Carbon monoxide-induced autophagy enhances human mesenchymal stromal cell function via paracrine actions in murine polymicrobial sepsis.

Sepsis is a life-threatening process due to organ dysfunction resulting from severe infections. Mesenchymal stromal cells (MSCs) are being investigated as therapy for sepsis, along with conditioning regimens to improve their function. Carbon monoxide (CO) gas, which is cytoprotective at low doses, induces autophagy and is a mediator of inflammation. We evaluated CO-induced autophagy in human MSCs (hMSCs), and its impact on cell function in murine cecal ligation and puncture. Conditioning of hMSCs with CO ex vivo resulted in enhanced survival and bacterial clearance in vivo, and neutrophil phagocytosis of bacteria in vitro. Decreased neutrophil infiltration and less parenchymal cell death in organs were associated with increased macrophage efferocytosis of apoptotic neutrophils, promoting resolution of inflammation. These CO effects were lost when the cells were exposed to autophagy inhibition prior to gas exposure. When assessing paracrine actions of CO-induced autophagy, extracellular vesicles (EVs) were predominantly responsible. CO had no effect on EV production, but altered their miRNA cargo. Increased expression of miR-145-3p and miR-193a-3p by CO was blunted with disruption of autophagy, and inhibitors of these miRNAs led to a loss of neutrophil phagocytosis and macrophage efferocytosis. Collectively, CO-induced autophagy enhanced hMSC function during sepsis via paracrine actions of MSC-derived EVs.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Clinical feature, GALC variant spectrum, and genotype-phenotype correlation in Korean Krabbe disease patients: Multicenter experience over 13 years.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by deficiency of the galactocerebrosidase (GALC) due to variants in the GALC gene. Here, we provide the first and the largest comprehensive analysis of clinical and genetic characteristics, and genotype-phenotype correlations of KD in Korean in comparison with other ethnic groups. From June 2010 to June 2023, 10 patients were diagnosed with KD through sequencing of GALC. Clinical features, and results of GALC sequencing, biochemical test, neuroimaging, and neurophysiologic test were obtained from medical records. An additional nine previously reported Korean KD patients were included for review. In Korean KD patients, the median age of onset was 2 years (3 months-34 years) and the most common phenotype was adult-onset (33%, 6/18) KD, followed by infantile KD (28%, 5/18). The most frequent variants were c.683_694delinsCTC (23%) and c.1901T>C (23%), while the 30-kb deletion was absent. Having two heterozygous pathogenic missense variants was associated with later-onset phenotype. Clinical features were similar to those of other ethnic groups. In Korean KD patients, the most common phenotype was the adult-onset type and the GALC variant spectrum was different from that of the Caucasian population. This study would further our understanding of KD.

Epigallocatechin-3-gallate restores mitochondrial homeostasis impairment by inhibiting HDAC1-mediated NRF1 histone deacetylation in cardiac hypertrophy.

Decompensated cardiac hypertrophy is accompanied by impaired mitochondrial homeostasis, whether histone acetylation is involved in this process is yet to be determined. The role of HDAC1-mediated NRF1 histone deacetylation was investigated in transverse aortic constriction (TAC)-induced hypertrophy in rats and phenylephrine (PE)-induced hypertrophic cardiomyocytes. Administration of epigallocatechin-3-gallate (EGCG), an inhibitor of HDAC1, restored cardiac function, decreased heart/body weight and fibrosis, increased the ratio of mtDNA/nDNA and the percentage of LysoTracker+ CMs in TAC, compared with TAC without receiving EGCG. In PE-treated hypertrophic H9C2 cells, EGCG attenuated cell hypertrophy and increased LC3B II+MitoTracker+ puncta, as well as the ratio of mtDNA/nDNA. Interestingly, NRF1 but not PGC-1α expression was decreased in TAC- or PE-induced hypertrophic hearts or cells, respectively, while EGCG upregulated both NRF1 and PGC-1α in vitro. EGCG treatment also increased the interaction between PGC-1α and NRF1. In addition to inhibiting HDAC1 expression, EGCG decreased the binding of HDAC1 and increased the binding of acH3K9 or acH3K14 in the promotor regions of PGC-1α and NRF1. In neonatal rat cardiomyocytes, restored NRF1, TFAM and FUNDC1 were abolished by the overexpression of HDAC1. Collectively, data suggest that NRF1 reduction was averted by EGCG via inhibiting HDAC1-mediated histone deacetylation. Acetylation of NRF1 histone may play a key role in maintaining mitochondrial homeostasis associated with cardiac hypertrophy.

