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Non-canonical autophagy pathways decorate single-membrane vesicles with Atg8-family proteins such as MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Phagosomes containing the bacterial pathogen Listeria monocytogenes (L.m.) can be targeted by a non-canonical autophagy pathway called LC3-associated phagocytosis (LAP), which substantially contributes to the anti-listerial activity of macrophages and immunity. We here characterized a second non-canonical autophagy pathway targeting L.m.-containing phagosomes, which is induced by damage caused to the phagosomal membrane by the pore-forming toxin of L.m., listeriolysin O. This pore-forming toxin-induced non-canonical autophagy pathway (PINCA) was the only autophagic pathway evoked in tissue macrophages deficient for the NADPH oxidase CYBB/NOX2 that produces the reactive oxygen species (ROS) that are required for LAP induction. Similarly, also bone marrow-derived macrophages (BMDM) exclusively targeted L.m. by PINCA as they completely failed to induce LAP because of insufficient production of ROS through CYBB, in part, due to low expression of some CYBB complex subunits. Priming of BMDM with proinflammatory cytokines such as TNF and IFNG/IFNγ increased ROS production by CYBB and endowed them with the ability to target L.m. by LAP. Targeting of L.m. by LAP remained relatively rare, though, preventing LAP from substantially contributing to the anti-listerial activity of BMDM. Similar to LAP, the targeting of L.m.-containing phagosomes by PINCA promoted their fusion with lysosomes. Surprisingly, however, this did not substantially contribute to anti-listerial activity of BMDM. Thus, in contrast to LAP, PINCA does not have clear anti-listerial function suggesting that the two different non-canonical autophagy pathways targeting L.m. may have discrete functions. Abbreviations: actA/ActA: actin assembly-inducing protein A; ATG: autophagy-related; BMDM: Bone marrow-derived macrophages; CALCOCO2/NDP52: calcium-binding and coiled-coil domain-containing protein 2; CYBA/p22phox: cytochrome b-245 light chain; CYBB/NOX2: cytochrome b(558) subunit beta; E. coli: Escherichia coli; IFNG/IFNγ: interferon gamma; L.m.: Listeria monocytogenes; LAP: LC3-associated phagocytosis; LGALS: galectin; LLO: listeriolysin O; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NCF1/p47phox: neutrophil cytosol factor 1; NCF2/p67phox: neutrophil cytosol factor 2; NCF4/p67phox: neutrophil cytosol factor 4; Peritoneal macrophages: PM; PINCA: pore-forming toxin-induced non-canonical autophagy; plc/PLC: 1-phosphatidylinositol phosphodiesterase; PMA: phorbol 12-myristate 13-acetate; RB1CC1/FIP200: RB1-inducible coiled-coil protein 1; ROS: reactive oxygen species; S. aureus: Staphylococcus aureus; S. flexneri: Shigella flexneri; SQSTM1/p62: sequestosome 1; S. typhimurium: Salmonella typhimurium; T3SS: type III secretion system; TNF: tumor necrosis factor; ULK: unc-51 like autophagy activating kinase; PM: peritoneal macrophages; WT: wild type.

Background: Chronic granulomatous disease (CGD), one of the phagocytic system defects, is the primary immunodeficiency caused by dysfunction of the NADPH oxidase complex which generates reactive oxygen species (ROS), which are essential for killing pathogenic microorganisms, especially catalase-positive bacteria and fungi. Objective: The objective of our study was to assess the clinical and laboratory characteristics, treatment modalities, and prognosis of patients with CGD. Methods: We retrospectively reviewed 63 patients with CGD who have been diagnosed, treated, and/or followed-up between 1984 and 2018 in Hacettepe University, Ankara, in Turkey, as a developing country. Results: The number of female and male patients was 26/37. The median age at diagnosis was 3.8 (IQR: 1.0–9.6) years. The rate of consanguinity was 63.5%. The most common physical examination finding was lymphadenopathy (44/63), growth retardation (33/63), and hepatomegaly (27/63). One adult patient had squamous cell carcinoma of the lung. The most common infections were lung infection (53/63), skin abscess (43/63), and lymphadenitis (19/63). Of the 63 patients with CGD, 6 patients had inflammatory bowel disease (IBD). Twelve of the 63 patients died during follow-up. CYBA, NCF1, CYBB, and NCF2 mutations were detected in 35%, 27.5%, 25%, and 12.5% of the patients, respectively. Conclusion: We identified 63 patients with CGD from a single center in Turkey. Unlike other cohort studies in Turkey, due to the high consanguineous marriage rate in our study group, AR form of CGD was more frequent, and gastrointestinal involvement were found at relatively lower rates. The rate of patients who treated with HSCT was lower in our research than in the literature. A majority of the patients in this study received conventional prophylactic therapies, which highlight on the outcome of individuals who have not undergone HSCT.

