Small Epithelial Cells Research Articles

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

A Model Based on Automated Urinalysis Parameters for Urothelial Carcinoma Risk Stratification in Suspected Patients.

The aim of this study was to develop and validate a risk stratification model for the screening of patients with suspected urothelial carcinoma (UC). We enrolled 671 consecutive patients with suspected UC and generated a risk stratification model based on urinary parameters by using an automated urinalysis analyzer (Sysmex UN-9000). All patients received urine cytology examination from January 1, 2019, to October 31, 2022. Out of the 671 patients, 191 (28.5%) were ultimately diagnosed with UC. The four features associated with the presence of malignancy on multivariable analysis can be summarized by using the mnemonic UC-PAAS: UC, protein vs. creatinine ratio (P/C), age, atypical cells (Atyp.C), and small round epithelial cell (SRC). Major criteria include Atyp.C ≥ 0.1/μL (2 points) and age ≥ 65 years (2 points); minor criteria include SRC ≥ 2.7/μL (1 point) and abnormal P/C results (1 point). The model evidenced good discrimination (area under the curve = 0.802, 95% confidence interval [0.756, 0.848]) in the training group. A UC-PAAS cutoff of more than 4 points identified a high-risk population, of whom 37 of 59 (62.7%) had UC; the negative predictive value was 0.867. The validation group yielded similar findings. We present a urinalysis-based screening model, the UC-PAAS, that may serve as an accessory clinical tool for the evaluation of patients with suspected UC, because the model identifies patients at higher risk who require closer follow-up than others or additional examinations.

Uncovering a unique pathogenic mechanism of SARS-CoV-2 omicron variant: selective induction of cellular senescence.

SARS-CoV-2 variants are constantly emerging with a variety of changes in the conformation of the spike protein, resulting in alterations of virus entry mechanisms. Solely omicron variants use the endosomal clathrin-mediated entry. Here, we investigate the influence of defined altered spike formations to study their impact on premature cellular senescence. In our study, in vitro infections of SARS-CoV-2 variants delta (B.1.617.2) and omicron (B.1.1.529) were analyzed by using human primary small alveolar epithelial cells and human ex vivo lung slices. We confirmed cellular senescence in human lungs of COVID-19 patients. Hence, global gene expression patterns of infected human primary alveolar epithelial cells were identified via mRNA sequencing. Solely omicron variants of SARS-CoV-2 influenced the expression of cell cycle genes, highlighted by an increased p21 expression in human primary lung cells and human ex vivo lungs. Additionally, an upregulated senescence-associated secretory phenotype (SASP) was detected. Transcriptomic data indicate an increased gene expression of p16, and p38 in omicron-infected lung cells. Significant changes due to different SARS-CoV-2 infections in human primary alveolar epithelial cells with an overall impact on premature aging could be identified. A substantially different cellular response with an upregulation of cell cycle, inflammation- and integrin-associated pathways in omicron infected cells indicates premature cellular senescence.

Refractory celiac disease and its mimickers: a review on pathogenesis, clinical-pathological features and therapeutic challenges.

Refractory celiac disease (RCD) and enteropathy-associated T-cell lymphoma (EATL) are rare, yet severe complications of celiac disease (CD). Over the last decades, several studies have addressed the biology and clinical-pathological features of such conditions, highlighting unique disease patterns and recurrent genetic events. Current classification proposals identify two forms of RCD, namely: (i) type 1 RCD (RCD-I), characterized by phenotypically normal intra-epithelial lymphocytes (IELs); and (ii) type 2 RCD (RCD-II), featuring phenotypically aberrant IELs. While RCD-I likely represents a gluten-independent dysimmune reaction against small bowel epithelial cells, RCD-II is better considered an in situ aggressive T-cell lymphoma, with high rates of progression to overt EATL. The diagnosis of RCD and EATL is often challenging, due to misleading clinical-pathological features and to significant overlap with several CD-unrelated gastro-intestinal disorders. Similarly, the treatment of RCD and EATL is an unmet clinical need for both gastroenterologists and hematologists. Moving from such premises, this review aims to provide a comprehensive view of RCD and EATL, specifically considering their pathogenesis and the many still open issues concerning their diagnosis and clinical management.

