What We Know (and Do not Know) Regarding the Pathogenesis of Pulmonary Thrombosis in COVID-19

The clinical course of coronavirus disease 2019 (COVID-19) is often complicated by the onset of venous thrombosis and thromboembolism (VTE), encompassing also pulmonary thrombosis. Recent statistics attests that the cumulative frequency of VTE can be as high as 30% in COVID-19 hospitalized patients, increasing to nearly 40 to 70% (depending on systematic screening) in those with severe illness, mechanical ventilation, or intensive care unit admission. The risk of venous thrombosis seems mostly limited to the active phase of disease, and is directly associated with some genetic (i.e., inherited prothrombotic predisposition) and demographical factors (male sex, overweight/obesity), disease severity (risk increasing progressively from hospitalization to development of severe illness, being the highest in patients needing mechanical ventilation and/or intensive care), presence and extent of pulmonary disease, coexistence of multiple risk factors (immobilization, mechanical ventilation, co- or superinfections), along with increased values of inflammatory and thrombotic biomarkers. At least three different phenotypes of pulmonary thrombosis may develop in COVID-19 patients, one caused by typical embolization from peripheral venous thrombosis (e.g., deep vein thrombosis), a second type triggered by local inflammation of nearby pulmonary tissue, and a third one mostly attributable to the prothrombotic state consequent to the pronounced systemic inflammatory response (i.e., the so-called cytokine storm) that is frequently observed in COVID-19. Although the pathogenesis of these three conditions has different features, their discrimination is essential for diagnostic and therapeutic purposes. The prognosis of COVID-19 patients who develop pulmonary thrombosis is also considerably worse than those who do not, thus probably needing frequent monitoring and more aggressive therapeutic management.

Specific features of the pathology of the respiratory system in SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus: Sarbecovirus) infected Syrian hamsters (Mesocricetus auratus)

Introduction. Verification of histological changes in respiratory system using Syrian (golden) hamsters (Mesocricetus auratus) as experimental model is an important task for preclinical studies of drugs intended for prevention and treatment of the novel coronavirus infection COVID-19.The aim of this work was to study pathological changes of pulmonary tissue in SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) experimental infection in Syrian hamsters. Material and methods. Male Syrian hamsters weighting 80–100 g were infected by intranasal administration of culture SARS-CoV-2 at dose 4 × 104 TCID50/ml (TCID is tissue culture infectious dose). Animals were euthanatized on 3, 7 and 14 days after infection, with gravimetric registration. The viral load in lungs was measured using the polymerase chain reaction (PCR). Right lung and trachea tissues were stained with hematoxylin-eosin and according to Mallory.Results and discussion. The highest viral replicative activity in lungs was determined 3 days after the infection. After 7 days, on a background of the decrease of the viral load in lungs, a pathologically significant increase of the organ’s gravimetric parameters was observed. Within 3 to 14 days post-infection, the lung histologic pattern had been showing the development of inflammation with a succession of infiltrative-proliferative, edematousmacrophagal and fibroblastic changes. It was found that initial changes in respiratory epithelium can proceed without paranecrotic interstitial inflammation, while in the formation of multiple lung parenchyma lesions, damage to the epithelium of bronchioles and acinar ducts can be secondary. The appearance of epithelioid large-cell metaplastic epithelium, forming pseudoacinar structures, was noted as a pathomorphological feature specific to SARS-CoV-2 infection in Syrian hamsters.Conclusion. As a result of the study, the specific features of the pathology of the respiratory system in SARSCoV-2 infected Syrian hamsters were described. These findings are of practical importance as reference data that can be used for preclinical studies to assess the effectiveness of vaccines and potential drugs.

Pulmonary choristoma in a new-born calf with multiple cranial and nervous malformations

ABSTRACT: Multiple congenital malformations can occur concomitantly in several species since the anomaly in one organ may lead directly to the malformation of another. Additionally, the etiology is not always clarified. Choristoma refers to an ectopic tissue that is histologically normal in an abnormal location. A case of pulmonary and nodal choristoma associated with cranioschisis, hydrocephalus, and syringomyelia in a new-born calf is reported here. Clinically, the calf had a mass in the frontal region of the head associated with local bone deformation. At necropsy, there was cranioschisis in the junction of the frontal bones and a 14 × 10 × 7 cm mass, grossly consistent with well-differentiated pulmonary tissue covered by skin, covering the opening between the frontal bones, and extending into the cranial cavity, leading to local cerebral compression. With the pulmonary choristoma, there was a well-differentiated lymphoid tissue. Additionally, in the central nervous system, there was severe hydrocephalus involving lateral ventricles and multiple areas of syringomyelia in the spinal cord.

