TNF-alpha Converting Enzyme Research Articles

Exploratory studies on chrysin via antioxidant, antimicrobial, ADMET, PASS and molecular docking evaluations

IntroductionChrysin, a flavonoid found in Feng Jiao (Chinese name for propolis), exhibits a wide array of pharmacological activities and is recognized for the significant roles it plays in traditional Chinese medicine. The present study aimed to explore the in vitro antioxidant and antimicrobial potentials of chrysin and provide mechanistic insights via in silico experiments. MethodsThe in vitro antioxidant and antimicrobial activities of chrysin, a natural flavone found in propolis was investigated. Molecular docking interactions, the Adsorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) and Prediction of Activity Spectra for Substances (PASS) were used to predict the biological activities of chrysin. ResultsChrysin exhibited a considerable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging activity and ferric reducing antioxidant capacity. Chrysin demonstrated inhibition against S. typhi, and B. subtilis with lowest MIC value of 1.25 mg/mL which was similar to that of chloramphenicol. Drug-likeness and ADMET study of the two compounds showed their physicochemical properties as non-toxic, non-mutagenic, high permeability, good solubility and absorption characteristics. DiscussionThe research below shows that chrysin and silymarin had antioxidant, free radical scavenging, antibacterial, hepatoprotective and anticarcinogenic properties based on PASS prediction of biological activities. Molecular docking of chrysin and silymarin, a standard hepatoprotective drug, with tumour necrosis factor alpha converting enzyme (TACE) and manganese-dependent superoxide dismutase (SOD 2) showed interactions with LEU 401, VAL 402, HIS 405 amino acid residues at the active sites of TACE. Chrysin non-covalently interacted with amino acids of SOD 2 at the active site while silymarin non-covalently interacted at a site different from the active site. Silymarin demonstrated a minimum binding energy of – 9.7 Kcal/mol and – 7.0 Kcal/mol for TACE and SOD 2, respectively while chrysin showed a minimal binding energy of – 9.0 Kcal/mol and – 6.8 Kcal/mol for TACE and SOD2 respectively. ConclusionThe study concluded that chrysin exhibited antioxidant and antimicrobial activity via inhibition and regulation of TACE and SOD2 and possibly offer protection against hepatotoxicity in animal experimental models.

Effects of T-PRF and A-PRF on the Osteogenic Biomarkers in Intrabony Defects of Periodontitis Patients

Background: Different derivatives of platelet-rich fibrin (PRF) have been developed but the efficacy of these derivatives in tissue healing and regeneration is still unclear. The aim of this study was to evaluate the effects of titanium-prepared PRF (T-PRF) and advanced PRF (A-PRF) on osteogenic biomarkers in gingival crevicular fluid (GCF) and clinical parameters. Methods: Seventeen systemically healthy participants with 30 bilateral infrabony defects were recruited. Following phase I periodontal therapy, intrabony defects were treated either with A-PRF+open flap debridement (OFD) or T-PRF+OFD. Plaque index (PI), gingival index (GI), pocket depth (PD), clinical attachment loss (CAL) was recorded at the baseline and 6th month after treatment. GCF samples were collected at the baseline and 3rd, 6th months after surgery. Nuclear factor receptor activator (RANK), receptor activator nuclear kappa-B ligand (RANKL), osteoprotegerin (OPG) and tumor necrosis factor alpha converting enzyme (TACE) in GCF samples were analyzed by human enzyme-linked immunosorbent assay (ELISA). Results: In both groups, statistically significant changes were observed in clinical parameters, however, there was no difference between the groups. In terms of osteogenic biomarkers in GCF, there were no statistically significant differences between and within the groups. Conclusion: Different derivatives of PRF can be used to enhance the clinical outcomes of intrabony defects in periodontitis.

Structural, computational and in silico studies of 4-bromo-3-flurobenzonitrile as anti-Alzheimer and anti-Parkinson agents

A novel dimer of the 4-bromo-3-fluorobenzonitrile was crystallized and studied using a spectroscopic method such as the scanning electron microscope method. The computational simulations substantiated the structural analysis findings. The Hirshfeld surface analysis has been performed for visualizing, exploring and quantifying the intra and inter-molecular interactions that stabilize the crystal packing of the compound. The NBO and QTAIM analyses were applied to study the nature and origin of the attractive forces involved in the crystal structure. Further, the pharmacokinetic properties of the compound were evaluated, indicating good brain-blood barrier and central nervous system penetration capability. Hence, in silico studies was carried out to explore the binding pattern of the titled compound against acetylcholinesterase and butyrylcholinesterase, and tumor necrosis factor-alpha converting enzyme proteins using molecular docking and molecular dynamics simulations approach. Further, the titled compound is compared with standard drugs through molecular docking studies. The in silico studies finally predicts that the compound under investigation may act as a good inhibitor for treating Alzheimer’s disease and further in vitro and in vivo studies may provide its therapeutic potential. Communicated by Ramaswamy H. Sarma

Interactions of the protein tyrosine phosphatase PTPN3 with viral and cellular partners through its PDZ domain: insights into structural determinants and phosphatase activity.