Distinct immune surveillance in primary biliary cholangitis and primary sclerosing cholangitis is linked with discrete cholangiocarcinoma risk.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are 2 major liver autoimmune diseases. PBC is common in women and primarily affects intrahepatic small bile duct epithelial cells, known as cholangiocytes. In contrast, PSC is dominant in men and primarily affects medium and big intrahepatic and extrahepatic bile duct epithelial cells. Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, and its incidence is increasing worldwide in both men and women. Numerous retrospective and clinical studies have suggested that PBC patients rarely develop CCA compared to PSC patients. CCA is accountable for the higher deaths in PSC patients due to ineffective therapies and our inability to diagnose the disease at an early stage. Therefore, it is paramount to understand the differences in immune surveillance mechanisms that render PBC patients more resistant while PSC patients are susceptible to CCA development. Here, we review several potential mechanisms contributing to differences in the susceptibility to CCA in PBC versus PSC patients.

Chac1 silencing mitigates hemorrhagic shock-induced intestinal injury by inhibiting oxidative stress and ferroptosis

Hemorrhagic shock (HS) is a common and significant cause of mortality and morbidity, often resulting in structural damage and dysfunction of the intestines. ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (Chac1) has been reported to be involved in the regulation of oxidative stress and ferroptosis in mammals. Herein, we investigate the effects of Chac1 on HS-induced intestinal injury induced by HS both in vitro and in vivo. Sprague-Dawley rat model with HS was established, and our investigations showed upregulation of the mRNA and protein levels of Chac1 in the model’s ileum tissues. Histopathological analysis revealed that knockdown of Chac1 attenuated the intestinal injury induced by HS. Depletion of Chac1 also reduced the increase in intestinal fatty acid binding protein (I-FABP) concentration. Immunofluorescence staining indicated that silencing Chac1 significantly suppressed the downregulation of occludin and zonula occludens-1 (ZO-1). HS-induced changes in lipid peroxidation (LPO), malondialdehyde (MDA), and glutathione (GSH) levels were reversed in the absence of Chac1, suggesting that downregulation of Chac1 alleviated HS-induced oxidative stress. Additionally, HS led to a decrease in glutathione peroxidase 4 (Gpx4) and ferritin heavy chain 1 (Fth1) expression, along with an increase in ferrous ion (Fe2+) concentration. Knockdown of Chac1 significantly inhibited ferroptosis by increasing Gpx4 and Fth1 expression while reducing the Fe2+ concentration. In vitro experiments using the rat small intestine crypt epithelial cells (IEC-6) demonstrated that depletion of Chac1 suppressed oxidative stress and ferroptosis induced by hypoxia/reoxygenation (H/R). In conclusion, our study provides evidence that downregulation of Chac1 mitigates HS-induced intestinal injury by inhibiting oxidative stress and ferroptosis.

BestCyte® primary screening of 500 ThinPrep Pap Test thin-layers: 3 Cytologists’ Interobserver diagnostic concordance with predicate manual microscopy relative to Truth Reference diagnoses defining NILM, ASCUS+, LSIL+, and ASCH+ thresholds for specificity, sensitivity, and equivalency grading