Striated preferentially expressed gene deficiency leads to mitochondrial dysfunction in developing cardiomyocytes.

A deficiency of striated preferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg-/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg-/- hearts. Imaging of E18.5 Speg-/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg-/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg-/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α.

Autophagy mediates an amplification loop during ferroptosis.

Ferroptosis, a programmed cell death, has been identified and associated with cancer and various other diseases. Ferroptosis is defined as a reactive oxygen species (ROS)-dependent cell death related to iron accumulation and lipid peroxidation, which is different from apoptosis, necrosis, autophagy, and other forms of cell death. However, accumulating evidence has revealed a link between autophagy and ferroptosis at the molecular level and has suggested that autophagy is involved in regulating the accumulation of iron-dependent lipid peroxidation and ROS during ferroptosis. Understanding the roles and pathophysiological processes of autophagy during ferroptosis may provide effective strategies for the treatment of ferroptosis-related diseases. In this review, we summarize the current knowledge regarding the regulatory mechanisms underlying ferroptosis, including iron and lipid metabolism, and its association with the autophagy pathway. In addition, we discuss the contribution of autophagy to ferroptosis and elucidate the role of autophagy as a ferroptosis enhancer during ROS-dependent ferroptosis.

Diagnostic Performance of Interferon Gamma Release Assay (IGRA) for Cell-Mediated Immune Responses to SARS-CoV-2.

BACKGROUND: As SARS-CoV-2 infection became a pandemic, much effort has been made to measure both antibody production and T cell response to SARS-CoV-2 to diagnose COVID-19 patients or find out their immune status. Authors tried to determine the optimal cutoff value and evaluate clinical performance of one interferon-γ release assay (IGRA) kit and compared their results with serological antibody assay in COVID-19 patients. METHODS: Study subjects included 100 patients confirmed as COVID-19 with RT-PCR method and 88 healthy volunteers who were PCR negative. IGRA tests were performed using STANDARDTM E Covi-FERON ELISA. Presence of SARS-CoV-2 antibodies was detected using STANDARD Q COVID-19 IgM/IgG Plus Test. Cutoff value was assessed using receiver operating characteristic (ROC) curve. RESULTS: The cutoff value was 0.24 IU/mL and the area under the curve (AUC) of the ROC curve was 0.973 with 95% confidence interval (CI) of 0.940 - 1.005. At this cutoff value, sensitivity and specificity were 91.7% and 100%, respectively. In addition, when compared with antibody test, concordance rate was 95%. CONCLUSIONS: STANDARDTM E Covi-FERON ELISA showed high sensitivity and specificity, when the cutoff value was 0.24 IU/mL. It was also consistent with the antibody test. IGRA test was a good indicator of cellular immune response in COVID-19 patients.

Evaluation of two SARS-CoV-2 IgG/IgM Rapid Tests in Capillary Blood Samples.

