Passage of SARS-CoV-2 in cells expressing human and mouse ACE2 selects for mouse-adapted and ACE2-independent viruses

Human ACE2 (hACE2) is required for cell attachment and entry of SARS-CoV-2. Mouse ACE2 (mACE2) does not support infection of early SARS-CoV-2 isolates. Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with a series of spike protein amino acid changes, all of which have been reported in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. Remarkably, one mouse-adapted virus was able to replicate efficiently in ACE2-negative cell lines, a characteristic not described for any SARS-CoV-2 variants. ACE2-independent entry by SARS-CoV-2 represents a new biology for SARS-CoV-2 with potential widespread implications for disease and intervention development.

Lung histopathologic clusters in severe COVID-19: a link between clinical picture and tissue damage

Background Autoptic pulmonary findings have been described in severe COVID-19 patients, but evidence regarding the correlation between clinical picture and lung histopathologic patterns is still weak. Methods This was a retrospective cohort observational study conducted at the referral center for infectious diseases in northern Italy. Full lung autoptic findings and clinical data of patients who died from COVID-19 were analyzed. Lung histopathologic patterns were scored according to the extent of tissue damage. To consider coexisting histopathologic patterns, hierarchical clustering of histopathologic findings was applied. Results Whole pulmonary examination was available in 75 out of 92 full autopsies. Forty-eight hospitalized patients (64%), 44 from ICU and four from the medical ward, had complete clinical data. The histopathologic patterns had a time-dependent distribution with considerable overlap among patterns. Duration of positive-pressure ventilation (p < 0.0001), mean positive end-expiratory pressure (PEEP) (p = 0.007), worst serum albumin (p = 0.017), interleukin 6 (p = 0.047), and kidney SOFA (p = 0.001) differed among histopathologic clusters. The amount of PEEP for long-lasting ventilatory treatment was associated with the cluster showing the largest areas of early and late proliferative diffuse alveolar damage. No pharmacologic interventions or comorbidities affected the lung histopathology. Conclusions Our study draws a comprehensive link between the clinical and pulmonary histopathologic findings in a large cohort of COVID-19 patients. These results highlight that the positive end-expiratory pressures and the duration of the ventilatory treatment correlate with lung histopathologic patterns, providing new clues to the knowledge of the pathophysiology of severe SARS-CoV-2 pneumonia.

The effect of Java Plum Fruit (Zyzygium cumini) extract on leucocyte and lung histopathology of mouse exposed cigarette smoke

Java Plum fruit can decrease free radical activities. The overwhelmed production of free radicals will reduce the immunity system in the body. This research aims to study the effects of java plum fruit extracts on leukocytes and histopathology of mouse lungs that have been exposed to cigarette smoke for 30 days. Frozen-dried java plum fruit is macerated with water. The mice are divided into Control groups (K-). The mice of the control group were given only water orally. (K+) are mice that are exposed to commercial cigarette smoke once a day. (RJ) were the mice exposed to cigarette smoke once a day as java plum fruit extract orally of 180 mg/kg body weight. (RF) were the mice exposed to cigarette smoke given with java plum fruit extract with 180 mg/kg of body weight that is applied on its filter. The results showed a significantly lower number of leukocytes (p < 0.05) in the K-,RF, and RJ groups compared to K+. Meanwhile, there was a significant increase in the number of lung pathological cells (p < 0.05) in the K+, RF, and RJ compared to the control group (K-). The results conclude that java plum fruit can reduce free radicals in animals affected by inflammation due to cigarette smoke particles in the lungs.

THE DYNAMICS OF LUNG HISTOPATHOLOGY IN ACUTE BACLOFEN POISONING

Baclofen (sold under the name Baclosan©, Lioresal©) is a muscle relaxant. This drug is chemically different from other muscle relaxants. Due to its pronounced psychotropic effect the drug is often a subject of abuse especially among young people. The article deals with the dynamics of lung histopathology in acute baclofen poisoning. Experimental studies were performed on 15 Wistar rats. The animals were divided into 3 groups (the controls and two experimental groups). The controls included 5 intact rats. Each experimental group included 5 animals. Both groups were treated with baclofen at a dosage of 85 mg/kg. The duration of the experiment was 3 and 24 hours, respectively. We revealed a complex of pathological changes in the lungs of the rats. There were circulatory disorders in all the elements of the microcirculatory bed, areas of emphysema, atelectasis and dystelectasis, WBC infiltration of intralveolar septa and their thickening due to edema. The changes were even more severe 24 hours after the drug administration. These results along with the results of chemical analysis will be useful in establishing the fact of baclofen intoxication and the exact moment of the intoxication.