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. We solved the X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE). We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the protein's phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.

The discovery of potential tumor necrosis factor alpha converting enzyme inhibitors via implementation of K Nearest Neighbor QSAR analysis

Tumor necrosis factor-alpha converting enzyme (TACE) is considered as a pro-inflammatory cytokine which catalyzes the formation of TNF-α from membrane bound TNF-α precursor protein. It has important role in various pathological diseases such as inflammation, anorexia, rheumatoid arthritis, cancer and viral replication. Despite the reporting of a variety of TACE inhibitors of diverse chemical scaffolds whether peptide, peptide-like compounds or others, the bioavailability and pharmacokinetic problems are reflected strongly on its clinical effectiveness. In this effort we employed a novel combination of k-nearest neighbor QSAR modeling to select the best critical ligand-TACE contacts capable of elucidation of TACE inhibitory bioactivity among a long list of inhibitors. The recurrence of one valid pharmacophore hypothesis among the successful models emerged the pharmacophore to be used as 3D search queries to screen the National Cancer Institute’s structural database for new TACE inhibitors. Eight potent TACE inhibitors were discovered with inhibition percentage exceeding 50% at 100 μM inhibitor concentration.

Oral Administration of Vitamin D3 Prevents Corneal Damage in a Knock-Out Mouse Model of Sjögren's Syndrome.

Vitamin D deficiency has been associated with dry eye development during Sjögren's syndrome (SS). Here, we investigated whether repeated oral vitamin D3 supplementation could prevent the corneal epithelium damage in an SS mouse model. 30 female mouse knock-out for the thrombospondin 1 gene were randomized (six per group) in untreated mice euthanized at 6 weeks as negative control (C-) or at 12 weeks as the positive control for dry eye (C+). Other mice were sacrificed after 6 weeks of oral vitamin D3 supplementation in the drinking water (1000, 8000, and 20,000 IU/kg/week, respectively). The C+ mice showed alterations in their corneal epithelial morphologies and thicknesses (p < 0.01 vs. C-), while the mice receiving 8000 (M) and 20,000 (H) IU/kg/week of vitamin D3 showed preservation of the corneal epithelium morphology and thickness (p < 0.01 vs. C+). Moreover, while the C+ mice exhibited high levels and activity of corneal tumor necrosis factor alpha converting enzyme (TACE), neovascularization and fibrosis markers; these were all reduced in the M and H mice. Oral vitamin D3 supplementation appeared to counteract the negative effect of TACE on corneal epithelium in a mouse model of SS-associated dry eye.

Probing the bioactive compounds of Kigelia africana as novel inhibitors of TNF-α converting enzyme using HPLC/GCMS analysis, FTIR and molecular modelling

Tumor Necrosis Factor Alpha Converting Enzyme (TACE) mediates inflammatory disorder and contributes to the pathophysiology of a variety of illnesses, such as chronic inflammation and cancer. This study identified metabolites in solvent extracts of Kigelia africana as putative TACE inhibitors due to the plant’s known anti-inflammatory properties. HPLC-MS/GCMS analysis was used to characterize tentative phytochemicals from K. africana. The identified metabolites (n = 123) were docked with TACE to reveal the lead compounds. Binding free energy, ADMET prediction, molecular dynamics simulation at 100 ns, and DFT calculation were further conducted. The results revealed that K. africana contains sterol, phenols, alkaloids, terpenes and flavonoids. The FTIR shows that the extracts had peaks that correspond to the presence of different functional groups. The quantum polarized ligand docking (QPLD) analysis identified compound (n = 3) with binding affinity higher than standard compound IK-682. The hits also had modest ADMET profiles, interacted with essential residues within TACE binding pockets, and formed stable complexes with the protein. The 100 ns MD simulation shows that the compounds formed fairly stable interactions and complex with the protein as evidenced through RMSF, RMSD and MM-GBA results. The HOMO/LUMO, global descriptive molecular electrostatic potential Fukui function aid in the identification of the compounds’ atomic sites prone to electrophilic/neutrophilic attacks, and non-covalent interactions. This study suggests that K. africana’s bioactive compounds are capable of mitigating inflammation by inhibiting TACE. Communicated by Ramaswamy H. Sarma

Protective effect of Ceiba pentandra (L) Gaertn on CCl4-induced oxidative stress and liver damage in rats