BackgroundThe BestCyte® Cell Sorter Imaging System (BestCyte) facilitates algorithmic discrimination of clinically relevant cells in Pap test cytopathology by classifying and projecting images of cells in galleries based on cytomorphology. Warranted is awareness of potential BestCyte advantages as measured through 3 cytologists’ interobserver diagnostic concordance, specificity and sensitivity differentials, and equivalency grading relative to manual microscopy (MM). ObjectivesUsing 500 MM-reported ThinPrep thin-layers, analyze: (1) cytologists’ blinded BestCyte screening to raise Bethesda diagnoses; (2) correlate BestCyte and MM diagnoses (i.e., predicate) to establish Truth Reference Diagnoses (TRDx) from concordance between 4 possible diagnoses; (3) analyze cytologists’ and MM predicate diagnoses through 4 diagnostic thresholds defined by TRDx: NILM (Negative) for specificity, and ASCUS+, LSIL+, and ASCH+ (Positive) for graded sensitivity (with abnormal cells decreasing in size with increasing dysplasia); and, (4) statistically determine cytologists’ equivalency grading to MM using 95% Confidence Interval (CI) ranges. Results500 TRDx breakdown (n/%): NILM (241/48.2), ASCUS (79/15.8), ASCH (9/1.80), AGUS (2/0.40), LSIL (86/17.2), HSIL (68/13.6), CA (2/0.40), UNSAT (13/2.60). TRDx breakdown (n/%) per 4 of 4, 3 of 4, 2 of 4 diagnostic concordances: 264 (52.8%), 182 (36.4%), 54 (10.8%), respectively. No cases of discordant diagnoses were recorded. HSIL TRDx were established from 66.2% of 4 of 4 concordances, followed by NILM (59.3%), LSIL (46.5%), ASCUS (41.8%); antithetically, from 4.40% of 2 of 4 concordances. Specificity for MM predicate (NILM): 67.08%; for Cytologists 1, 2, and 3: 89.71%, 82.30%, 97.53%, respectively. For NILM threshold, cytologists revealed Significantly Superior equivalency to MM. Sensitivity for ASCUS+, LSIL+, and ASCH+ thresholds: MM (91.36%, 86.67%, 74.36%); Cytologist 1 (95.88%, 96.97%, 94.87%); Cytologist 2 (95.47%, 95.76%, 93.59%), Cytologist 3 (94.65%, 95.15%, 98.72%), respectively. Cytologists revealed Significantly Superior equivalency to MM for graded Positive thresholds; with Cytologist 3 for ASCUS+ being: Superior. ConclusionsBestCyte detects and efficiently displays abnormal cells in strategic galleries standardizing objectivity by systematizing mosaics of cell-types for cytologists’ consideration. BestCyte fosters consistent, enhanced cytologists’ sensitivity values for the ASCUS+, LSIL+, and ASCH+ Positive thresholds relative to MM. Also, BestCyte facilitates improved specificity and superior equivalency grading to MM reflecting efficient screening, and reduced labor. Confident interpretations of small dysplastic epithelial cells characteristic of HSIL led to exceptional interobserver diagnostic concordance inferring BestCyte is primed for effective cervical cancer screening practice.

IDF21-0230 Anti-inflammatory activity of glyoxalase-1 inducer supports further evaluation for prevention of COVID-19

Background: Pre-clinical and clinical studies suggest an essential role of proteases, plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and furin, in virion fusion with lung alveolar epithelial cells for viral infectivity of coronavirus SARS-CoV-2. Angiotensin converting enzyme-2 (ACE2) and glucose regulated protein-78 (GRP78) are considered to be receptors for cell surface binding of the spike protein of SARS-CoV-2. Risk of progression of associated COVID-19 disease to severe symptoms and risk of mortality is linked to increased levels of inflammatory mediators, including monocyte chemoattract protein-1 (MCP-1) and interleukin-8 (IL8), which are also likely risk factors for severe, life-threatening disease. Increased MCP-1 and IL8 in obese subjects and patients with diabetes may contribute to poor clinical outcomes when infected with coronavirus. In a double blind placebo-controlled clinical crossover study in overweight and obese subjects, treatment with Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP), once daily by oral capsule dietary supplement for 8 weeks, decreased expression of MCP-1, IL8, the receptor for advanced glycation endproducts (RAGE) and cyclo-oxygenase-2 (COX-2) in peripheral blood mononuclear cells (PBMCs). Placebo had no effect. We hypothesized that tRES-HESP may decrease protease and inflammatory gene expression linked to SARS-CoV-2 infection and COVID-19 progression. If so, the anti-inflammatory activity of Glo1 inducer in PBMCs found in clinical trial may also be evidenced in human small airway alveolar epithelial cells in primary culture. Aim: The aim of this project was to assess the anti-inflammatory effect of tRES-HESP on human alveolar epithelial cells in primary culture, assessing effects on protease and inflammatory gene expression considered essential for SARS-CoV-2 infectivity and progression of COVID-19 severity. Method: Human small airway epithelial cells (SAECs) were purchased from Lonza. SAECs were cultured in Small Airway Growth Media with supplements, according to the supplier’s instructions. SAECs (10,000 cells/cm2) were cultured with and without 5 µM tRES-HESP and the effect of basal gene expression studied after 72 h for mRNA (qRT-PCR) and protein (Western blotting); and significance of effect assessed by Student’s t-test, n = 3 or 4. The concentration of glucose in the medium was 6 mM. Results: In SAEC primary cultures, tRES-HESP decreased basal protein levels of TMPRSS2 (- 58%; P<0.001) and furin protease (- 43%; P<0.01) after 72 h. tRES-HESP also decreased mRNA of TMPRSS2 (- 62%, P<0.01), GRP78 (- 67%, P<0.001), COX-2 (-34%, P<0.001) and RAGE mRNA (- 56%, P<.001) and decreased basal secretion of MCP-1 and IL8 (-29% and - 25%, P<0.001). Discussion: tRES-HESP decreased basal expression of proteases essential for SARS-CoV-2 infection, GRP78 and secretion of inflammatory mediator risk predictors of severe COVID-19. These effects are likely due to activation of Nrf2 by tRES-HESP and decrease of unfolded protein response activation by suppression of dicarbonyl stress. These outcomes suggest previous anti-inflammatory response of tRES-HESP in overweight and obese subjects found in PBMCs may also occur concomitantly in the lung. TMPRSS2 is an antioxidant response element-linked gene down regulated by activation of Nrf2. These anti-proteolytic and anti-inflammatory responses of tRES-HESP suggest it is suitable for further evaluation for prevention of COVID-19, particularly in obesity and diabetes.

