Inhibition Of Phagocytosis Research Articles

Cigarette Smoke Exposure Inhibits Early Phase of Antibody Production through Inhibition of Immune Functions in Alveolar Macrophage

Background::Cigarette smoke (CS) is inhaled into the lung. Alveolar macrophage (AM) is known to play an important role in the lung immune system. However, the relationship between AM functions and antibody production by CS is not fully investigated.Objective::Therefore, we investigated the effects of AM from CS exposed mice on antibody production. Mice were exposed to 20 cigarettes/day for 10 days. AM were obtained by broncho-alveolar lavage. Antibody production was analyzed by plaque-forming cell assay using seep red blood cell (SRBC) as antigen.Methods::B cell proliferation was analyzed by 3H-thymidine incorporation. Phagocytic activity using fluorescein isothiocyanate-labeled SRBC and expressions of surface antigens on AM were analyzed by flow cytometry. Cytokines and NF-κB mRNA expressions of AM were analyzed by RTPCR.Results and Discussion:Antibody production was decreased at the induction phase, but not at the expression phase by AM from smoked mice (SM) compared with non-smoked mice (NSM). B cell proliferation was decreased by cigarette extracts dose-dependently. Phagocytic activity of AM was decreased in SM compared with NSM. Expression of surface antigens on AM was decreased in SM compared with NSM. Cytokines or NF-κB mRNA expressions of AM were decreased in SM compared with NSM.Conclusion::These results suggest that the inhibition of antibody production by cigarette smoking is caused by the inhibition of phagocytosis and expressions of surface antigens associated with antigen presentation. Such inhibition of AM functions may increase the risk of bacterial and virus infections.

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.

CR3 ruffles FcγR’s claim over phagocytic cups

Phagocytosis plays diverse roles in biology, but our understanding of the purpose, interplay, and cell signaling mechanisms associated with different modes of phagocytosis is limited, without being able to capture and visualize each step in this rapid process from the beginning to end. A new study by Walbaum et al. uses stunning time-lapse 3D imaging of the engulfment of erythrocytes by macrophages via sinking, ruffling, and cup formation, unequivocally confirming a visionary 44-year-old theory derived from still electron microscopy photos that phagocytosis mediated by complement receptor CR3 occurs via a sinking mechanism and antibody-mediated phagocytosis occurs via phagocytic cup formation. The article also challenges the dogma, showing that phagocytic cup formation is not unique to antibody receptor phagocytosis, rather CR3 plays a complex role in different modes of phagocytosis. For example, inhibition of antibody-mediated phagocytosis leads to a compensatory upregulation of CR3-mediated sinking phagocytosis. These findings animate, in vivid colors, processes previously only captured as stills, exposing interactions between different phagocytic mechanisms and altering our basic understanding of this important process.

Inhibition of LC3-associated phagocytosis in COPD and in response to cigarette smoke

Introduction/Rationale:In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD.Methods:Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence.Results:Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis.Conclusion:LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

Calreticulin – a multifaced protein

Summary Calreticulin (CALR) is a highly conserved multi-function protein that primarily localizes within the lumen of the endoplasmic reticulum (ER). It participates in various processes in the cells, including glycoprotein chaperoning, regulation of Ca2+ homeostasis, antigen processing and presentation for adaptive immune response, cell adhesion/migration, cell proliferation, immunogenic cell death, gene expression and RNA stability. The role of CALR in the assembly, retrieval and cell surface expression of MHC class I molecules is well known. A fraction of the total cellular CALR is localized in the cytosol, following its retro-translocation from the ER. In the cell stress conditions, CALR is also expressed on the cell surface via an interaction with phosphatidylserine localized on the inner leaflet of the plasma membrane. The abovementioned mechanism is relevant for the recognition of the cells, as well as immunogenicity and phagocytic uptake of proapoptotic and apoptotic cells. Lastly, the presence of CALR exon 9 gene mutations was confirmed in patients with myeloproliferative neoplasms. Their presence results in an abnormal CALR structure due to the loss of its ER-retention sequence, CALR extra-ER localisation, the formation of a complex with thrombopoietin receptor, and oncogenic transformation of hematopoietic stem cells. It is also known that CALR exon 9 mutants are highly immunogenic and induce T cell response. Despite this fact, CALR mutant positive hematopoietic cells emerge. The last phenomenon is probably the result of the inhibition of phagocytosis of the cancer cells exposing CALR mutant protein by dendritic cells.

Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model.

The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Effect of Cytochalasin B on Human Monocyte Binding and Sphering of IgG-coated Human Erythrocytes

The ability of human mononuclear phagocytic cells to bind IgG-coated human erythrocytes (EA) and to cause bound EA to become osmotically fragile (sphered) was investigated in the presence of cytochalasin B, a known inhibitor of phagocytosis. Cytochalasin B inhibited the binding of EA to mononuclear cells in a dose-dependent fashion; 80% inhibition of binding was observed at a concentration of 5 mug/ml. This profound effect on EA binding together with presently available data suggested a role for IgG receptor mobility in the macrophage binding of IgG-coated erythrocytes. Cytochalasin B, however, had a minimal effect on the capacity of mononuclear cells to sphere adherent EA, suggesting that the processes involved in macrophage-induced spherocytosis may differ from those operable in phagocytosis.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Sensitivity of Rodent Microglia to Kynurenines in Models of Epilepsy and Inflammation In Vivo and In Vitro: Microglia Activation Is Inhibited by Kynurenic Acid and the Synthetic Analogue SZR104

Kynurenic acid is an endogenous modulator of ionotropic glutamate receptors and a suppressor of the immune system. Since glutamate and microglia are important in the pathogenesis of epilepsy, we investigated the possible action of the synthetic kynurenic acid analogue, SZR104, in epileptic mice and the action of kynurenic acid and SZR104 on the phagocytotic activity of cultured microglia cells. Pilocarpine epilepsy was used to test the effects of SZR104 on morphological microglia transformation, as evaluated through ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemistry. Microglia-enriched rat secondary cultures were used to investigate phagocytosis of fluorescent microbeads and Iba1 protein synthesis in control and lipopolysaccharide-challenged cultures. SZR104 inhibited microglia transformation following status epilepticus. Kynurenic acid and SZR104 inhibited lipopolysaccharide-stimulated phagocytotic activity of microglia cells. Although kynurenic acid and its analogues proved to be glutamate receptor antagonists, their immunosuppressive action was dominant in epilepsy. The inhibition of phagocytosis in vitro raised the possibility of the inhibition of genes encoding inflammatory cytokines in microglial cells.

T cell co-stimulator inducible co-stimulatory (ICOS) exerts potential anti-atherosclerotic roles through downregulation of vascular smooth muscle phagocytosis and proliferation.

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease. The role of the immune system in the etiology of the disease, particularly T cells, has been widely studied and is well established. T cell activation directly regulates co-signaling molecules present in immune synapses. Targeting one or several of these co-signaling molecules can inhibit T cell-mediated inflammation and delay or reduce AS. In recent years, this strategy has increasingly become a research focus. As such, we explored the role and therapeutic potential of the T cell co-stimulatory molecule inducible co-stimulatory (ICOS) in AS.MethodsWe compared the expression of ICOS in early AS lesions occurring in ApoE-deficient (ApoE-KO) rats fed a fat-diet and wild type (WT) rats fed the same diet. Eight-week old ApoE-KO and WT rats [ApoE-KO(0) and WT(0)] were fed a high-fat diet for 16 weeks [ApoE-KO(16) and WT(16)]. ICOS expression in aortic tissues was analyzed by quantitative real-time PCR, western blot, and confocal microscopy. The effect of ICOS overexpression in a transfected human T cell line on the phagocytosis and proliferation of co-cultured human aortic smooth muscle cells (HASMCs) was studied in vitro.ResultsCompared with WT(0), ApoE-KO(0), and WT(16) rats, ICOS expression in ApoE-KO(16) rats was significantly down-regulated both at the mRNA and protein levels. In vitro experiments indicated that ICOS overexpression reduces phagocytosis and proliferation by HASMCs, and may therefore produce an anti-atherosclerotic effect.ConclusionsThe immune synaptic co-signaling molecule ICOS has an anti-atherosclerotic effect through inhibition of HASMC phagocytosis and proliferation, and can be used to delay plaque formation during the early stages of AS.