Myelodysplastic syndrome with myelofibrosis (MDS-MF) has been associated with an inferior prognosis compared with MDS without MF. However, MDS-MF is not listed independently as a subtype of MDS, and its clinical and genetic characteristics remain poorly understood. We retrospectively compared 53 patients with MDS-MF (44 MF grade 1/MF1; 9 MF grade 2–3/MF2 − 3) and 31 with de novo MDS without MF (MDS). The leukemic transformation risks of both MDS-MF2 − 3 and MDS-MF1 were increased compared with the MDS group. To identify the potential mechanisms responsible for the leukemic transformation of MDS-MF, we performed single-cell sequencing for one MDS-MF2 − 3 patient before and after leukemic transformation to explore the variations in gene expression levels. In addition to upgraded expression levels of acute myeloid leukemia-related genes during leukemic transformation, expression levels of some inflammation-related genes (such as S100s, RNASE3, and CYBB) were also increased, and inflammation-related pathways were up-regulated. These results suggest that inflammation-related genes and pathways may play an important role in the leukemic transformation of MDS-MF.

BACKGROUND AND PURPOSE Skeletal muscle dysfunction is a major comorbidity of chronic obstructive pulmonary disease (COPD). This type of muscle dysfunction may be a direct consequence of oxidative insults evoked by cigarette smoke (CS) exposure. The present study examined the effects of a potent Nox inhibitor and reactive oxygen species (ROS) scavenger, apocynin, on CS-induced muscle dysfunction. EXPERIMENTAL APPROACH Male BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks, with or without the coadministration of apocynin (5 mg·kg-1 , i.p.). C2C12 myotubes exposed to either hydrogen peroxide (H2 O2 ) or water-soluble cigarette smoke extract (CSE) with or without apocynin (500 nM) were used as an experimental model in vitro. KEY RESULTS Eight weeks of CS exposure caused muscle dysfunction in mice, reflected by 10% loss of muscle mass and 54% loss of strength of tibialis anterior which were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2 O2 or CSE caused myofibre wasting, accompanied by ~50% loss of muscle-derived insulin-like growth factor (IGF)-1 and two-fold induction of Cybb, independent of cellular inflammation. Expression of myostatin and MAFbx, negative regulators of muscle mass, were up-regulated under H2 O2 but not CSE conditions. Apocynin treatment abolished CSE-induced Cybb expression, preserving muscle-derived IGF-1 expression and signalling pathway downstream of mammalian target of rapamycin (mTOR), thereby preventing myofibre wasting. CONCLUSION AND IMPLICATIONS Targeted pharmacological inhibition of Nox-derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.

Introduction Early-onset preeclampsia is a pregnancy complication associated with high maternal and perinatal morbidity, mortality. Intense efforts have been made to elucidate the pathogenesis, but the molecular mechanism is still elusive. This study aimed to identify potential key genes related to early-onset preeclampsia, and to obtain a better understanding of the molecular mechanisms of this disease. Methods We performed a multi-step integrative bioinformatics analysis of microarray dataset GSE74341 downloaded from Gene Expression Omnibus (GEO) database including 7 early-onset preeclampsia and 5 gestational age matched normotensive controls. The differentially expressed genes (DEGs) were identified using the “limma” package, and their potential functions were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Furthermore, the protein–protein interaction network (PPI) was obtained from the STRING database and the PPI network was visualized by Cytoscape software. Then, hub modules and hub genes were screened out from the PPI network, and enrichment analysis was performed for them. Also, validation of hub genes expression in early-onset PE was down by using microarray dataset GSE44711. Results A total of 628 DEGs (256 down- and 372 up-regulated) were identified in early-onset PE compared to controls. A total of 4 significant hub modules and 26 significant hub genes were identified. Conclusion In conclusion, the DEGs related to cell-cell or cell-extracellular matrix interaction (ITGA5, SPP1, LUM, VCAN, APP), placenta metabolic or oxidative stress (CCR7, NT5E, CYBB) were predicted to be newly potential crucial genes that may play significant roles in the pathogenesis of early-onset PE.