BACKGROUND: As COVID-19 has spread rapidly around the world, it has become essential to detect the virus quickly and accurately for disease prevention and control. Therefore, in response to the COVID-19 pandemic, the need for rapid serological point-of-care test has increased. Recently, many antibody tests have been developed to detect IgM and/or IgG to SARS-CoV-2 in human blood. The authors conducted a prospective study to evaluate the performance of a rapid chromatographic immunoassay and a fluorescent immunoassay for the qualitative detection of specific antibodies, IgM and IgG to SARS-CoV-2 in capillary blood samples, compared to the real-time RT-PCR. METHODS: The subjects included 70 patients who were confirmed positive by real-time RT-PCR and 70 people who were negative. STANDARD Q COVID-19 IgM/IgG Plus Test (chromatographic immunoassay) and Fluorescent immunoassay for IgM and IgG to SARS-CoV-2 (fluorescent immunoassay) were performed using capillary blood samples. Based on the results of real-time RT-PCR assay, clinical sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of two rapid tests were investigated. And the agreement rate between two rapid tests was also presented. RESULTS: Sensitivity, specificity, PPV and NPV of the chromatographic immunoassay were 82.9%, 98.6%, 98.3%, and 85.2%, respectively. At more than 7 days after the onset of symptoms, sensitivity increased to 87.3%. Sensitivity, specificity, PPV, and NPV were 81.4%, 100.0%, 100.0%, and 84.3%, respectively, for the fluorescent immunoassay. At more than 7 days after the onset of symptoms, sensitivity increased to 85.7%. The agreement rate of the two tests was 97.1%. CONCLUSIONS: STANDARD Q COVID-19 IgM/IgG Plus Test and STANDARD F COVID-19 IgM/IgG Combo FIA turned out very specific and sensitive enough to detect individuals infected to SARS-CoV-2. Also, these tests were simple, fast, visually interpretable, and required a small amount of capillary whole blood.

Comprehensive Evaluation of the NeoBase 2 Non-derivatized MSMS Assay and Exploration of Analytes With Significantly Different Concentrations Between Term and Preterm Neonates.

Background: Despite the popularity of the NeoBase 2 Non-derivatized MSMS assay (PerkinElmer, Turku, Finland), there are no reports of its comprehensive evaluation, including the ability to distinguish transient tyrosinemia of the newborn (TTN) from tyrosinemia type 1 (TYR 1) using succinylacetone (SUAC). No newborn screening (NBS) cutoffs for preterm neonates in the Korean population have been suggested. We evaluated the NeoBase 2 assay and identified analytes requiring different cutoffs in preterm neonates. Methods: Residual NBS dried blood spot samples and proficiency testing (PT) materials of the Newborn Screening Quality Assurance Program and the Korean Association of External Quality Assessment Service were used. Precision, accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), linearity, recovery, carryover, and performance of SUAC were evaluated. Cutoffs were determined, and analytes requiring different cutoffs in preterm neonates were investigated. Results: Mean CVs for within-run and between-day precision were within 15%. Accuracy analysis indicated high agreement with in-house derivatized assay results and results of other PT participants. All analytes demonstrated acceptable LOD, LLOQ, and linearity. Recoveries were acceptable, except for SUAC. Carryover was negligible. Cutoffs were established for all analytes; Tyr, adenosine, and C20:0-lysophosphatidylcholine required different cutoffs in preterm neonates. Differential diagnosis of TYR 1 and TTN was successful with simultaneous Tyr and SUAC measurement. Conclusions: The NeoBase 2 assay demonstrated satisfactory performance. The additional analytes provide a wider diagnostic coverage, and the simultaneous measurement of Tyr and SUAC is efficient in excluding TYR 1. The new cutoffs for preterm neonates may decrease false-positive rates, without compromising diagnostic sensitivity.

Severe congenital neutropenia mimicking chronic idiopathic neutropenia: a case report.