Survey the Immuno-pharmacology Effect of Botulinum Toxin in Cell Signaling of Bronchial Smooth Muscle Cells of the Allergic Asthma

BackgroundAsthma is the lung disease that influenced more than 350 million people in worldwide. ASM spasm leads to AHR and bronchial obstruction that are acute symptoms of asthma attack. BTX is bacteria toxin that acts as muscle relaxant and may have therapeutic effect on AHR and asthma. Therefore, the effect of BTX on the AHR and related gene expression was evaluated.MethodsAfter producing of asthma mice model, which were treated with BTX in two ways; IN and N (0.01, 0.1, 1 and 10 U/ml). AHR was measured on day 24, 26, 28, 30 and gene expression of TrkA, TrkB, M1-M5, α7nAChR, TNF-α and ERK2 were evaluated. At least, in lung histopathology, perivascular and peribronchial inflammation, mucus production and goblet cell hyperplasia were studied. ResultsOn day 24, treatment with BTX for all dosages had no significant effect on AHR but, on day 26 and 28, AHR was decreased and it was continued on day 30 for all treated groups. Treatment with BTX had no significant effect on the expressions of TrkA, TrkB, M1, M2, M3, M4, M5, α7nAChR, TNF-α and ERK2 genes, perivascular inflammation, peribronchial inflammation, hyperplasia of the goblet cell and production of mucus. Also, the mice have been received BTX 10 mg/ml, were died. ConclusionsBTX therapy controlled asthma attacks via decreasing of AHR and induction of relaxation in the ASMs. But, it has no significant effect on inflammation and mucus production. In using of BTX, attention to the safe dose and prevention of dangerous side effects are necessary.

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3–1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

Case Report: SARS-CoV-2 Mother-to-Child Transmission and Fetal Death Associated With Severe Placental Thromboembolism

SARS-CoV-2 infection during pregnancy is not usually associated with significant adverse effects. However, in this study, we report a fetal death associated with mild COVID-19 in a 34-week-pregnant woman. The virus was detected in the placenta and in an unprecedented way in several fetal tissues. Placental abnormalities (MRI and anatomopathological study) were consistent with intense vascular malperfusion, probably the cause of fetal death. Lung histopathology also showed signs of inflammation, which could have been a contributory factor. Monitoring inflammatory response and coagulation in high-risk pregnant women with COVID-19 may prevent unfavorable outcomes, as shown in this case.

Automated Digital Quantification of Pulmonary Fibrosis in Human Histopathology Specimens

Pulmonary fibrosis is characterized by abnormal interstitial extracellular matrix and cellular accumulations. Methods quantifying fibrosis severity in lung histopathology samples are semi-quantitative, subjective, and analyze only portions of sections. We sought to determine whether automated computerized imaging analysis shown to continuously measure fibrosis in mice could also be applied in human samples. A pilot study was conducted to analyze a small number of specimens from patients with Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF) or idiopathic pulmonary fibrosis (IPF). Digital images of entire lung histological serial sections stained with picrosirius red and alcian blue or anti-CD68 antibody were analyzed using dedicated software to automatically quantify fibrosis, collagen, and macrophage content. Automated fibrosis quantification based on parenchymal tissue density and fibrosis score measurements was compared to pulmonary function values or Ashcroft score. Automated fibrosis quantification of HPSPF lung explants was significantly higher than that of IPF lung explants or biopsies and was also significantly higher in IPF lung explants than in IPF biopsies. A high correlation coefficient was found between some automated quantification measurements and lung function values for the three sample groups. Automated quantification of collagen content in lung sections used for digital image analyses was similar in the three groups. CD68 immunolabeled cell measurements were significantly higher in HPSPF explants than in IPF biopsies. In conclusion, computerized image analysis provides access to accurate, reader-independent pulmonary fibrosis quantification in human histopathology samples. Fibrosis, collagen content, and immunostained cells can be automatically and individually quantified from serial sections. Robust automated digital image analysis of human lung samples enhances the available tools to quantify and study fibrotic lung disease.