Lymphocyte-Rich Spindle Cell Thymoma: Clinicopathologic, Immunohistochemical, Ultrastructural and Molecular Genetic Study of 80 Cases.

A study of 80 cases of spindle cell thymoma in which the spindle cell component was overshadowed by massive numbers of stromal lymphocytes is presented. The patients were 38 women and 42 men, aged 8 to 81 years (mean=54 y). All tumors presented as an anterior mediastinal mass; 5 patients had myasthenia gravis and one had Good syndrome. The tumors were well-circumscribed, encapsulated, and measured 2.9 to 26.0 cm in greatest diameter (mean=7.3 cm). Using modified Masaoka staging, 66 tumors were stage I, 10 were stage IIa, 2 were stage III and 1 was stage IV. Histologically the tumors were characterized by a predominant lymphocytic population admixed with scattered small spindle epithelial cells. The neoplastic spindle cells in these tumors demonstrated 2 major growth patterns: in 33 cases, the tumors were exclusively composed of dense sheets of lymphocytes containing scattered spindle cells resembling a lymphocyte-rich thymoma (WHO type B1); in the remaining cases the tumors showed admixtures of a predominantly lymphocytic component with areas that were lymphocyte-poor and contained a pure spindle cell population similar to WHO type A. Immunohistochemical stains and electron microscopy corroborated the spindle cell morphology in both types. The GTF2I p.L424H variant was identified in 53 of 63 (84%) cases analyzed. Clinical follow-up in 27 cases showed that most of the tumors behaved in an indolent manner. Our study expands the spectrum of spindle cell thymoma by demonstrating the existence of cases that are predominantly composed of lymphocyte-rich elements and lack areas with a pure (lymphocyte poor) spindle cell morphology.

Solid Cell Nests Hyperplasia (Ultimobranchial Body Remnants) of the Thyroid: A Pitfall

Abstract Introduction/Objective Solid cell nests (SCN) are small epithelial cell nests interspersed within thyroid parenchyma, resembling squamous/transitional epithelium. SCNs, which are ultimobranchial remnants, are popularly considered pluripotent stem-cells responsible for developing follicular and C-cells. While SCNs are not an uncommon incidental findings in thyroid, solid cell nest hyperplasia is rare. SCNs are often mistaken for benign entities such as C-cell hyperplasia (CCH) or malignant lesions such as papillary thyroid carcinoma (PTC), metastatic squamous cell carcinoma, or medullary thyroid microcarcinoma (MTC). Methods/Case Report To reiterate this diagnostic dilemma, we present a case of a 57-year-old male with a six-year history of Hashimoto thyroiditis and multiple bilateral thyroid nodules. Ultrasonography revealed two nodules, one in each lobe, measuring 2.0x1.9x1.8cm(right) and 1.6x1.5x1.5cm(left). Both were solid, hypoechoic nodules with smooth margins and no echogenic foci. Fine-needle aspiration of right nodule was suspicious for follicular neoplasm, Hürthle- cell type, and the left nodule was atypia of unknown significance. Right-hemithyroidectomy specimen revealed follicular adenoma and oncocytic adenomatous nodules in a background of florid lymphocytic thyroiditis (Hashimoto). In two blocks, small solid nests of cells were identified, largest focus measuring 0.5 cm. The cells were polygonal to epithelioid with moderate amphophilic cytoplasm. Nuclei were centrally located, irregular to oval with occasional grooves. While nests had a squamoid appearance, they did not have intercellular bridges. Although nuclear grooves and evenly dispersed chromatin and chromocenters were noted, they lacked optical clearing or intra-nuclear inclusions characteristic for PTC. Thus, excluding these two possibilities, primary diagnostic considerations were SCN versus CCH. Immunohistochemical analysis showed cells positive for P63 and CK5/6 and negative for PAX-8, TTF-1, thyroglobulin, CEA, and calcitonin. Results (if a Case Study enter NA) NA Conclusion If wrongly diagnosed as CCH, patients may be placed in a high-risk category for possible development of MTC. It is, thus, necessary to be aware of SCN, which can occasionally become hyperplastic, to prevent misdiagnosis.