Abstract 12889: Clathrin-mediated Endocytosis Plays a Key Role in Stem Cell Therapy for Ischemic Heart Disease

Background and Aim: Cell therapy represents a potential frontier to transform the treatment and prognosis of heart failure and acute myocardial infarction (AMI). Extracellular vesicles (EVs) have come out as key mediators of intercellular communication to improve cardiac function in cell therapy. However, the mechanisms by which cardiomyocytes respond to EVs remain unclear. Here, we sought to clarify the effect of cardiomyocyte endocytosis on AMI. Methods and Results: Treatment with the culture supernatant of adipose-derived regenerative cells (ADRCs) prevented cardiomyocyte cell damage under conditions of hypoxia in vitro. In vivo, the injection of ADRCs in the heart simultaneous with coronary artery ligation decreased infarct area in the heart in a dose-dependent manner. In addition, the injection of ADRCs prevented cardiac rupture after AMI and significantly reduced apoptosis in cardiomyocytes at 1 and 2 days. To examine the efficacy of cardiomyocyte endocytosis, cardiomyocytes were cultured with EVs previously isolated from ADRC culture supernatant and labeled with the fluorescent dye, PKH67. Notably, hypoxic culture conditions increased the intensity of the labeled EVs in cardiomyocytes. An inhibitor of clathrin-mediated endocytosis (chlorpromazine), but not inhibitors of caveolae-mediated endocytosis, micropinocytosis, and phagocytosis (genistein, cytochalasin D, or amiloride), significantly suppressed the ADRC culture supernatant-induced decreases of hypoxia-damaged cardiomyocytes. Chlorpromazine treatment also decreased hypoxia-induced cardiomyocyte capture of labeled EVs secreted from ADRCs. To investigate whether communication between cardiomyocytes and ADRCs affords cardiac protection through clathrin-mediated endocytosis in vivo, mice were treated with ADRCs alone or in combination with chlorpromazine following coronary artery ligation. Chlorpromazine treatment failed to show ADRC-induced cardiac protection such as prevention of cardiac rupture and anti-apoptotic effect. Conclusions: Our results indicate that EVs secreted from ADRCs prevent cardiac rupture after AMI and that clathrin-mediated endocytosis plays a critical role in transporting the EVs into cardiomyocytes.

Abstract PO-009: Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system

Abstract Introduction: Exosomes are emerging as promising nanocarriers for genes and drugs; however, major challenges exist including the absence of active tumor targeting and a high proportion of clearance by mononuclear phagocytic system (MPS) in liver and spleen. To overcome these obstacles currently preventing the effective application of nanodelivery technology in the field of cancer biology, we generate a novel exosome that recognizes both pancreatic ductal adenocarcinoma (PDAC) specific cell surface marker avb3 and the signal regulatory protein (SIRPa) on macrophages. Using this novel exosome, we have achieved active PDAC targeting and MPS evasion. Methods: We engineered the CD9, an exosome marker, to display a HA tagged RGD peptide at the E174 site of CD9 (CD9-HA-RGD). We overexpressed CD9-HA-RGD and a Flag tagged CD47 (CD47-Flag) in HEK 293 cells. CD47, a “don’t eat me” protein signal, mediates inhibition of phagocytosis through interacting with SIRPa on macrophages. Exosomes overexpressing CD9-HA-RGD and CD47-Flag were harvested from HEK 293 cell culture media by ultracentrifugation followed by purification with size exclusion chromatography (SEC). Exosomes displaying RGD and CD47 (Exo-RGD/CD47) were characterized in vitro using ELISA, western blot (WB), flow cytometry (FCM), PDAC cell uptake, and macrophage phagocytosis assays. Exo-RGD/CD47 was also evaluated in vivo in patient-derived PDAC cell line (PDCL5) orthotopic xenograft mouse model. Results: RGD and CD47 were demonstrated to be successfully displayed on exosome surface by analysis of HA and Flag expression using ELISA and FCM. Engineered Exo-RGD/CD47 exosomes achieved enhanced cell binding and increased uptake capacities in an avb3 expression dependent manner in PDAC cells. This was confirmed using excess RDG peptide in a competition assay. Control exosomes demonstrated low uptake efficiency by PDAC cells. Exo-RGD/CD47 resulted in 71.5% reduction of macrophage phagocytosis when compared to control exosomes as evaluated by THP1 cells phagocytosis assay. Using DiR stained exosomes, we demonstrated that Exo-RGD/CD47 systemic delivery significantly reduced liver accumulation up to 80.6% and 65.8% after 1h and 6h after injection, respectively, as compared to control group (p<0.05). Similarly, Exo-RGD/CD47 delivery resulted in 70.1% reduction of splenic accumulation as compared to control at 6h after application (p<0.05). Exo-RGD/CD47 systemic delivery also showed significant tumor accumulation in the PDCL5 xenograft tumor model as compared to control (p<0.05). Conclusions: These data demonstrate that CD9 is an editable exosome biomarker by which peptides can be effectively displayed on the surface of an exosome. Displaying RGD and CD47 on the exosome surface results in a novel exosome that enables active PDAC targeting and evasion of phagocytosis by MPS. These findings suggest the newly designed exosome may have a better translational potential in PDAC diagnosis and therapy than traditional exosomes. Citation Format: Shi-He Liu, Justin Creeden, F. Charles Brunicardi, JianTing Zhang. Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-009.