BackgroundCeiba pentandra (L.) Gaertn, popularly known as the white silk-cotton tree and popularly used in Chinese and Indian traditional medicine. The bark is used to treat diarrhea, pain, fever, cardiac problems, asthma, and gastrointestinal problems. The present study aimed to evaluate the hepatoprotective and antioxidant effects of ethanol extract of C. pentandra bark (EECP-BK) against CCl4-induced hepatic damage in rats. MethodsPreliminary phytochemical analysis was performed to identify the bioactive compounds. CCl4 (40 mM) induced enzymatic changes at HepG2 cells were adopted for the in-vitro model. HepG2 cells were pretreated with EECP-BK (25, 50, 100, and 200 µg/ml) and standard (silymarin 12.5 µg/ml) respectively. Rat model of CCl4-induced liver damage for adopted for the in-vivo model. Rats were pretreated orally with EECP-BK 200 & 400 mg/kg and standard (silymarin 100 mg/kg) for 7 days. Rats were injected with 1 ml/kg of 50% CCl4 on day 8 to induce hepatic damage and were observed for antioxidant levels and serum biomarkers. In addition, in-silico studies have been performed to explore the binding properties of compounds from EECP-BK with tumor necrosis factor-alpha converting enzyme (TACE) targeting hepatic inflammation ResultsThe phytochemical analysis confirmed the presence of glycoside, steroids, phenols, tannins, and saponins. EECP-BK 200 µg/ml showed the highest protection against CCl4 in HepG2 cells. Similarly, silymarin 12.5 µg/ml showed good protection against CCl4. In rats, EECP-BK up to 2000 mg/kg did produce any toxic signs or mortality. CCl4 injection in rats showed elevated liver enzymatic and bilirubin levels and causes liver damage. It also decreased the SOD, CAT, and GSH levels. Pre-treatment with EECP-BK 400 mg/kg showed a significant reduction in SGPT, SGOT, ALP, and total bilirubin levels and increased SOD, CAT, and GSH levels. Similar results were observed with, silymarin treatment also. Furthermore, EECP-BK showed protection over hepatocyte necrosis induced by CCl4 in rats which is evident by histopathology. Molecular docking studies revealed that acacetin 7-rutinoside binds favorably with the active site of TACE with a binding affinity of -8.8 kJ/mol. ConclusionOur findings supported the antioxidative capability of EECP-BK by showing that it protected the liver from CCl4-induced hepatic damage. Additional research is needed to determine its safety profile and to find its molecular mechanism for therapeutic application.

NOTCH and Tumor Necrosis Factor-Alpha Converting Enzyme Levels Could be used in COVID-19 for Risk Stratification

Background: Novel coronavirus disease-2019 (COVID-19) has become a public emergency that is characterized by a dysregulated immune response and hypercoagulable state. The purpose of the present study was to evaluate NOTCH and tumor necrosis factor-alpha converting enzyme (TACE) levels in COVID-19-infected patients and assess their predictive value on the severity of the disease. Methods: A total of 116 severe-critical COVID-19 patients who were interned intensive care were included in the study. The severity of the disease was evaluated according to the WHO classification system. Patients were divided into two groups according to their cTroponin T (cTnT) levels. Patients who had cTnT levels at least five times the upper limit of normal constituted Group 1 (n = 58); patients who had normal cTnT levels constituted Group 2 (n = 58). Besides, 62 age- and sex-matched healthy controls, who applied to cardiology outward clinic were taken as a control group (Group 3). All patients underwent echocardiographic examination. NOTCH and TACE levels were assessed using enzyme-linked immunosorbent assay. Results: The average age of the patients was 59.96 ± 15.46 years, 92 (51.7%) were female and 86 (48.3%) were male. The mean length of hospital stay was 16.35 ± 10.97 days. NOTCH levels were significantly higher in Group 1 patients compared to Group 2 and control group of patients P = 0.001). NOTCH levels of Group 2 were significantly higher compared to the control group (P = 0.002). Similarly, the TACE levels of Group 1 were significantly higher than that of Group 2 and the control group (P = 0.001). Mortality and length of hospital stay were significantly higher in Group 1 patients compared to Group 2 patients (P = 0.002 and P = 0.004, respectively). TACE levels of deceased patients were significantly higher than that of live patients (P = 0.004). There was a positive relationship between the length of hospital stay and NOTCH levels in Group 1 patients (r = 0.527, P = 0.003). TACE and NOTCH levels were positively correlated with troponin levels (r = 0.627 and r = 0.671, respectively P &lt; 0.001 for both). NOTCH value of 0.34 nmol/L and TACE value of 6.53 μg/mL predicted inhospital mortality with a sensitivity of 90.30% and 63.6% and specificity of 91.5% and 78.6%, respectively. Conclusion: Measurement of NOTCH and TACE levels during severe acute respiratory syndrome coronavirus infection could be helpful for risk stratification.