The role of micronutrient and immunomodulation effect in the vaccine era of COVID-19.

Different dietary nutrients have distinct effects, including enhancing immune response activity and supporting mucous membrane integrity. These effects are critical in fighting against pathogenic agents, which cover coronavirus disease 2019 (COVID-19), the coronavirus disease that shuts down globally. Recent researches have shown that micronutrient deficiency is commonly associated with compromised immune responses, respiratory tract infections, or even susceptibility to COVID-19. The relationship between Vit A and infection is its role in mucosal epithelium integrity (skin and mucous membrane), the supplementation could be an option for assisted-treating the SARS-CoV-2 virus and a possible prevention of lung infection. Vit C/ascorbic acid stimulates oxygen radical scavenging activity of the skin and enhances epithelial barrier function. Ascorbic acid alone or with other natural compounds (baicalin and theaflavin) may inhibit the expression of angiotensin-converting enzyme II in human small alveolar epithelial cells and limited the entry of SARS-CoV-2. Vitamin D receptors can be expressed by immune cells, and different immune cells (macrophages, monocytes, dendritic cells, T cells, and B cells) can convert Vit D into its active form 1,25-(OH)2 D. Oral vitamin D intake can be a readily way to restrict the viral infection through downregulation of ACE2 receptor and to attenuate the disease severity by decreasing the frequency of cytokine storm and pulmonary pro-inflammatory response. Vit E supports T-cell mediated functions, optimization of Th1 response, and suppression of Th2 response. Vitamin E supplementation can lower the production of superoxides and may favors the antioxidants and benefit the progress of COVID-19 treatment. Zinc plays an essential role in both innate and adaptive immune systems and cytokine production, and Zinc-dependent viral enzymes to initiate the infectious process have proved the Zinc levels are directly associated with symptoms relieved of COVID-19. Iron is an essential component of enzymes involved in the activation of immune cells, lower iron levels predispose to severe symptoms of SARS-CoV-2, and monitoring the status can predict the disease severity and mortality. Selenium participates in the adaptive immune response by supporting antibody production and development. Deficiency can reduce antibody concentration, decreased cytotoxicity of NK cells, compromised cellular immunity, and an attenuated response to vaccination. The COVID-19 vaccines including three broad categories, protein-based vaccines, gene-based vaccines (mRNA vaccines and DNA vaccines), combination of gene and protein-based vaccines. Micronutrients are involved in immunity from the virus entering the human to innate immune response and adaptive immune response. Micronutrients are indispensable in immune response of vaccination.

Cultivation of fractionated cells from a bioactive-alkaloid-bearing marine sponge Axinella sp.

Sponges are among the most primitive multicellular organisms and well-known as a major source of marine natural products. Cultivation of sponge cells has long been an attractive topic due to the prominent evolutionary and cytological significance of sponges and as a potential approach to supply sponge-derived compounds. Sponge cell culture is carried out through culturing organized cell aggregates called 'primmorphs.' Most research culturing sponge cells has used unfractionated cells to develop primmorphs. In the current study, a tropical marine sponge Axinella sp., which contains the bioactive alkaloids, debromohymenialdisine (DBH), and hymenialdisine (HD), was used to obtain fractionated cells and the corresponding primmorphs. These alkaloids, DBH and HD, reportedly show pharmacological activities for treating osteoarthritis and Alzheimer's disease. Three different cell fractions were obtained, including enriched spherulous cells, large mesohyl cells, and small epithelial cells. These cell fractions were cultivated separately, forming aggregates that later developed into different kinds of primmorphs. The three kinds of primmorphs obtained were compared as regards to appearance, morphogenesis, and cellular composition. Additionally, the amount of alkaloid in the primmorphs-culture system was examined over a 30-d culturing period. During the culturing of enriched spherulous cells and developed primmorphs, the total amount of alkaloid declined notably. In addition, the speculation of alkaloid secretion and some phenomena thatoccurred during cell culturing are discussed.