Severe chronic neutropenia is classified as severe congenital, cyclic, autoimmune, or idiopathic. However, there is a lot of uncertainty regarding the diagnosis of severe congenital neutropenia (SCN) and chronic idiopathic neutropenia, and this uncertainty affects further evaluations and treatments. A 20-year-old man presented with fever and knee abrasions after a bicycle accident. On admission, his initial absolute neutrophil count (ANC) was 30/µL. He had no medical history of persistent severe neutropenia with periodic oscillation of ANC. Although his fever resolved after appropriate antibiotic therapy, ANC remained at 80/µL. Bone marrow (BM) aspiration and biopsy were performed, and a BM smear showed myeloid maturation arrest. Moreover, genetic mutation test results showed a heterozygous missense variant in exon 4 of the neutrophil elastase ELANE: c597+1G>C (pV190-F199del). The patient was diagnosed with SCN. After discharge, we routinely checked his ANC level and monitored any signs of infection with minimum use of granulocyte colony-stimulating factor (G-CSF), considering its potential risk of leukemic transformation. Considering that SCN can be fatal, timely diagnosis and appropriate management with G-CSF are essential. We report the case of a patient with SCN caused by ELANE mutation who had atypical clinical manifestations. For a more accurate diagnosis and treatment of severe chronic neutropenia, further studies are needed to elucidate the various clinical features of ELANE.

Prevalence of Group B Streptococcus Colonization in Pregnant Women at a University Hospital in Korea.

BACKGROUND: Group B Streptococcus (GBS) colonization in pregnant women is a risk factor for causing infection in neonates; therefore, GBS screening tests are performed on them. Culture methods and molecular diagnostics are mainly performed for GBS detection; however, culture methods differ in the detection rate for GBS depending on the procedure of culture. The authors intended to confirm the difference in GBS colonization rate in the conventional culture method, enrichment culture method, and molecular genetic test as screening tests for GBS. METHODS: Duplicate vagino-rectal swabs were collected from 371 pregnant women between the 35th and 37th week of gestation; one was used for conventional culture method and the other was frozen at -80℃, followed by enrichment culture method and molecular genetic test. RESULTS: The prevalence of GBS colonization identified by conventional culture, enrichment culture, and molecular genetic test was 4.35% (17/391), 8.95% (35/391), and 22.25% (87/391), respectively. The detection rate by enrichment culture method was 2.06 times higher (17/391 vs. 35/391) than that by conventional culture method. It was identified that there was a significant difference in the detection rates of GBS between the two methods (p < 0.001). The detection rate identified in molecular genetic test was much higher at 22.25% (87/391). The concordance rate of the results from three detection methods for GBS was 80.05% (313/391). All pregnant women colonized with GBS were given intrapartum antibiotic prophylaxis using cefazolin and their neonates were confirmed not to be infected with GBS. CONCLUSIONS: Prevalence of GBS colonization in pregnant women is shown to vary depending on detection method. Particularly, it differs greatly depending on the use of enrichment media in the culture method. Therefore, it is necessary that the microbiological laboratory implements the culture method with supplementary procedures such as selective or enrichment media in order to improve the detection rate of GBS.

Changes in the Distribution of Respiratory Microorganisms before and during the COVID-19 Pandemic, Daegu, Korea.

BACKGROUND: Acute respiratory infection (ARI) is the most common infectious disease in all ages and genders worldwide. Respiratory microorganisms such as respiratory viruses, are commonly responsible for causing ARI. COVID-19 is still prevalent in Korea. The implementation of lockdown and strict control measures, the mandatory wearing of masks, and social distancing are critical steps for controlling the risk of COVID-19 spread. This study was conducted to find out how these changes in daily lives impacted the distribution of respiratory microorganisms. METHODS: A retrospective study was conducted to identify the incidence and distribution patterns of ARI-causing respiratory microorganisms before (Period Ⅰ) and during the COVID-19 pandemic (Period Ⅱ) in terms of detection method, age, month, and season. In particular, data in Periods Ⅰ and Ⅱ were compared for eight major kinds of respiratory microorganisms: adenovirus (AdV), human metapneumovirus (HMPV), human rhinovirus/enterovirus (Rhino/Entero), influenza virus (Flu) A, Flu B, human parainfluenza virus (HPIV) 3, respiratory syncytial virus, and Mycoplasma pneumoniae. RESULTS: A total of 27,191 respiratory specimens were tested, of which 5,513 were obtained from children and adolescents (age groups 1 ⁓ 5) and 21,678 from adults (age group 6). The overall positive rates for at least one respiratory microorganism in Periods Ⅰ and Ⅱ were 23.1% (1,199/5,193) and 4.9% (1,070/21,998), respectively (p < 0.001). The overall positive rates in male and female patients were significantly different (8.7% vs. 7.9%; p = 0.016). On the FilmArray™ RP assay, positive rates in all age groups decreased significantly in Period Ⅱ compared with Period Ⅰ. AdV, Rhino/Entero, and Flu A were detected in all four seasons, but HMPV and HPIV3 were not detected. The overall positive rates on FilmArray and the Flu antigen test in Period Ⅱ were significantly decreased. In the COVID-19 test, the positive rates were high in March and April 2020, and decreased thereafter, but these increased again in the winter of 2020/2021. CONCLUSIONS: Life changes due to COVID-19 pandemic have had a significant impact on the distribution of respiratory microorganisms; our study results might provide useful information on respiratory virus epidemiology.