Fostamatinib for the Treatment of Warm Antibody Autoimmune Hemolytic Anemia (wAIHA): A Phase 3, Randomized, Double-Blind, Placebo-Controlled, Global Study

Fostamatinib for the Treatment of Warm Antibody Autoimmune Hemolytic Anemia (wAIHA): A Phase 3, Randomized, Double-Blind, Placebo-Controlled, Global Study

Apicomplexan co-infections impair with phagocytic activity in avian macrophages

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte–derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles (“Zymosan”) by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella–infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to “Zymosan” was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

CD47-SIRPα Axis as a Biomarker and Therapeutic Target in Cancer: Current Perspectives and Future Challenges in Nonsmall Cell Lung Cancer.

CD47 is a cell surface protein in the immunoglobulin superfamily which is normally expressed at low levels in every healthy cell. It´s main physiologic function is to act as an inhibitor of phagocytosis; this occurs throughout interaction with SIRPa expressed on macrophages. Interaction between CD47 and SIRPa leads to activation of tyrosine phosphatases that inhibit myosin accumulation at the submembrane assembly site of the phagocytic synapse, resulting in phagocytosis blockade. In this way CD47 acts as a “don´t eat me signal” for healthy self-cells; accordingly, loss of CD47 leads to phagocytosis of aged or damaged cells. Taking advantage of this anti-phagocytic signal provided by CD47, many types of tumors overexpress this protein, thereby avoiding phagocytosis by macrophages and aiding in the survival of cancer cells. The aim of this review is to describe the physiologic the pathophysiologic role of CD47; summarize the available high-quality information about this molecule as a potential biomarker and/or therapeutic target in cancer; finally, we present an in-depth analysis of the available information about CD47 in association with nonsmall cell lung cancer, EGFR mutations, and tumor microenvironment.

Serum From Melioidosis Survivors Diminished Intracellular Burkholderia pseudomallei Growth in Macrophages: A Brief Research Report.

Melioidosis is a neglected tropical disease with high mortality rate. It is caused by the Gram-negative, CDC category B select agent Burkholderia pseudomallei (B. ps) that is intrinsically resistant to first-line antibiotics. An antibody-based vaccine is likely to be the most effective control measure. Previous studies have demonstrated significant mechanistic roles of antibodies in protection against death in animal models, but data from human melioidosis is scarce. Herein, we used in-vitro antibody-dependent cellular phagocytosis and growth inhibition assays to assess the mechanism of protective antibodies in patients with acute melioidosis. We found that serum from patients who survived the disease enable more live B. ps to be engulfed by THP-1 derived macrophages (median 1.7 × 103 CFU/ml, IQR 1.1 × 103-2.5 × 103 CFU/ml) than serum from patients who did not survive (median 1.2 × 103 CFU/ml, IQR 0.7 × 103-1.8 × 103, p = 0.02). In addition, the intracellular growth rate of B. ps pre-opsonized with serum from survivors (median 7.89, IQR 5.58–10.85) was diminished when compared with those with serum from non-survivors (median 10.88, IQR 5.42–14.88, p = 0.04). However, the difference of intracellular bacterial growth rate failed to reach statistical significance when using purified IgG antibodies (p = 0.09). These results provide new insights into a mechanistic role of serum in protection against death in human melioidosis for antibody-based vaccine development.