Respiratory syncytial virus-induced high mobility group box 1 secretion modulates innate immune response to promote inflammation

Abstract Respiratory syncytial virus (RSV) is the single most common human pathogen responsible for ~ 60% of all acute lower respiratory tract infections in young children, and is a major cause of severe respiratory morbidity and mortality in the elderly, and the immunocompromised. Yet, no vaccine or efficacious treatment is available. Although the mechanisms of RSV-induced acute airway disease are not fully understood, lung inflammatory and immune events are key. Our studies show that RSV induces increased extracellular secretion of high-mobility group box 1 (HMGB1), a prominent damage-associated molecular pattern family member that alerts the immune system to potential damage and trigger immediate response. The goal of this study is to investigate the molecular mechanisms of RSV-induced HMGB1 release in the activation of innate immune response in promoting lung inflammation. Studies were conducted in BALB/c mice as well as human lung epithelial cells including A549 and normal small alveolar epithelial cells. To determine the role of HMGB1 in the activation of innate immune signaling, BALB/c mice and airway epithelial cells were infected with RSV in the presence or absence of recombinant HMGB1 or HMGB1 monoclonal antibody and measured RIG-I-dependent interferon-mediated innate antiviral immune response. Our studies demonstrated an important role of HMGB1 in cellular protection and the activation of innate immune signaling cascades to promote inflammation. Our studies indicate that HMGB1 acts as a signaling molecule to induce an increased inflammatory response in the lung, thus contributing to RSV pathogenesis. Modulation of proinflammatory function of HMGB1 may attenuate RSV-induced lung inflammation.

Inhibition of ACE2 Expression by Ascorbic Acid Alone and its Combinations with Other Natural Compounds.

Background:Angiotensin-converting enzyme II or ACE2 is an integral membrane protein present on many types of cells, including vascular endothelial cells and lung alveolar epithelial cells. This receptor serves as the entry point for SARS-coronaviruses (SARS-CoVs), including a novel coronavirus 2019-nCoV. Limited availability of these receptors can thwart cellular entry of this virus.Methods:We tested the effects of ascorbic acid (vitamin C) on cellular expression of ACE2 at the protein and RNA levels in human small alveolar epithelial cells and microvascular endothelial cells. In addition, we investigated whether combinations of ascorbic acid with other natural compounds can affect ACE2 expression.Results:The results show that ascorbic acid itself has moderate but consistent lowering effects on ACE2 expression at the cellular, protein, and RNA levels. Some natural compounds were effective in lowering ACE2 cellular expression, with the highest inhibitory effects observed for baicalin (75%) and theaflavin (50%). Significantly, combinations of these and other test compounds with ascorbic acid further decreased ACE2 expression. The highest impact of ascorbate on ACE2 expression was noted when combined with theaflavin (decrease from 50% to 87%), zinc (decrease from 22% to 62%), and with 10-undecenoic acid (from 18% to 53%). Ascorbic acid showed moderate additional benefits in decreasing ACE2 expression when combined with N-acetylcysteine and baicalin.Conclusion:Our study provides valuable experimental confirmation of the efficacy of micronutrients in controlling ACE2 expression—the coronavirus cellular “entry” point. It further validates the importance of nutrient interactions in various aspects of cellular metabolism and in considering potential therapeutic applications of nutrient-based approaches. The study shows that ascorbic acid and its combination with some natural compounds could be included in developing preventive and therapeutic approaches toward the current pandemic.

HIF-1α Modulates Core Metabolism and Virus Replication in Primary Airway Epithelial Cells Infected with Respiratory Syncytial Virus.