Mesenchymal stromal cell-derived syndecan-2 regulates the immune response during sepsis to foster bacterial clearance and resolution of inflammation.

Sepsis is a life-threatening process related to a dysregulated host response to an underlying infection, which results in organ dysfunction and poor outcomes. Therapeutic strategies using mesenchymal stromal cells (MSCs) are under investigation for sepsis, with efforts to improve cellular utility. Syndecan (SDC) proteins are transmembrane proteoglycans involved with cellular signaling events including tissue repair and modulating inflammation. Bone marrow-derived human MSCs express syndecan-2 (SDC2) at a level higher than other SDC family members; thus, we explored SDC2 in MSC function. Administration of human MSCs silenced for SDC2 in experimental sepsis resulted in decreased bacterial clearance, and increased tissue injury and mortality compared with wild-type MSCs. These findings were associated with a loss of resolution of inflammation in the peritoneal cavity, and higher levels of proinflammatory mediators in organs. MSCs silenced for SDC2 had a decreased ability to promote phagocytosis of apoptotic neutrophils by macrophages in the peritoneum, and also a diminished capability to convert macrophages from a proinflammatory to a proresolution phenotype via cellular or paracrine actions. Extracellular vesicles are a paracrine effector of MSCs that may contribute to resolution of inflammation, and their production was dramatically reduced in SDC2-silenced human MSCs. Collectively, these data demonstrate the importance of SDC2 for cellular and paracrine function of human MSCs during sepsis.

Patients presenting high fever with lymphadenopathy after COVID-19 vaccination were diagnosed with hemophagocytic lymphohistiocytosis.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still continuing worldwide. Currently, two mRNA-based vaccines and two DNA vaccines using an adenovirus vector are representative vaccines. Since the SARS-CoV-2 vaccines began to be administered, a significant decrease in new infections and COVID-19-associated death has been reported. However, various adverse events from mild symptoms to death have also been described after vaccination. CASE DESCRIPTION: Patients with high fever and lymphadenopathy who are diagnosed with hemophagocytic lymphohistiocytosis (HLH) after COVID-19 vaccination are very rare, and there is no standard management guideline for these patients thus far. Herein, we described two cases of HLH after the administration of an mRNA-based vaccine and adenovirus vector vaccine. DISCUSSION: HLH is a life-threatening hyperinflammatory syndrome that occurs due to persistent stimulation of lymphocytes and histiocytes in various underlying conditions at all ages. Although the exact mechanisms and risk factors of COVID-19 vaccination-related HLH are still unknown, vigorous immune stimulation may trigger a huge cytokine storm, rarely resulting in HLH. It is important to note that early suspicion by clinicians can lower the mortality rate.

Prenatal diagnosis of combined methylmalonic acidemia and homocystinuria cobalamin C type using clinical exome sequencing and targeted gene analysis.