Deficiency in Inactive Rhomboid Protein2 (iRhom2) Alleviates Alcoholic Liver Fibrosis by Suppressing Inflammation and Oxidative Stress.

Chronic alcohol exposure can lead to liver pathology relating to inflammation and oxidative stress, which are two of the major factors in the incidence of liver fibrosis and even liver cancer. The underlying molecular mechanisms regarding hepatic lesions associated with alcohol are not fully understood. Considering that the recently identified iRhom2 is a key pathogenic mediator of inflammation, we performed in vitro and in vivo experiments to explore its regulatory role in alcohol-induced liver fibrosis. We found that iRhom2 knockout significantly inhibited alcohol-induced inflammatory responses in vitro, including elevated expressions of inflammatory cytokines (IL-1β, IL-6, IL-18, and TNF-α) and genes associated with inflammatory signaling pathways, such as TACE (tumor necrosis factor-alpha converting enzyme), TNFR1 (tumor necrosis factor receptor 1), and TNFR2, as well as the activation of NF-κB. The in vivo results confirmed that long-term alcohol exposure leads to hepatocyte damage and fibrous accumulation. In this pathological process, the expression of iRhom2 is promoted to activate the TACE/NF-κB signaling pathway, leading to inflammatory responses. Furthermore, the deletion of iRhom2 blocks the TACE/NF-κB signaling pathway and reduces liver damage and fibrosis caused by alcohol. Additionally, the activation of the JNK/Nrf2/HO-1 signaling pathway caused by alcohol exposure was also noted in vitro and in vivo. In the same way, knockout or deleting iRhom2 blocked the JNK/Nrf2/HO-1 signaling pathway to regulate the oxidative stress. Therefore, we contend that iRhom2 is a key regulator that promotes inflammatory responses and regulates oxidative stress in alcoholic liver fibrosis lesions. We posit that iRhom2 is potentially a new therapeutic target for alcoholic liver fibrosis.

Investigation of the antileishmanial activity and mechanisms of action of acetyl-thiohydantoins

The currently available treatment options for leishmaniasis are associated with high costs, severe side effects, and high toxicity. In previous studies, thiohydantoins demonstrated some pharmacological activities and were shown to be potential hit compounds with antileishmanial properties.The present study further explored the antileishmanial effect of acetyl-thiohydantoins against Leishmania amazonensis and determined the main processes involved in parasite death. We observed that compared to thiohydantoin nuclei, acetyl-thiohydantoin treatment inhibited the proliferation of promastigotes. This treatment caused alterations in cell cycle progression and parasite size and caused morphological and ultrastructural changes. We then investigated the mechanisms involved in the death of the protozoan; there was an increase in ROS production, phosphatidylserine exposure, and plasma membrane permeabilization and a loss of mitochondrial membrane potential, resulting in an accumulation of lipid bodies and the formation of autophagic vacuoles on these parasites and confirming an apoptosis-like process. In intracellular amastigotes, selected acetyl-thiohydantoins reduced the percentage of infected macrophages and the number of amastigotes/macrophages by increasing ROS production and reducing TNF-α levels. Moreover, thiohydantoins did not induce cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), or sheep erythrocytes. In silico and in vitro analyses showed that acetyl-thiohydantoins exerted in vitro antileishmanial effects on L. amazonensis promastigotes in apoptosis-like and amastigote forms by inducing ROS production and reducing TNF-α levels, indicating that they are good candidates for drug discovery studies in leishmaniasis treatment. Additionally, we carried out molecular docking analyses of acetyl-thiohydantoins on two important targets of Leishmania amazonensis: arginase and TNF-alpha converting enzyme. The results suggested that the acetyl groups in the N1-position of the thiohydantoin ring and the ring itself could be pharmacophoric groups due to their affinity for binding amino acid residues at the active site of both enzymes via hydrogen bond interactions. These results demonstrate that thiohydantoins are promising hit compounds that could be used as antileishmanial agents.

TNFα and Immune Checkpoint Inhibition: Friend or Foe for Lung Cancer?

Tumor necrosis factor-alpha (TNFα) can bind two distinct receptors (TNFR1/2). The transmembrane form (tmTNFα) preferentially binds to TNFR2. Upon tmTNFα cleavage by the TNF-alpha-converting enzyme (TACE), its soluble (sTNFα) form is released with higher affinity for TNFR1. This assortment empowers TNFα with a plethora of opposing roles in the processes of tumor cell survival (and apoptosis) and anti-tumor immune stimulation (and suppression), in addition to angiogenesis and metastases. Its functions and biomarker potential to predict cancer progression and response to immunotherapy are reviewed here, with a focus on lung cancer. By mining existing sequencing data, we further demonstrate that the expression levels of TNF and TACE are significantly decreased in lung adenocarcinoma patients, while the TNFR1/TNFR2 balance are increased. We conclude that the biomarker potential of TNFα alone will most likely not provide conclusive findings, but that TACE could have a key role along with the delicate balance of sTNFα/tmTNFα as well as TNFR1/TNFR2, hence stressing the importance of more research into the potential of rationalized treatments that combine TNFα pathway modulators with immunotherapy for lung cancer patients.