Metabolic reprogramming of host cells is key to the foundation of a successful viral infection. Hypoxia inducible factors (HIFs) mediate oxygen utilization by regulating cellular metabolism and redox homeostasis. Under normoxic conditions, HIF proteins are synthesized and subsequently degraded following ubiquitination to allow for normal metabolic activities. Recent studies suggest that respiratory syncytial virus (RSV) has the ability to induce HIF-1α stabilization and accumulation through non-hypoxic mechanisms. This makes the HIF pathway a potential avenue of approach for RSV therapeutic development. Using a model of primary human small alveolar epithelial cells, we demonstrate RSV infections to greatly alter cellular metabolism in favor of the glycolytic and pentose phosphate pathways. Additionally, we show RSV infections to stabilize HIF-1α and HIF-2α expression in these cells. Inhibition of HIF-1α, but not HIF-2α, was found to significantly reduce RSV replication as well as the glycolytic pathway, as measured by the expression of hexokinase II. Our study contributes to the understanding of RSV-mediated changes to cellular metabolism and supports further investigation into anti-HIF-1α therapeutics for RSV infections.

Effect of tobacco smoke on respiratory syncytial virus-induced high-mobility group box 1-mediated innate antiviral signaling

Abstract Exposure to tobacco smoke (TS), either direct or secondhand, is associated with an increase in the severity of lower respiratory tract infections, including bronchiolitis, mainly caused by respiratory syncytial virus (RSV). No vaccine or effective treatment is available for RSV. Although the mechanism of TS-induced RSV severity remains largely unknown, lung immune responses are key. High Mobility Group Box-1 (HMGB1) is a multifunctional nuclear protein that is released to the extracellular space during infection and regulates inflammatory responses as damage-associated molecular pattern. The goal of this study is to dissect the role of HMGB1 in the activation of antiviral signaling in RSV and TS-induced airway inflammation. Studies were conducted in airway epithelial cells, including A549 and normal small alveolar epithelial cells. We show that RSV and TS-induced oxidative stress post-translationally modifies HMGB1 including hyperacetylation, phosphorylation, and oxidation, leading to its enhanced extracellular secretion from lung epithelial cells. Secreted HMGB1 activates signaling cascades to activate p38 MAPK and NF-κB to induce proinflammatory mediator release from immune cells and induce inflammation. We also show that hmgb1 knockdown exacerbates inflammation in RSV-infected and TS-exposed cells and that treatment with recombinant HMGB1 activates immune cells to induce an inflammatory response, promotes the activation of antiviral pathways and inhibits viral replication. Our studies suggest an important role for HMGB1 in TS-induced RSV pathogenesis and guide the development of novel therapeutic strategies against respiratory infections that are caused or exacerbated by environmental toxicants such as TS.

The role of high-mobility group box 1 protein in respiratory syncytial virus-induced autophagy

Abstract Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections in infants and young children worldwide, for which no efficacious treatments or vaccines are currently available. Although the pathogenic mechanisms of RSV-induced acute airway disease are still unclear, lung immune and inflammatory responses are key. High-mobility group box 1 (HMGB1) is a redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular signaling molecule that mediates the activation of inflammatory responses to infection/injury. HMGB1 has been implicated in mitochondrial function and quality control. The objective of this study is to investigate the role of HMGB1 in autophagosome formation and mitochondrial quality control in RSV infection. Studies were conducted in lung epithelial cells including A549 and normal small alveolar epithelial cells. Our studies show that RSV induces HMGB1 secretion in lung epithelial cells via NFκB and TLR4 pathways, and activates innate immune cells to promote inflammation. We show that RSV-induces LC3B and promote autophagy whereas recombinant HMGB1 (rHMGB1) treatment inhibits RSV-induced autophagy. We also show that RSV infection inhibits the inflammasome pathway in lung epithelial cells but rHMGB1 treatment activates inflammasomes. Our results indicate that RSV-induced epithelial HMGB1 secretion triggers inflammasome activation in innate immune cells to promote inflammation. Understanding HMGB1 and associated cellular signaling in RSV-induced lung inflammation may pave the way for the development of novel therapeutic interventions to treat not only RSV, but also other respiratory infections that affect humans of all ages.

