laboratory tests showed elevated inflammatory indexes including leukocyte count, C-reactive protein, and interleukin-6 in most of the patients with COVID-19 (Table S2).

OCT indicated clear bronchiole in healthy controls (Figure 1A) and mucus retention in the bronchiole of patients with COVID-19 (Figure 1B). A volume of 1-8 mL white to gray sputum with high viscosity (Figure 1C) was aspirated from the respiratory tract of 16 patients with COVID-19. Induced sputum from healthy control was clear and transparent with low viscosity. Compared to healthy control, airway mucus from patients with COVID-19 had a higher level of MUC5AC (Figure 1D), MUC1 (Figure 1E), and MUC1-CT fragment (Figure 1F). However, there were no significant differences in the concentration of total protein, sodium, or chloride in the airway mucus from patients with COVID-19 when compared to healthy control (Table S3).

Representative cross-sectional images of bronchioles in (A) healthy and (B) critical ill COVID-19 patients were obtained with optical coherence tomography and bronchoscopy. C, Airway mucus aspirated by bronchoscopy. D, MUC1 and (E) MUC5AC protein levels as measured by ELISA, and (F) MUC1-CT level as measured by Western blot analysis in the airway mucus from COVID-19 patients and healthy control (HC). The levels of MUC5AC and MUC1 were normalized to their average signal reading of healthy control group. COVID-19, coronavirus disease 2019; CT, cytoplasmic tail; ELISA, enzyme-linked immunosorbent assay; MUC, mucin

Although more than half of patients with COVID-19 presented with a dry cough,6 this study provided direct evidence showing mucus retention in the small airway of patients with COVID-19, and patients were not able to expectorate by themselves and need bronchoscopy aspiration to help them to clean respiratory tract. The sputum from these patients with COVID-19 was viscous, which is not surprising as MUC5AC levels are extremely high, hyperconcentration of this gel-forming MUC dehydrates airway surfaces and causes mucus adhesion, which may contribute to airflow obstruction and respiratory distress. Clearance of airway mucus is an important way to increase oxygen and carbon dioxide exchange, bronchoscopy aspiration of airway mucus was used in all our patients to relieve hypoxia. In our center, all the 16 critical ill COVID-19 patients recovered and were discharged from hospitalization, which may attribute to our aggressive clearance of the respiratory tract. It is of note that bedside bronchoscopy may not be available in some hospitals as the medical resources are limited during the COVID-19 pandemic, carbocisteine has been reported to inhibit airway MUC5AC secretion, which could be used to reduce sputum viscosity and elasticity in patients with COVID-19. In addition, hydration of sputum by aerosolized hypertonic saline solutions or mannitol, and dilation of bronchi via aerosolized salbutamol may facilitate sputum expectoration.

MUC1 is a membrane-tethered MUC expressed on the apical surface of epithelial cells.78 Since MUC1-CT fragment is on the cytoplasmic side of the cells, the elevated sputum CT fragment in patients with COVID-19 could come from detached and disrupted epithelial cells, which is evidenced by the pathological findings of diffuse alveolar damage with fibromyxoid exudates and macrophage infiltration in the lung tissue from patients with COVID-19.9

The limitation of this study is that induced sputum was used in the control group to compare airway mucus aspirated via bronchoscopy from patients with COVID-19, because it was very difficult for the patients with COVID-19 to expectorate sputum even with hypertonic saline solution inhalation.

The findings may suggest that increased level of MUCs in the airway mucus may contribute to the high viscosity of airway mucus and sputum retention in the small airway of patients with COVID-19, airway mucus clearance may be indicated to relieve respiratory distress, and MUC5AC may serve as a target for mucolytic agents in treating COVID-19. And MUC1-CT may serve as an indicator reflecting the severity of airway and alveolar epithelial cell damage.

ACKNOWLEDGMENTS

This study was supported by grants from the National Key R&D Project (2016YFC0903700 and 2016YFC1304102), the National Natural Science Foundation of China (81520108001 and 81770043), and grant specific for COVID-19 study from Guangzhou Institute of Respiratory Health. The authors would like to thank Dr Kwang Chul Kim (University of Arizona) for the invaluable assistance with the manuscript.

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

Source : https://onlinelibrary.wiley.com/doi/10.1002/jmv.26406

Bill Munro Vs The Coronavirus

Bill brought inhalation of H2O2 to the publics attention, he used it 5-6 pumps, 6 or 7x a day. To keep his Oxygen levels high, to keep the Viruses out, which come when Oxygen is down.

This was something I tried many years ago, but didn’t feel a direct or urgent need to do, so put it into my memory locker.

However, after studying this new Viral Epidemic which increasingly surrounds us, I pulled the information back to my short term memory to mix with the pH principles I had been reflecting on.

My daughter has suffered from Chronic Lung Disease as a result of being born at 24weeks gestation, so Oxygen and Viral susceptibility has been something I am used to reflecting deeply upon.

Anyway, the data shows that if you are of a lower pH, this virus group (Corona) is 10x more forceful and you are 10x more susceptible (at a pH of 6 Vs 7), so after ensuring I got my diet plans and supplements into order, I began to reflect over and over how else I can best prepare.

This is when Bill Munro came back to my mind, so I ordered 2x Nasal Vaporiser’s for about £3, and I decided that I would start taking a similar number of rounds of H2O2 as Bill did, but diluted to 1.5% with 50% Distilled water and 50% Food Grade H2O3 at 3%.