Pulmonary Guardians and Special Regulatory Devices in the Lung of Nile Monitor Lizard (Varanus niloticus) with Special Attention to the Communication Between Telocyte, Pericyte, and Immune Cells

Monitor lizards are acclimatized to a variety of environments. Most of the monitor species are terrestrial, although there are arboreal and semiaquatic monitors. Such accommodation requires unique cellular structure and regulatory devices in various organs, particularly their lungs. This study aimed to report the pulmonary guardians and special regulatory devices that may guard and promote the function of the lungs of the Nile monitor lizards (Varanus niloticus). Specially structured vessels were recorded in the pulmonary tissue involving atypical glomus vessels, vessels with variable wall thickness, and a venule with specialized internal elastic membrane. Moreover, numerous lung resident guardians could be identified including both alveolar and interstitial macrophages, dendritic cells, mast cells, and B- and T-lymphocytes. Pericytes were demonstrated surrounding the capillary endothelium with a characteristic direct hetero-cellular junction with telocytes. Telocytes established a microenvironment through an indirect hetero-cellular junction with the interstitial macrophage, dendritic cells, and pneumocyte type II. Collectively, these data indicate a significant role played by the specially structured vessels and the resident immune cells in guarding the pulmonary tissue of the Nile monitor lizards and promoting its function. Telocytes are suggested to play a key role in angiogenesis and cellular communication to promote the function of the immune cells.

Exome sequence analysis of siblings affected with Niemann-Pick type C disease

<p>Mutations in either the Niemann-Pick type C1 or C2 (NPC1/NPC2) gene result in a fatal lysosomal storage disorder, Niemann-Pick type C (NP-C) disease, for which there is no effective cure. The disease is characterized by systemic and neurodegenerative symptoms arising from toxic accumulation of unesterified cholesterol within the late endosome and lysosome, with a common cause of death for patients being respiratory failure or recurrent infection of pulmonary tissue. Interestingly, the disease symptoms are heterogeneous, with age of onset and severity varied, even among siblings with the same mutations in the NPC1 or NPC2 gene causing this monogenic disease. To date there is no clear explanation for disease severity in siblings with the same mutation. As siblings are raised in the same environment, the major hypothesis of this thesis is that there are genetic modifiers that explain variation in disease severity within siblings. To determine if there are genetic variants associated with disease severity, exomes were sequenced from five sibling pairs exhibiting divergent onset and progression of NPC disease. Out of 23,105 genes, 26 variants were identified that were predicted to have functional consequences in NP-C patients, of which homozygous MUC5B and MARCH8 variants segregated across siblings that exhibited increased and decreased severity of disease, respectively. A cluster of variants was discovered on chromosome 11 belonging to the matrix metalloproteinase (MMP) family. Further investigation of one of these variants, a frameshift insertion in MMP-12, confirmed that this locus regulates the accumulation of unesterified cholesterol in primary neurons derived from a murine model of NPC disease. However, this region on chromosome 11 did not have any statistically significant copy number alteration detectable through a depth of coverage analysis. Overall, these results provide groundwork into the sequence variants mediating disease severity, which with further investigations, may be novel pharmacological targets to treat NPC disease.</p>