BACKGROUND: Combined methylmalonic acidemia and homocystinuria is a rare inherited disorder of intracellular cobalamin metabolism caused by biallelic variants in one of the following genes: MMACHC (cblC), MMADHC (cblD), LMBRD1 (cblF), ABCD4 (cblJ), THAP11 (cblX-like), and ZNF143 (cblX-like), or a hemizygous variant in HCFC1 (cblX). Prenatal diagnosis of combined methylmalonic acidemia with homocystinuria is crucial for high-risk couples since the disorder can be life-threatening for offspring. We would like to describe two infant deaths both of which are likely attributable to cblC despite not having a genetic confirmation, and subsequent pregnancy and prenatal genetic testing. METHODS: Parental clinical exome sequencing and targeted Sanger sequencing of MMACHC gene in amniotic fluid was performed to check the carrier status of the fetus. RESULTS: Parental clinical exome sequencing revealed a heterozygous pathogenic variant [NM_015506.2:c.217C>T (p.Arg73*)] in the MMACHC gene of the mother and [NM_015506.2:c.609G>A (p.Trp203*)] in the MMACHC gene of the father. Targeted Sanger sequencing of MMACHC gene in amniotic fluid revealed that the fetus carried only one nonsense variant [NM_015506.2:c.609G>A (p.Trp203*)], which was inherited from the father. The mother delivered a healthy baby and the neonate did not show any symptoms or signs of combined methylmalonic acidemia and homocystinuria after birth. CONCLUSION: We present a case of prenatal diagnosis with parental exome sequencing, which successfully diagnosed the carrier status of the fetus and parents in a combined methylmalonic acidemia and homocystinuria family.

Application of an LC-MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.

Sulfasalazine attenuates tamoxifen-induced toxicity in human retinal pigment epithelial cells.

Tamoxifen, a nonsteroidal estrogen receptor (ER) antagonist, is used routinely as a chemotherapeutic agent for ER-positive breast cancer. However, it is also causes side effects, including retinotoxicity. The retinal pigment epithelium (RPE) has been recognized as the primary target of tamoxifen-induced retinotoxicity. The RPE plays an essential physiological role in the normal functioning of the retina. Nonetheless, potential therapeutic agents to prevent tamoxifen-induced retinotoxicity in breast cancer patients have not been investigated. Here, we evaluated the action mechanisms of sulfasalazine against tamoxifen- induced RPE cell death. Tamoxifen induced reactive oxygen species (ROS)-mediated autophagic cell death and caspase-1-mediated pyroptosis in RPE cells. However, sulfasalazine reduced tamoxifen-induced total ROS and ROS-mediated autophagic RPE cell death. Also, mRNA levels of tamoxifen-induced pyroptosis-related genes, IL-1ß, NLRP3, and procaspase-1, also decreased in the presence of sulfasalazine in RPE cells. Additionally, the mRNA levels of tamoxifen-induced AMD-related genes, such as complement factor I (CFI), complement factor H (CFH), apolipoprotein E (APOE), apolipoprotein J (APOJ), toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4), were downregulated in RPE cells. Together, these data provide novel insight into the therapeutic effects of sulfasalazine against tamoxifen-induced RPE cell death. [BMB Reports 2020; 53(5): 284-289].

Nucleotide-binding oligomerization domain protein 2 deficiency enhances CHOP expression and plaque necrosis in advanced atherosclerotic lesions.