Simultaneous monitoring assay for T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface

Isolation of antigen (Ag)-specific T cells is an important step in the investigation of T-cell immunity. Activation-induced markers (AIMs), such as CD154/tumor necrosis factor (TNF)/CD107A/CD134/CD137 enable the sorting of Ag-specific T cells without using human leukocyte antigen (HLA)-multimers. However, optimal conditions suitable for simultaneous detection of both Ag-specific CD4 and CD8 T cells have not been investigated. Here, conditions were optimized to simultaneously detect the maximum number of activated CD4 and CD8 T cells in a TCR-dependent manner. First, the frequency of total pools of AIM-positive cells induced by superantigen, staphylococcal enterotoxin B (SEB), stimulation in various culture conditions was monitored and compared side-by-side. The total amount of AIM-positive CD4 T cells, but not CD8 T cells, was significantly abrogated by addition of brefeldin A. TNF-alpha converting enzyme inhibitor treatment effectively increased the TNF-positive population, without affecting other markers’ positivity. AIM-positive CD4 T cells and CD8 T cells were detected at least 3 h after stimulation. Furthermore, examination of the multiple combination of each marker revealed that minimum contribution of CD134 on the total pool of AIM-positive cells at this setting, suggesting the essential and non-essential AIMs to maximize the detected number of AIM-positive cells.Taken together, this optimized method will be a useful tool for the simultaneous monitoring the T-cell receptor stimulation-dependent activation of CD4 and CD8 T cells using inducible markers on the cell surface including Ag-specific T cells.

Implanted 3D gelatin microcryogel enables low-dose cell therapy for osteoarthritis by preserving the viability and function of umbilical cord MSCs

Mesenchymal stem cells (MSCs) have shown promise as a therapy for osteoarthritis (OA). However, to date there is no standardized cell dosage, and the mechanism of action of MSCs in OA is still not completely understood. Biomaterials are considered to improve seeded cell function, so we established biocompatible and biodegradable gelatin microcryogels (GMs) as the carrier for umbilical cord–derived MSCs (UCMSCs) in this study. First, we generated UCMSC-GM complexes, optimized the pore size and cell density, and discovered that GMs enhanced cell viability and growth factor secretion, especially epidermal growth factors (EGF), under different biomechanical conditions, which is possibly mediated by tumor necrosis factor-alpha converting enzyme (TACE), a metalloproteinase crucial for EGF mature and release. In addition, Luciferase tracing and GFP fluorescence assay showed that GMs enhanced the retention of UCMSCs in the knee joint of mice. Then, we treated mice with OA knee joints with either low-dose free MSCs (3 × 104 cells), UCMSC-loaded GMs (3 × 104 cells) or high-dose free MSCs (3 × 105 cells), and found that UCMSC-GMs (3 × 104 cells) promoted cartilage regeneration and inhibited macrophage-mediated synovitis better than low-dose free UCMSCs, and achieved a similar effect to that of high-dose (3 × 105 cells) treatment. Further transwell assay showed that GMs promoted chondrocyte anabolism in vitro, which is greatly blocked by Gefitinib, an EGF receptor (EGFR) signalling pathway inhibitor. In conclusion, this study provides evidence that the UCMSC-GM complex exhibited a significantly stronger effect in attenuating OA progression compared to UCMSCs alone even at a low dose, and these results shed light on the clinical application of cell therapy for OA.