Female carriers with X-linked chronic granulomatous disease (XL-CGD) who have

Accurate diagnosis of complete inactivation of tuberculosis lesions is still a challenge with respect to sputum-negative tuberculosis. RNA-sequencing was conducted to uncover potential lncRNA indicators of metabolic activity in tuberculosis lesions. Lung tissues with high metabolic activity and low metabolic activity demonstrated by fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography were collected from five sputum-negative tuberculosis patients for RNA-sequencing. Differentially-expressed mRNAs and lncRNAs were identified. Their correlations were evaluated to construct lncRNA-mRNA co-expression network, in which lncRNAs and mRNAs with high degrees were confirmed by quantitative real-time PCR using samples collected from 11 patients. Prediction efficiencies of lncRNA indicators were assessed by receiver operating characteristic curve analysis. Bioinformatics analysis was performed for potential lncRNAs. 386 mRNAs and 44 lncRNAs were identified to be differentially expressed. Differentially-expressed mRNAs in lncRNA-mRNA co-expression network were significantly associated with fibrillar collagen, platelet-derived growth factor binding, and leukocyte migration involved in inflammatory response. Seven mRNAs (C1QB, CD68, CCL5, CCL19, MMP7, HLA-DMB, and CYBB) and two lncRNAs (ENST00000429730.1 and MSTRG.93125.4) were validated to be significantly up-regulated. The area under the curve of ENST00000429730.1 and MSTRG.93125.4 was 0.750 and 0.813, respectively. Two lncRNAs ENST00000429730.1 and MSTRG.93125.4 might be considered as potential indicators of metabolic activity in tuberculosis lesions for sputum-negative tuberculosis.

Glioblastoma multiforme (GBM) is a highly heterogeneous disease with a mesenchymal subtype tending to exhibit more aggressive and multitherapy-resistant features. Glioblastoma stem-cells derived from mesenchymal cells are reliant on iron supply, accumulated with high reactive oxygen species (ROS), and susceptible to ferroptosis. Temozolomide (TMZ) treatment is the mainstay drug for GBM despite the rapid development of resistance in mesenchymal GBM. The main interconnection between mesenchymal features, TMZ resistance, and ferroptosis are poorly understood. Herein, we demonstrated that a subunit of NADPH oxidase, CYBB, orchestrated mesenchymal shift and promoted TMZ resistance by modulating the anti-ferroptosis circuitry Nrf2/SOD2 axis. Public transcriptomic data re-analysis found that CYBB and SOD2 were highly upregulated in the mesenchymal subtype of GBM. Accordingly, our GBM cohort confirmed a high expression of CYBB in the GBM tumor and was associated with mesenchymal features and poor clinical outcome. An in vitro study demonstrated that TMZ-resistant GBM cells displayed mesenchymal and stemness features while remaining resilient to erastin-mediated ferroptosis by activating the CYBB/Nrf2/SOD2 axis. The CYBB maintained a high ROS state to sustain the mesenchymal phenotype, TMZ resistance, and reduced erastin sensitivity. Mechanistically, CYBB interacted with Nrf2 and consequently regulated SOD2 transcription. Compensatory antioxidant SOD2 essentially protected against the deleterious effect of high ROS while attenuating ferroptosis in TMZ-resistant cells. An animal study highlighted the protective role of SOD2 to mitigate erastin-triggered ferroptosis and tolerate oxidative stress burden in mice harboring TMZ-resistant GBM cell xenografts. Therefore, CYBB captured ferroptosis resilience in mesenchymal GBM. The downstream compensatory activity of CYBB via the Nrf2/SOD2 axis is exploitable through erastin-induced ferroptosis to overcome TMZ resistance.

Most of known primary immunodeficiencies (PID) are monogenic diseases, mainly due to the point defects in the immune system genes. However, in some rare cases the immunodeficiency is caused by various chromosomal aberrations. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. A retrospective analysis was performed on a group of 15 patients with clinical and laboratory signs of immunodeficiency, whose chromosomal aberrations were detected by various methods. In six patients from four different families, microdeletions with inclusion of a gene ( CTLA4 , NFKB1 ) or a part of a known PID gene ( NBAS , DCLRE1C ) were detected. Four patients had a complex phenotype, where a mutation in the known PID genes ( BTK , CYBB , STAT1 GOF , ATM ) was combined with a chromosomal defect. In one case, a homozygous damage in the USB1 gene was confirmed by detection of dysomy chromosome 16 from the father`s side. Two patients were diagnosed with known chromosomal defects – DiGeorge2 and Jacobsen syndrome. Two other patients had various chromosome abnormalities not previously described in PID`s patients. 12/15 (80%) patients had syndromic features – various skeletal dysmorphisms, malformations, and developmental delay. Immunodeficiency genes can be damaged within the chromosomal aberrations. The combination of the various methods of genetic testing is important for patients with PID. Even in PID patients without syndromic features the chromosomal analysis methods or PID diagnosis are necessary.