Endoplasmic reticulum (ER) stress-induced cell death of vascular smooth muscle cells (VSMCs) is extensively involved in atherosclerotic plaque stabilization. We previously reported that nucleotide-binding oligomerization domain protein 2 (NOD2) participated in vascular homeostasis and tissue injury. However, the role and underlying mechanisms of NOD2 remain unknown in ER stress-induced cell death of VSMC during vascular diseases, including advanced atherosclerosis. Here, we report that NOD2 specifically interacted with ER stress sensor activating transcription factor 6 (ATF6) and suppressed the expression of proapoptotic transcription factor CHOP (C/EBP homologous protein) during ER stress. CHOP-positive cells were increased in neointimal lesions after femoral artery injury in NOD2-deficient mice. In particular, a NOD2 ligand, MDP, and overexpression of NOD2 decreased CHOP expression in wild-type VSMCs. NOD2 interacted with an ER stress sensor molecule, ATF6, and acted as a negative regulator for ATF6 activation and its downstream target molecule, CHOP, that regulates ER stress-induced apoptosis. Moreover, NOD2 deficiency promoted disruption of advanced atherosclerotic lesions and CHOP expression in NOD2-/- ApoE-/- mice. Our findings indicate an unsuspected critical role for NOD2 in ER stress-induced cell death.

Myeloperoxidase Deficiency Manifesting as Pseudoneutropenia with Low Mean Peroxidase Index and High Monocyte Count in 4 Adult Patients.

Myeloperoxidase (MPO) deficiency, one of the most common inherited phagocyte defects, and may exist as a transient phenomenon in combination with some clinical condition. Hematological analyzer ADVIA 2120i is used to identify the different types of leukocytes based on their size and staining properties, and by mean peroxidase index (MPXI). When MPO deficiency is present, neutrophils may be incorrectly counted as monocytes with lower MPXI values. We encountered a few cases of MPO deficiency with abnormally high monocytes counts resulting in pseudoneutropenia. These abnormal reports could lead to a mistaken diagnosis of severe neutropenia, which could result in unnecessary therapy. Manual differential count exhibited the normal differential count in every case. Every case yielded a markedly low MPXI value below -20. In conclusion, we suggest that MPO deficiency must be considered in patients especially when abnormally high monocyte counts combined with low MPXI values are observed.

Oxidative Stress-Induced Pentraxin 3 Expression Human Retinal Pigment Epithelial Cells is Involved in the Pathogenesis of Age-Related Macular Degeneration.

: (1) Background: Age-related macular degeneration (AMD) is closely related with retinal pigment epithelial (RPE) cell dysfunction. Although the exact pathogenesis of AMD remains largely unknown, oxidative stress-induced RPE damage is believed to be one of the primary causes. We investigated the molecular mechanisms of pentraxin 3 (PTX3) expression and its biological functions during oxidative injury. (2) Methods: Using enzyme-linked immunosorbent assays and real-time reverse transcription-polymerase chain reaction, we analyzed mRNA and protein levels of PTX3 in the presence or absence of oxidative stress inducer, sodium iodate (NaIO3), in primary human H-RPE and ARPE-19 cells. Furthermore, we assessed cell death, antioxidant enzyme expression, and AMD-associated gene expression to determine the biological functions of PTX3 under oxidative stress. (3) Results: NaIO3 increased PTX3 expression, in a dose- and time-dependent manner, in H-RPE and ARPE-19 cells. We found phosphorylated Akt, a downstream target of the PI3 kinase pathway, phosphor- mitogen-activated protein kinase kinase 1/2 (ERK), and intracellular reactive oxygen species (ROS) were predominantly induced by NaIO3. NaIO3-induced PTX3 expression was decreased in the presence of phosphoinositide 3 (PI3) kinase inhibitors, ERK inhibitors, and ROS scavengers. Furthermore, NaIO3 enhanced mRNA expression of antioxidant enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), catalase (CAT), and glutathione S-reductase (GSR) in the control shRNA expressing RPE cells, but not in hPTX3 shRNA expressing RPE cells. Interestingly, NaIO3 did not induce mRNA expression of AMD marker genes, such as complement factor I (CFI), complement factor H (CFH), apolipoprotein E (APOE), and toll-like receptor 4 (TLR4) in hPTX3 shRNA expressing RPE cells. 4) Conclusions: These results suggest that PTX3 accelerates RPE cell death and might be involved in AMD development in the presence of oxidative stress.