Hypothesis: Alpha-1-antitrypsin is a promising treatment option for COVID-19

No definitive treatment for COVID-19 exists although promising results have been reported with remdesivir and glucocorticoids. Short of a truly effective preventive or curative vaccine against SARS-CoV-2, it is becoming increasingly clear that multiple pathophysiologic processes seen with COVID-19 as well as SARS-CoV-2 itself should be targeted. Because alpha-1-antitrypsin (AAT) embraces a panoply of biologic activities that may antagonize several pathophysiologic mechanisms induced by SARS-CoV-2, we hypothesize that this naturally occurring molecule is a promising agent to ameliorate COVID-19. We posit at least seven different mechanisms by which AAT may alleviate COVID-19. First, AAT is a serine protease inhibitor (SERPIN) shown to inhibit TMPRSS-2, the host serine protease that cleaves the spike protein of SARS-CoV-2, a necessary preparatory step for the virus to bind its cell surface receptor ACE2 to gain intracellular entry. Second, AAT has anti-viral activity against other RNA viruses HIV and influenza as well as induces autophagy, a known host effector mechanism against MERS-CoV, a related coronavirus that causes the Middle East Respiratory Syndrome. Third, AAT has potent anti-inflammatory properties, in part through inhibiting both nuclear factor-kappa B (NFκB) activation and ADAM17 (also known as tumor necrosis factor-alpha converting enzyme), and thus may dampen the hyper-inflammatory response of COVID-19. Fourth, AAT inhibits neutrophil elastase, a serine protease that helps recruit potentially injurious neutrophils and implicated in acute lung injury. AAT inhibition of ADAM17 also prevents shedding of ACE2 and hence may preserve ACE2 inhibition of bradykinin, reducing the ability of bradykinin to cause a capillary leak in COVID-19. Fifth, AAT inhibits thrombin, and venous thromboembolism and in situ microthrombi and macrothrombi are increasingly implicated in COVID-19. Sixth, AAT inhibition of elastase can antagonize the formation of neutrophil extracellular traps (NETs), a complex extracellular structure comprised of neutrophil-derived DNA, histones, and proteases, and implicated in the immunothrombosis of COVID-19; indeed, AAT has been shown to change the shape and adherence of non-COVID-19-related NETs. Seventh, AAT inhibition of endothelial cell apoptosis may limit the endothelial injury linked to severe COVID-19-associated acute lung injury, multi-organ dysfunction, and pre-eclampsia-like syndrome seen in gravid women. Furthermore, because both NETs formation and the presence of anti-phospholipid antibodies are increased in both COVID-19 and non-COVID pre-eclampsia, it suggests a similar vascular pathogenesis in both disorders. As a final point, AAT has an excellent safety profile when administered to patients with AAT deficiency and is dosed intravenously once weekly but also comes in an inhaled preparation. Thus, AAT is an appealing drug candidate to treat COVID-19 and should be studied.

Caveolae-Associated Protein 3 (Cavin-3) Influences Adipogenesis via TACE-Mediated Pref-1 Shedding.

Abnormal adipogenesis regulation is accompanied by a variety of metabolic dysfunctions and disorders. Caveolae play an important role in the regulation of fat production, modulated by caveolae-associated proteins (Cavin-1 to 4). Here, we investigated the role of Cavin-3 in lipogenesis and adipocyte differentiation, as the regulatory functions and roles of Cavin-3 in adipocytes are unknown. A Cavin-3 knockdown/overexpression stable cell line was established, and adipogenesis-related gene and protein expression changes were investigated by real-time quantitative PCR and Western blot analysis, respectively. Additionally, confocal immune-fluorescence microscopy was used to verify the intracellular position of the relevant factors. The results showed that Cavin-3 mRNA and protein expression were elevated, along with physiological factors such as lipid droplet formation, during adipogenesis. Cavin-3 silencing resulted in retarded adipocyte differentiation, and its overexpression accelerated this process. Furthermore, Cavin-3 knockdown resulted in decreased expression of adipogenesis-related genes, such as PPAR-γ, FAS, aP2, and Adipoq, whereas preadipocyte factor-1 (Pref-1) was markedly increased during adipocyte maturation. Overall, Cavin-3 influences caveolar stability and modulates the tumor necrosis factor-alpha-converting enzyme (TACE)-mediated Pref-1 shedding process in both mouse and human adipocytes. The Cavin-3-dependent shedding mechanism appears to be an important process in adipocyte maturation, providing a potential therapeutic target for obesity-related disorders.

EGFR-Dependent IL8 Production by Airway Epithelial Cells After Exposure to the Food Flavoring Chemical 2,3-Butanedione.

2,3-Butanedione (DA), a component of artificial butter flavoring, is associated with the development of occupational bronchiolitis obliterans (BO), a disease of progressive airway fibrosis resulting in lung function decline. Neutrophilic airway inflammation is a consistent feature of BO across a range of clinical contexts and may contribute to disease pathogenesis. Therefore, we sought to determine the importance of the neutrophil chemotactic cytokine interleukin-8 (IL-8) in DA-induced lung disease using in vivo and in vitro model systems. First, we demonstrated that levels of Cinc-1, the rat homolog of IL-8, are increased in the lung fluid and tissue compartment in a rat model of DA-induced BO. Next, we demonstrated that DA increased IL-8 production by the pulmonary epithelial cell line NCI-H292 and by primary human airway epithelial cells grown under physiologically relevant conditions at an air-liquid interface. We then tested the hypothesis that DA-induced epithelial IL-8 protein occurs in an epidermal growth factor receptor (EGFR)-dependent manner. In these in vitro experiments we demonstrated that epithelial IL-8 protein is blocked by the EGFR tyrosine kinase inhibitor AG1478 and by inhibition of tumor necrosis factor-alpha converting enzyme using the small molecule inhibitor, TAPI-1. Finally, we demonstrated that DA-induced IL-8 is dependent upon ERK1/2 and Mitogen activated protein kinase kinase activation downstream of EGFR signaling using the small molecule inhibitors AG1478 and PD98059. Together these novel in vivo and in vitro observations support that EGFR-dependent IL-8 production occurs in DA-induced BO. Further studies are warranted to determine the importance of IL-8 in BO pathogenesis.