Age is a critical risk factor for many neurologic conditions, including progressive multiple sclerosis. Yet the mechanisms underlying the relationship are unknown. Using lysolecithin-induced demyelinating injury to the mouse spinal cord, we characterized the acute lesion and investigated the mechanisms of increased myelin and axon damage with age. We report exacerbated myelin and axon loss in middle-aged (8–10 months of age) compared with young (6 weeks of age) female C57BL/6 mice by 1–3 d of lesion evolution in the white matter. Transcriptomic analysis linked elevated injury to increased expression ofCybb, the gene encoding the catalytic subunit of NADPH oxidase gp91phox. Immunohistochemistry in male and femaleCx3cr1CreER/+:Rosa26tdTom/+mice for gp91phox revealed that the upregulation in middle-aged animals occurred primarily in microglia and not infiltrated monocyte-derived macrophages. Activated NADPH oxidase generates reactive oxygen species and elevated oxidative damage was corroborated by higher malondialdehyde immunoreactivity in lesions from middle-aged compared with young mice. From a previously conducted screen for generic drugs with antioxidant properties, we selected the antihypertensive CNS-penetrant medication indapamide for investigation. We report that indapamide reduced superoxide derived from microglia cultures and that treatment of middle-aged mice with indapamide was associated with a decrease in age-exacerbated lipid peroxidation, demyelination and axon loss. In summary, age-exacerbated acute injury following lysolecithin administration is mediated in part by microglia NADPH oxidase activation, and this is alleviated by the CNS-penetrant antioxidant, indapamide.SIGNIFICANCE STATEMENTAge is associated with an increased risk for the development of several neurologic conditions including progressive multiple sclerosis, which is represented by substantial microglia activation. We demonstrate that in the lysolecithin demyelination model in young and middle-aged mice, the latter group developed greater acute axonal and myelin loss attributed to elevated oxidative stress through NADPH oxidase in lineage-traced microglia. We thus used a CNS-penetrant generic medication used in hypertension, indapamide, as we found it to have antioxidant properties in a previous drug screen. Following lysolecithin demyelination in middle-aged mice, indapamide treatment was associated with decreased oxidative stress and axon/myelin loss. We propose indapamide as a potential adjunctive therapy in aging-associated neurodegenerative conditions such as Alzheimer's disease and progressive multiple sclerosis.

Summary Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910, X91− or X91+), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910-CGD and two X91−-CGD). One X910-CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91−-CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91−-CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91−-CGD patients correlates with mild clinical forms of CGD, whereas X910-CGD and X91+-CGD cases remain the most clinically severe forms.

Background Patients with chronic granulomatous disease (CGD) develop severe infections, including Bacillus Calmette-Guérin (BCG). Although the autosomal recessive CGD (AR-CGD) patients should hypothetically develop relatively fewer infections compared to the X-linked CGD (X-CGD) patients due to more residual reactive oxygen intermediates, the impacts of BCG vaccination on AR-CGD and X-CGD patients are unclear. Herein, we demonstrated the clinical features of BCG infections, treatments, and genetic factors in CGD patients after BCG vaccination under the Japanese immunization program. Methods We collected data retrospectively from 43 patients with CGD and assessed their history of initial infection, age at diagnosis of CGD, BCG vaccination history, clinical course, treatment for BCG infections, and genetic mutations associated with CGD. Results Fourteen CGD patients avoided BCG vaccination because of other preceding infections and family history. Of 29 patients with CGD who received BCG vaccination, 20 patients developed BCG infections. Although the age at onset of initial infection in X-CGD patients was significantly younger than that in AR-CGD patients (P Conclusions Although X-CGD patients develop severe infections at a younger age than AR-CGD patients, our data suggested that BCG infections develop at high frequency in both AR-CGD and X-CGD patients, regardless of genotype and mutant forms.