Comparative study on the antituberculous effect and mechanism of the traditional Chinese medicines NiuBeiXiaoHe extract and JieHeWan

Background The traditional Chinese medicine NiuBeiXiaoHe (NBXH) extract and Chinese medicine preparation JieHeWan (JHW) exhibit anti-tuberculosis effects. The anti- tuberculosis effect of NBXH was compared with that of JHW to elucidate the mechanism of action of NBXH. Methods BALB/c mice aged 6-8 weeks were randomly divided into a normal control group, Tuberculosis (TB) model group, JHW treatment group, and NBXH treatment group. After 3 and 13 weeks of treatment, the therapeutic effect in each group was evaluated by comparing lung histopathology, lung and liver colony counts, the number of spots representing effector T cells secreting IFN-γ in an ELISPOT, and the levels of Th1, Th2, and Th17 cytokines, which were measured by a cytometric bead array (CBA). Mouse RNA samples were subjected to transcriptome sequencing. Results After 13 weeks of treatment, the mean histopathological lesion area of the NBXH group was significantly smaller than that of the TB model group (P < 0.05). Compared with those in the TB model group, the lung colony counts in the JHW and NBXH groups were significantly decreased (P < 0.05), and the IL-2 and IL-4 levels in the NBXH group were significantly increased (P < 0.05). NBXH partly restored significant changes in gene expression caused by Mycobacterium tuberculosis (M. tuberculosis) infection. According to GO and KEGG analyses, the changes in biological process (BP), cell composition (CC) and molecular function (MF) terms and in signaling pathways caused by NBXH and JHW treatment were not completely consistent, but they were mainly related to the immune response and inflammatory response in the mouse TB model. Conclusions NBXH had therapeutic effects similar to those of JHW in improving lung histopathology, reducing lung colony counts, and regulating the levels of cytokines. NBXH restored significant changes in gene expression and repaired cell damage caused by M. tuberculosis infection by regulating immune-related pathways, which clarified the mechanism of action of NBXH.

Antibodies Against Angiotensin II Receptor Type 1 and Endothelin A Receptor Are Associated With an Unfavorable COVID19 Disease Course

BackgroundLung histopathology demonstrates vasculopathy in a subset of deceased COVID19 patients, which resembles histopathology observed in antibody-mediated lung transplant rejection. Autoantibodies against angiotensin II type 1 receptor (AT1R) and Endothelin receptor Type A (ETAR) have been demonstrated in antibody-mediated rejection and may also be associated with severe COVID19 infection. Objective To assess AT1R and ETAR auto-antibodies in COVID19 patients and controls, and explore their association with disease course.Methods65 hospitalized patients with COVID19 infection were included. Clinical and laboratory findings were retrospectively assessed. Patients with unfavorable disease course, admitted at the intensive care unit and/or deceased during hospital admission (n=33) were compared to admitted COVID19 patients with favorable disease course (n=32). The presence of antinuclear antibodies (ANA) and auto-antibodies against AT1R or ETAR in peripheral blood were compared between COVID19 with unfavorable and favorable disease course and age matched controls (n=20).ResultsThe presence of ANA was not significantly different between COVID19 patients with unfavorable (n=7/33; 21%) and favorable disease course (n=6/32; 19%) (p= 0.804) and controls (n=3/20; 15%). Auto-antibodies against AT1R were significantly increased in unfavorable disease course (median 14.59 U/mL, IQR 11.28 – 19.89) compared to favorable disease course (median 10.67 U/mL, IQR 8.55 – 13.0, p&lt; 0.01). ETAR antibody titers were also significantly increased in unfavorable disease course (median 7.21, IQR 5.0 – 10.45) as compared to favorable disease course (median 4.0, IQR 3.0 – 6.0, p &lt;0.05).ConclusionAuto-antibodies against AT1R and ETAR are significantly increased in COVID19 patients with an unfavorable disease course.