Issue Highlights - March 2019.

This issue features, among others, two reviews and four original manuscripts in the field of clinical cytometry, all of them in the area of leukemia/lymphoma immunophenotyping. These may be summarized as follows: Jevremovic and colleagues reviewed the position of flow cytometry in the diagnosis, prognosis and follow-up of T- and NK-cell lymphoproliferations 1. The diagnosis of these entities is generally based on the demonstration of immunophenotypic abnormalities and demonstration of clonality. The latter studies involve V-beta T-cell receptor repertoire analysis and killer immunoglobulin like receptors (KIR). In this context it is important to realize that a T-cell clone does not need to be synonymous with “malignancy” but could also reflect the transient clonal proliferation of “reactive” T cells. Among the mature T-cell lymphoproliferative disorders, T-cell large granular lymphocytic leukemia (T-LGLL), anaplastic large T-cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), mycosis fungoides or Sezary syndrome (MF/SS), T-cell prolymphocytic leukemia (T-PLL), hepatosplenic T-cell lymphoma (HSTCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) are distinguished. Within the NK-cell disorders, chronic lymphoproliferative disorder of NK cells (CLPD-NKs), extranodal NL/T-cell lymphoma, nasal type, aggressive NL-cell leukemia/lymphoma and NK-cell enteropathy are defined. Nowadays, flow cytometry is a sensitive and specific approach to detect T and NK-cell neoplasms but requires a thorough knowledge of the normal T-cell and NK-cell immune spectrum. Jan W. Gratama, Deputy Editor [Color figure can be viewed at wileyonlinelibrary.com] CD123, the alpha chain of IL3 receptor, is expressed in variable intensities on human leukocytes; the strongest expression is seen on plasmacytoid dendritic cells and basophils, whilst the marker is absent on nucleated red blood cells; a range of intermediate expression levels are seen on T cells, mature B cells, granulocytes and eosinophils. CD123 is expressed on AML blasts and most B-cell precursor ALL blasts whilst it is negative on most T-ALL blasts. The teams of the Laboratory for Medical Immunology at Erasmus MC (Rotterdam, Netherlands) in collaboration with the Dutch Childhood Oncology Group (The Hague, Netherlands) set out to study CD123 expression levels in 846 patients with acute leukemia in comparison to normal cell populations obtained in 1,252 unique bone marrow and peripheral blood samples 6. Immunophenotyping was performed using standardized procedures as developed by the EuroFlow collaborative project 7. Whilst published information on this topic was relatively scarce and difficult to compare due to different methodologies, Bras and colleagues confirmed most observations as they extensively document in this report 6. Importantly, they observed that in paired B-cell precursor ALL, CD123 expression was considerably higher at relapse than at initial diagnosis, whilst in AML, the leukemic stem cells (CD34pos, CD38neg fraction of tumor cells) expressed similar CD123 levels as the remainder of AML blasts. These observations make CD123 an interesting target for antibody-targeted therapy 8. Early positive minimal residual disease (MRD) is a strong predictor of response to treatment in ALL and AML. In an observational study of 125 children with B-cell precursor ALL, Loosveld and colleagues 9 investigated the prognostic value of flow cytometric assessment of MRD in peripheral blood on days 8 and 15 after start of cytoreductive therapy, compared to day 35 MRD on bone marrow using molecular techniques. Children with detectable PB MRD on day 15 and negative day 35 BM results had lower disease-free survival at 4 years follow-up, indicating the prognostic potential of this less invasive, blood-based procedure early after start of therapy as compared to conventional bone marrow studies. Mora and colleagues 10 studied 120 consecutive patients with chronic B-lymphoproliferations (81 within the CLL spectrum and 39 with other monoclonal B-cell disorders) to establish whether CD200, a member of the Ig receptors membrane proteins superfamily, would have added value to the immunological scoring system already developed by Matutes et al. 11. CD200 had already been suggested to be rather specific for B-CLL in earlier studies. This study confirmed the value of CD200 as diagnostic marker and revealed that it's addition to the Matutes score increased the accuracy of that ranking algorithm. Beke Debreceni et al. 12 analyzed the regulation of expression of the adhesion receptor L-selectin (CD62L) in a cohort of untreated patients with B-CLL. Expression of surface-bound CD62L and tumor necrosis factor alpha-converting enzyme (TACE), as well as total phosphatase and protein phosphatase-2A activities, were lower on B-CLL than on normal B cells whilst the reverse was true for soluble CD62L and phosphorylated mitogen-activated protein kinase (pp38MAPK). In separate in vitro experiments confirmed that a phosphatase inhibitor (calyculin A) could reduce the effect of pp38MAPK inhibitor on CD62L shedding. The authors concluded that the reduction of phosphatase activity in B-CLL has resulted in a downstream signaling cascade with subsequent reduction of surface-bound CD62L which is mediated by enhanced phosphorylation of p38MAPK and reduced TACE expression. Jan W. Gratama, Deputy Editor Email: j.w.gratama@erasmusmc.nl