Exome sequence analysis of siblings affected with Niemann-Pick type C disease

<p>Mutations in either the Niemann-Pick type C1 or C2 (NPC1/NPC2) gene result in a fatal lysosomal storage disorder, Niemann-Pick type C (NP-C) disease, for which there is no effective cure. The disease is characterized by systemic and neurodegenerative symptoms arising from toxic accumulation of unesterified cholesterol within the late endosome and lysosome, with a common cause of death for patients being respiratory failure or recurrent infection of pulmonary tissue. Interestingly, the disease symptoms are heterogeneous, with age of onset and severity varied, even among siblings with the same mutations in the NPC1 or NPC2 gene causing this monogenic disease. To date there is no clear explanation for disease severity in siblings with the same mutation. As siblings are raised in the same environment, the major hypothesis of this thesis is that there are genetic modifiers that explain variation in disease severity within siblings. To determine if there are genetic variants associated with disease severity, exomes were sequenced from five sibling pairs exhibiting divergent onset and progression of NPC disease. Out of 23,105 genes, 26 variants were identified that were predicted to have functional consequences in NP-C patients, of which homozygous MUC5B and MARCH8 variants segregated across siblings that exhibited increased and decreased severity of disease, respectively. A cluster of variants was discovered on chromosome 11 belonging to the matrix metalloproteinase (MMP) family. Further investigation of one of these variants, a frameshift insertion in MMP-12, confirmed that this locus regulates the accumulation of unesterified cholesterol in primary neurons derived from a murine model of NPC disease. However, this region on chromosome 11 did not have any statistically significant copy number alteration detectable through a depth of coverage analysis. Overall, these results provide groundwork into the sequence variants mediating disease severity, which with further investigations, may be novel pharmacological targets to treat NPC disease.</p>

Ambroxol Hydrochloride Loaded Gastro-Retentive Nanosuspension Gels Potentiate Anticancer Activity in Lung Cancer (A549) Cells

This study aimed to develop gastro-retentive sustained-release ambroxol (ABX) nanosuspensions utilizing ambroxol-kappa-carrageenan (ABX-CRGK) complexation formulations. The complex was characterized by differential scanning calorimetry, powder x-ray diffractometer, and scanning electron microscopy. The prepared co-precipitate complex was used for the development of the sustained-release formulation to overcome the high metabolic and poor solubility problems associated with ABX. Furthermore, the co-precipitate complex was formulated as a suspension in an aqueous floating gel-forming vehicle of sodium alginate with chitosan, which might be beneficial for targeting the stomach as a good absorption site for ABX. The suspension exhibited rapid floating gel behaviour for more than 8 h, thus confirming the gastro-retentive effects. Particle size analysis revealed that the optimum nanosuspension (ABX-NS) had a mean particle size of 332.3 nm. Afterward, the ABX released by the nanoparticles would be distributed to the pulmonary tissue as previously described. Based on extensive pulmonary distribution, the developed nanosuspension-released ABX nanoparticles showed significant cytotoxic enhancement compared to free ABX in A549 lung cancer cells. However, a significant loss of mitochondrial membrane potential (MMP) also occurred. The level of caspase-3 was the highest in the ABX-NS-released particle-treated samples, with a value of 416.6 ± 9.11 pg/mL. Meanwhile, the levels of nuclear factor kappa beta, interleukins 6 and 1 beta, and tumour necrosis alpha (NF-kB, IL-6, IL-1β, and TNF-α, respectively) were lower for ABX-NS compared to free ABX (p < 0.05). In caspase-3, Bax, and p53, levels significantly increased in the presence of ABX-NS compared to free ABX. Overall, ABX-NS produced an enhancement of the anticancer effects of ABX on the A549 cells, and the developed sustained-release gel was successful in providing a gastro-retentive effect.

Di-Tyrosine Crosslinking and NOX4 Expression as Oxidative Pathological Markers in the Lungs of Patients with Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a noninflammatory progressive lung disease. Oxidative damage is a hallmark of IPF, but the sources and consequences of oxidant generation in the lungs are unclear. In this study, we addressed the link between the H2O2-generating enzyme NADPH oxidase 4 (NOX4) and di-tyrosine (DT), an oxidative post-translational modification in IPF lungs. We performed immunohistochemical staining for DT and NOX4 in pulmonary tissue from patients with IPF and controls using validated antibodies. In the healthy lung, DT showed little or no staining and NOX4 was mostly present in normal vascular endothelium. On the other hand, both markers were detected in several cell types in the IPF patients, including vascular smooth muscle cells and epithelium (bronchial cells and epithelial cells type II). The link between NOX4 and DT was addressed in human fibroblasts deficient for NOX4 activity (mutation in the CYBA gene). Induction of NOX4 by Transforming growth factor beta 1 (TGFβ1) in fibroblasts led to moderate DT staining after the addition of a heme-containing peroxidase in control cells but not in the fibroblasts deficient for NOX4 activity. Our data indicate that DT is a histological marker of IPF and that NOX4 can generate a sufficient amount of H2O2 for DT formation in vitro.