Vascular Endothelial Growth Factor Inhibits Phagocytosis of Apoptotic Cells by Airway Epithelial Cells.

Professional phagocytes such as dendritic cells and macrophages can ingest particles larger than 0.5 μm in diameter. Epithelial cells are nonprofessional phagocytes that cannot ingest pathogenic microorganisms, but they can ingest apoptotic cells. Inhibition of the engulfment of apoptotic cells by the airway epithelium can cause severe airway inflammation. Vascular endothelial growth factor (VEGF) is an angiogenesis-promoting factor that can mediate allergic airway inflammation and can promote airway epithelial cells (AECs) proliferation, but it is not clear whether it affects the engulfment of apoptotic cells by AECs. In the present study, VEGF inhibited engulfment of apoptotic cells by AECs via binding to VEGF receptor(R)2. This inhibitory effect of VEGF was not influenced by masking of phosphatidylserine (PS) on the surface of apoptotic cells and was partially mediated by the PI3K-Akt signaling pathway. VEGF inhibition of phagocytosis involved polymerization of actin and downregulation of the expression of the phagocytic-associated protein Beclin-1 in AECs. Since engulfment of apoptotic cells by AECs is an important mechanism for airway inflammation regression, VEGF inhibition of the engulfment of apoptotic cells by airway epithelial cells may be important for mediating allergic airway inflammation.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

Microglia Do Not Take Up Soluble Amyloid-beta Peptides, But Partially Degrade Them by Secreting Insulin-degrading Enzyme

Microglia play important roles in the pathogenesis of Alzheimer’s disease (AD), in part, by affecting the clearance of amyloid-β (Aβ) peptides. Most studies, however, used synthetic soluble Aβ (sAβ) at higher concentrations. The exact mechanisms underlying microglia-mediated clearance of physiological sAβ at very low concentrations remain unclear. Here we reported that there were much more Iba-1- and CD68-positive microglia and significantly less sAβ left in the brain of adult mice 5 days after the surgery of sAβ microinjection compared to 2 h after the surgery (p < 0.05). However, very few Iba-1- and CD68-positive microglia co-localized with microinjected fluorescently labeled sAβ (FLsAβ42) 5 days after the surgery. Also, there was no co-localization of FLsAβ42 with a lysosomal marker (LAMP-1) 5 days after the surgery. There was no significant difference in the percentage of Aβ+/PE-CD11b+/APC-CD45low microglia between the control group and the group microinjected with TBS-soluble Aβ extracted from the brains of AD patients (p > 0.05). The degradation of physiological sAβ was prevented by a highly selective insulin-degrading enzyme inhibitor (Ii1) but not by a phagocytosis inhibitor (polyinosinic acid) or pinocytosis inhibitor (cytochalasin B) in vitro. Furthermore, the reduction of synthetic and physiological sAβ in the brain was partially prevented by the co-injection of Ii1 in vivo (p < 0.05). Our results demonstrate that microglia do not take up synthetic or physiological sAβ, but partially degrade it via the secretion of insulin-degrading enzyme, which will be beneficial for understanding how sAβ is removed from the brain by microglia.