L-Selectin Expression is Influenced by Phosphatase Activity in Chronic Lymphocytic Leukemia.

Adhesion receptors have important role in cellular invasiveness and L-selectin is a primary determinant in the binding of chronic lymphocytic leukemia (CLL) cells to several glycated proteins on endothelial cells. We investigated L-selectin expression on CLL cells and explored the mechanisms that lead to their shedding. Surface and soluble L-selectin expression levels were studied by flow cytometry and immunoassay, respectively. Magnetically isolated B-cells from patients and controls were investigated for total and protein phosphatase-2A activities. Flow cytometry of permeabilized cells was utilized for the determination of phosphorylated mitogen-activated protein kinase (pp38MAPK) and surface tumor necrosis factor alpha-converting enzyme expression (TACE). In CLL patients elevated absolute lymphocyte cell counts, high soluble and low surface L-selectin expression were observed. Similarly, TACE surface expression was significantly lower on B-CLL cells compared to normal B-cells. Both total phosphatase and protein phosphatase-2A activities were also significantly lower in B-CLL cells compared to normal B-cells and we found a consequently higher level of pp38 MAPK in B-CLL cells. Based on in vitro experiments a MAPK inhibitor could attenuate the phosphatase inhibitor's effect on L-selectin shedding. The lower phosphatase activity detectable in chronic lymphocytic leukemia, results in a downstream signaling cascade with subsequent reduction of surface L-selectin expression and this effect is mediated by enhanced phosphorylation of p38MAPK and an altered TACE expression. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Enhanced expression of TACE contributes to elevated levels of sVCAM-1 in endometriosis.

Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activity in endometriosis? Here we provide the first functional evidence that induced TACE activity in human endometriotic epithelial cells is at least in part responsible for the enhanced release of sVCAM-1 from these cells. We and others have shown that serum-soluble (s)VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. This study was conducted at the Tertiary Endometriosis Referral Center of the Medical University of Vienna. Samples from a total number of 97 women were collected between July 2013 and September 2014. After complete surgical exploration of the abdominopelvic cavity, 49 women with histologically proven endometriosis and 48 endometriosis-free control women were enrolled. Each participating woman contributed only one sample of eutopic endometrium and normal peritoneum, and some of the women with endometriosis contributed samples of diverse types of endometriotic lesions (in total 52 ectopic samples). Among the 49 women with endometriosis, 36 matched samples of endometriotic lesions and corresponding eutopic endometrium were collected. In order to detect sVCAM-1 and TACE protein by ELISA, peritoneal fluid (PF) samples were collected from 44 cases and 32 controls during surgery. Expression of TACE mRNA was analyzed by qRT-PCR in 111 endometrium tissue samples (28 eutopic control samples, 33 eutopic samples from women with endometriosis, 50 ectopic samples from lesions) and 37 healthy peritoneum samples. Immunohistochemistry was performed in 123 tissue samples (39 eutopic control samples, 42 eutopic samples from women with endometriosis, 42 ectopic samples from lesions) and the relation between tissue TACE protein levels and sVCAM-1 secretion was examined. PMA-induced sVCAM-1 release, and TACE- and VCAM-1-transcripts or proteins were measured in an immortalized endometriotic epithelial cell line (11Z) pre-incubated either with TACE inhibitors or following TACE siRNA knockdown. Here, we demonstrate that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) and that the overexpression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Additionally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. Albeit well characterized, our control population included women with other gynecologic diseases, which may have impacted the levels of sVCAM-1 and tissue TACE expression levels, e.g. benign ovarian cysts or uterine fibroids. Thus, the results of our analysis have to be interpreted carefully and in the context of the current experimental settings. The dysregulation of TACE substrate shedding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease. This work was supported by the Ingrid Flick Foundation. The authors have no competing interests to declare.