Effects of cannabis oil extract on immune response gene expression in human small airway epithelial cells (HSAEpC): implications for chronic obstructive pulmonary disease (COPD)

BackgroundChronic obstructive pulmonary disease (COPD) is commonly associated with both a pro-inflammatory and a T-helper 1 (Th1) immune response. It was hypothesized that cannabis oil extract can alleviate COPD symptoms by eliciting an anti-inflammatory Th2 immune response. Accordingly, the effects of cannabis oil extract on the expression of 84 Th2 and related immune response genes in human small airways epithelial cells (HSAEpC) were investigated.MethodsHSAEpC from a single donor were treated with three dilutions of a standardized cannabis oil extract (1:400, 1:800 and 1:1600) along with a solvent control (0.25% [2.5 ul/ml] ethanol) for 24 h. There were four replicates per treatment dilution, and six for the control. RNA isolated from cells were employed in pathway-focused quantitative polymerase chain reaction (qPCR) microarray assays.ResultsThe extract induced significant (P < 0.05) changes in expression of 37 tested genes. Six genes (CSF2, IL1RL1, IL4, IL13RA2, IL17A and PPARG) were up-regulated at all three dilutions. Another two (CCL22 and TSLP) were up-regulated while six (CLCA1, CMA1, EPX, LTB4R, MAF and PMCH) were down-regulated at the 1:400 and 1:800 dilutions. The relationship of differentially-expressed genes of interest to biologic pathways was explored using the Database for Annotation, Visualization and Integrated Discovery (DAVID).ConclusionsThis exploratory investigation indicates that cannabis oil extract may affect expression of specific airway epithelial cell genes that could modulate pro-inflammatory or Th1 processes in COPD. These results provide a basis for further investigations and have prompted in vivo studies of the effects of cannabis oil extract on pulmonary function.Trial registrationNONE (all in vitro experiments).

Metanephric adenoma of the right kidney: a challenging diagnosis in a 49-year-old female

Background: Metanephric adenoma of the kidney is a rare, usually solitary, and benign tumor of the kidney, predominately affecting female patients in the fifth or sixth decade of life. It is considered by some specialists to be the hyperdifferentiated benign end of the Wilm’s tumor spectrum. Case Presentation: The case was a 49-year-old woman with abdominal pain, a palpable mass in the right abdomen, and mild hematuria, without any other symptoms or laboratory findings. An ultrasonography and computed tomography revealed a tumor in the upper pole of the right kidney with a diameter of 5.3 cm. There was no infiltration of the neighboring structures and tissues. The differential diagnosis included papillary renal cell carcinoma, adult Wilm’s tumor, and metanephric adenoma. A partial nephrectomy was followed. The histological examination of the tumor revealed relatively small unvarying basophilic epithelial cells with scant cytoplasm, uniform nuclei, and some areas with nuclear grooves, delicate chromatin, and indistinct nucleoli in a loose non-cellular stroma and developed in a tightly packed alveolar, tubular, and rarely papillary pattern. The mitotic rate was extremely low. Tumor cells were positive for paired box gene 8, cluster differentiation 57, and Wilm’s tumor 1 and negative for racemase, cytokeratin 7, and EMA. The cell proliferation rate Ki-67 was extremely low, and there was diffuse, strong cytoplasmic positivity for BRAF V600E staining. Based on morphology and immunohistochemistry, the diagnosis of metanephric adenoma was made. Conclusion: Although metanephric adenomas may be difficult to diagnose clinically, the histological examination and the immunohistochemistry assay, including BRAF V600 staining, can make a safe diagnosis, avoiding the administration of incorrect treatment.

Release of transglutaminase 2 from respiratory syncytial virus-infected airway epithelial cells and its role in disease pathogenesis

Abstract Respiratory syncytial virus (RSV) is the common cause of severe lower respiratory tract infections in young children, the elderly and the immunocompromised, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to both the development and severity of asthma. Oxidative stress plays a major role in the pathogenesis of many lung inflammatory diseases. Our studies have shown that oxidative stress responses in the airways play a major role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium levels, which in turn activate calcium dependent enzymes, including transglutaminase 2 (TG2). TG2 is a transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications and has been implicated in the pathogenesis of many diseases. This study’s objective was to investigate the release of TG2 in airway epithelial cells in response to RSV infection and its potential mechanism. Studies were conducted in airway epithelial cells, a human lung carcinoma cell line and normal small alveolar epithelial cells. The changes in the TG2 levels were measured by Western blot and RT-PCR after cells were pretreated with antioxidants and infected with RSV. TG2 levels were significantly increased in the secretome with a concomitant decrease in the cell lysates, cytoplasmic and nuclear extracts in response to RSV infection. Antioxidants treatment significantly inhibited RSV-induced TG2 release. TG2 release was induced only by RSV but not RSV-induced cytokines or chemokines. These results suggest that modulation of oxidative stress represents a potential novel pharmacological approach to ameliorate virus-induced acute airway disease through TG2 release blockade.