The literature for killing the Virus shows that 0.5% H2O2 kills the virus in 1 Minute, so 1.5% should be effective in 20 Seconds or thereabouts. So, to my unqualified mind, this leads me to presume that several rounds of this daily may safeguard me against this threat, if not preventing it from entering my airways entirely, then at least killing it off 5-6 times a day, minimising the intensity of the infection as best I may.

When I have talked of this method to others, peoples minds immediately seem to jump to “you are swallowing bleach”, with a shocked reaction, yet I explain that this is simply water with an extra Oxygen molecule attached, which people regularly treat their teeth with at 7% some 450% stronger than I am using. People seem to not bat an eyelid at using 4.5x stronger bleach for their teeth and vanity, yet to stave of seemingly very harsh virus people react like I am the crazy one!

This method along with Alkalising my terrain via diet and supplements are the best methods I have encountered to protect myself against this horrid viral outbreak.

If the outbreak worsens in the UK, I am intending to upgrade to the full 3%

Thank you Bill.

Whole blueberry powder modulates the growth and metastasis of MDA-MB-231 triple negative breast tumors in nude mice.

Authors – Adams LS,  Kanaya N,  Phung S,  Liu Z,  Chen S.

Abstract

Previous studies in our laboratory demonstrated that blueberry (BB) extract exhibited antitumor activity against MDA-MB-231 triple negative breast cancer (TNBC) cells and decreased metastatic potential in vitro.

The current study tested 2 doses of whole BB powder, 5 and 10% (wt:wt) in the diet, against MDA-MB-231 tumor growth in female nude mice. In this study, tumor volume was 75% lower in mice fed the 5% BB diet and 60% lower in mice fed the 10% BB diet than in control mice (P ≤ 0.05).

Tumor cell proliferation (Ki-67) was lower in the 5 and 10% BB-fed mice and cell death (Caspase 3) was greater in the 10% BB-fed mice compared to control mice (P ≤ 0.05).

Gene analysis of tumor tissues from the 5% BB-fed mice revealed significantly altered expression of genes important to inflammation, cancer, and metastasis, specifically, Wnt signaling, thrombospondin-2, IL-13, and IFNγ. To confirm effects on Wnt signaling, analysis of tumor tissues from 5% BB-fed mice revealed lower β-catenin expression and glycogen synthase kinase-3β phosphorylation with greater expression of the β-catenin inhibitory protein adenomatous polyposis coli compared to controls.

A second study tested the ability of the 5% BB diet to inhibit MDA-MB-231-luc-D3H2LN metastasis in vivo. In this study, 5% BB-fed mice developed 70% fewer liver metastases (P = 0.04) and 25% fewer lymph node metastases (P = 0.09) compared to control mice.

This study demonstrates the oral antitumor and metastasis activity of whole BB powder against TNBC in mice.

MDA-MB-231 tumor volume (A), proliferation (B), and apoptosis (C) in female nude mice fed control, 5% BB powder, or 10% BB powder diet for 8 wk. Data are means ± SEM, n = 6. In A, labeled means at a time without a common letter differ, P ≤ 0.05. In B and C, asterisks indicate different from control: *P ≤ 0.05, **P ≤ 0.01. BB, blueberry.
In situ tumor growth and metastasis monitored by Xenogen IVIS imaging at different time points after MDA-MB-231 tumor implantation in female nude mice fed control or 5% BB diet for 7 wk. (A) Orthotopic breast tumor growth from baseline to wk 5 postimplantation. (B) In situ and ex-vivo imaging of RLN and LLN lymph node metastasis at wk 5 postimplantation. (C) Frequency of metastasis and mean intensity of liver and LN metastases analyzed by contingency table. Intensity data are mean ± SEM for the number of mice with metastases. *Different from control, P ≤ 0.05. BB, blueberry; LLN, left lymph node; RLN, right lymph node.

Source : https://www.ncbi.nlm.nih.gov/pubmed/21880954

Protective effects of blueberry- and strawberry diets on neuronal stress following exposure to (56)Fe particles.

Authors – Poulose SM,  Bielinski DF,  Carrihill-Knoll KL,  Rabin BM,  Shukitt-Hale B.

Abstract

Particles of high energy and charge (HZE particles), which are abundant outside the magnetic field of the Earth, have been shown to disrupt the functioning of neuronal communication in critical regions of the brain. Previous studies with HZE particles, have shown that irradiation produces enhanced indices of oxidative stress and inflammation as well as altered neuronal function that are similar to those seen in aging.

Feeding animals antioxidant-rich berry diets, specifically blueberries and strawberries, countered the deleterious effects of irradiation by reducing oxidative stress and inflammation, thereby improving neuronal signaling. In the current study, we examined the effects of exposure to (56)Fe particles in critical regions of brain involved in cognitive function, both 36h and 30 days post irradiation.

We also studied the effects of antioxidant-rich berry diets, specifically a 2% blueberry or strawberry diet, fed for 8 weeks prior to radiation as well as 30 days post irradiation. (56)Fe exposure caused significant differential, neurochemical changes in critical regions of the brain, such as hippocampus, striatum, frontal cortex, and cerebellum, through increased inflammation, and increased oxidative stress protein markers. (56)Fe exposure altered the autophagy markers, and antioxidant-rich berry diets significantly reduced the accumulation of p62 in hippocampus, a scaffold protein that co-localizes with ubiquitinated protein at the 30 days post irradiation time-point.

Exposure to (56)Fe particles increased the accumulation of disease-related proteins such as PHF-tau in the hippocampus of animals fed the control diet, but not in the irradiated animals fed the blueberry diet. These results indicate the potential protective effects of antioxidant-rich berry diets on neuronal functioning following exposure to HZE particles.

Source : https://www.ncbi.nlm.nih.gov/pubmed/25451098