Torsades de Pointes in Coronavirus Disease 2019 Infection

A 73-YEAR-OLD MAN (weight 69 kg, height 175 cm) was transferred to the authors’ critical care unit with respiratory and acute renal failure secondary to coronavirus disease 2019 (COVID-19) infection. His past medical history included hypertension, diabetes, and previous stroke, from which he had made a full recovery. The patient was treated with mechanical ventilation and peritoneal dialysis. He was recruited into the hydroxychloroquine arm of the RECOVERY (Randomised Evaluation of COVID-19 Therapy) trial. Baseline electrocardiogram (ECG) and subsequent ECG monitoring were normal and did not demonstrate any QT prolongation. Three days later, the patient developed a ventricular tachycardia with a circulatory collapse. Immediate cardiopulmonary resuscitation was initiated and defibrillator pads attached; however, the arrhythmia self-terminated within 2 minutes (Fig 1). After resuscitation, another ECG showed a normal QT interval. Arterial blood gas analysis did not show any signs of respiratory insufficiency or an acid-base disturbance. There were no signs of myocardial ischemia. A full panel of blood tests was performed, which did not demonstrate any relevant abnormalities in full blood count, biochemical studies, or coagulation. Hypomagnesemia and hypokalemia were absent. Echocardiography was normal. What is the diagnosis? Diagnosis: Torsades de pointes ventricular tachycardia presumably resulting from hydroxychloroquine therapy. Review of the telemetry data demonstrated a polymorphic ventricular tachycardia in the pattern of torsades de pointes, with a spontaneous onset and termination. A cardiac electrophysiology review was requested and confirmed the diagnosis of torsades de pointes. Review of ECGs, blood test results, and medication history allowed the authors to conclude that the arrhythmia had most likely been caused by hydroxychloroquine. Recommendations were made for stringent monitoring of serum electrolytes (to prevent hypomagnesemia and hypokalemia) and daily ECGs. No further antiarrhythmic therapy was recommended at this stage. Hydroxychloroquine was discontinued, and the patient's participation in the RECOVERY trial was terminated. No further arrhythmias were reported during the patent's stay. An adverse drug reaction report was submitted to the UK Medicines and Healthcare products Regulatory Agency (UK MHRA) to register the hydroxychloroquine-related life-threatening arrhythmia. In early 2020 during the COVID-19 pandemic, a number of studies reported possible therapeutic effects of hydroxychloroquine, and it was therefore included as 1 of the drugs in the platform UK RECOVERY trial.1Randomised evalution of COVID-19 Therapy (RECOVERY) Availible at: https://www.recoverytrial.net/files/protocol-archive/recovery-protocol-v4-0-2020-04-14.pdfGoogle Scholar However, preliminary data showed no benefit in hospitalized patients with COVID-19, which led to the UK MHRA instruction to stop recruitment into trials using hydroxychloroquine to treat patients with COVID-19.2UK Medicines & Healthcare products Regulatory AgencyMHRA suspends recruitment to COVID-19 hydroxychloroquine trials.2020https://www.gov.uk/government/news/mhra-suspends-recruitment-to-covid-19-hydroxychloroquine-trialsGoogle Scholar Hydroxychloroquine was introduced in 1955 as an anti-malarial drug. It has immunomodulatory properties that include inhibition of chemotaxis and phagocytosis, reduction of cytokine synthesis, inhibition of phospholipase A2, and signaling in lymphocytes.3Ben-Zvi I Kivity S Langevitz P et al.Hydroxychloroquine: From malaria to autoimmunity.Clin Rev Allerg Immunol. 2012; 42: 145-153Crossref PubMed Scopus (398) Google Scholar,4Kalia S Dutz JP. New concepts in antimalarial use and mode of action in dermatology.Dermatol Ther. 2007; 20: 160-174Crossref PubMed Scopus (187) Google Scholar Hydroxychloroquine has a large spectrum of side effects including cardiomyopathy and arrhythmias associated with the prolongation of the QT interval.3Ben-Zvi I Kivity S Langevitz P et al.Hydroxychloroquine: From malaria to autoimmunity.Clin Rev Allerg Immunol. 2012; 42: 145-153Crossref PubMed Scopus (398) Google Scholar Between 1964 and 2020, the UK MHRA registered 916 patients with adverse drug reactions, of whom 679 were classified as serious and 15 as fatal. The most commonly reported adverse drug reactions involved skin (454 cases) and the nervous system, including eye (285) and gastrointestinal tract (165). Cardiac adverse drug reactions were reported in 48 patients and included myocardial and/or pericardial disorders, arrhythmias, and heart failure.5UK Medicines & Healthcare products Regulatory AgencyInteractive drug analysis profile for hydroxychloroquine.2020https://info.mhra.gov.uk/drug-analysis-profiles/dap.html?drug=./UK_EXTERNAL/NONCOMBINED/UK_NON_000544442035.zip&agency=MHRAGoogle Scholar Cardiac arrhythmias were reported in 18 patients. Anesthesiologists should continue to be aware of the importance of a comprehensive drug history and are reminded of potential life-threatening arrhythmias when hydroxychloroquine is used. We declare that we have no known competing interests or relationships that could have influenced the work reported in this paper.