X-linked chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the CYBB gene (located on Xp21.1). Patients with large deletions on chromosome Xp21.1 can present with the McLeod phenotype and also Duchenne muscular dystrophy or retinitis pigmentosa. The objective of the present study was to describe a series of French patients with CGD and the McLeod phenotype. We retrospectively collected data from the medical records of 8 patients with CGD and the McLeod phenotype registered at the French National Reference Center for blood types. The median age at diagnosis of CGD was 1.2 years, the median age at diagnosis of the McLeod phenotype was 4.5 years, and the median length of follow-up was 15.2 years. Four patients displayed allo-immunization, with anti-KEL20 and anti-XK1 (formerly known as anti-KL) antibodies. Five of the 6 patients with available blood smears had acanthocytosis. Neuropsychiatric, muscle-related, and ocular manifestations were present in 4, 2, and 1 of the patients, respectively. Three of the 4 patients having undergone allogeneic hematopoietic stem cell transplantation (HSCT) are alive. Overall, 5 patients are alive, and 3 are alive and well. This is the largest yet descriptive study of a series of patients with X-linked CGD and the McLeod phenotype. Although this disease combination is rare, the timely, accurate diagnosis of the McLeod phenotype is critical because of the serious post-transfusion complications. However, HSCT can be considered in these patients.

Chronic granulomatous disease (CGD) is a primary immunodeficiency of phagocyte function due to defective NADPH oxidase (phox). Compared with the common types of CYBB/gp91phox, NCF1/p47phox, and CYBA/p22phox deficiency, NCF4/p40phox deficiency is a mild and atypical form of CGD without invasive bacterial or fungal infections. It can be diagnosed using serum-opsonized E.coli as a stimulus in dihydrorhodamine (DHR) assay. Patients with CYBC1/Eros deficiency, a new and rare form of CGD, present as loss of respiratory burst and gp91phox expression in phagocytes. Neutrophils from patients with CGD are deficient in neutrophil extracellular traps (NETosis), autophagy, and apoptosis. The hyper-activation of NF-ĸB and inflammasome in CGD phagocytes also lead to long-lasting production of pro-inflammatory cytokines and inflammatory manifestations, such as granuloma formation and inflammatory bowel disease-like colitis. Patients with CGD and X-linked female carriers also have a higher incidence of autoimmune diseases. The implementation of antimicrobial, anti-fungal, and interferon-γ prophylaxis has greatly improved overall survival. Residual NADPH oxidase activity is significantly associated with disease severity and the chance of survival of the patient. New therapeutic approaches using immunomodulators for CGD-related inflammatory manifestations are under investigation, including pioglitazone, tamoxifen, and rapamycin. Hematopoietic stem cell transplantation (HSCT) is the curative treatment. Outcomes of HSCT have improved substantially over the last decade with overall survival more than 84-90%, but there are debates about designing optimal conditioning protocols using myeloablative or reduced-intensity regimens. The gene therapy for X-linked CGD using hematopoietic stem and progenitor cells transduced ex vivo by lentiviral vector encoding the human gp91phox gene demonstrated persistence of adequate oxidase-positive neutrophils in a small number of patients. Gene therapy using genome-editing technology such as CRISPR/Cas9 nucleases is a promising approach for patients with CGD in the future.

Chronic granulomatous disease (CGD) is an inherited defect of phagocyte function due to defective NADPH oxidase. Patients with CGD are not able to effectively clear the infections because of the defect in the phagocyte production of oxygen free radicals and are prone to recurrent bacterial and fungal infections. Inflammatory complications are also noted in CGD such as colitis, non-infective granulomas causing gastrointestinal or urinary tract obstruction, hemophagocytic lymphohistiocytosis, and arthritis. Studies on toll-like receptor pathways and neutrophil extracellular traps in CGD have shed light on the role of NADPH oxidase in the innate immunity and pathogenesis of infections in CGD. Some reports also indicate a reduction of memory B cells and defective production of functional antibodies in CGD. Though the exact mechanisms for non-infective inflammatory complications in CGD are not yet clear, studies on efferocytosis and defective autophagy with inflammasome activation have made a substantial contribution to our understanding of the pathogenesis of inflammation in CGD. We also discuss the clinical and molecular features of p40phox defects and a newer genetic defect, EROS. Clinical phenotypes of X-linked carriers of CYBB are also discussed. Keywords: Chronic granulomatous disease, Colitis, EROS, Genetics, Infections, Inflammation, p40phox

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder marked by abnormal phagocytic function. CGD affects primarily neutrophils and manifests as an early predisposition to severe life-threatening infections. Additionally, patients with CGD are predisposed to unique autoimmune manifestations. While generally spared from infectious complications, heterozygous carriers of the abnormal genes implicated in CGD pathogenesis can still present with autoimmune disorders. A mutation in the CYBB gene is the only X-linked variant of this disease. This article describes a family with the CYBB mutation, its heterogenous presentation, and reviews the literature discussing disease management.

The reperfusion after an acute ischemic stroke can lead to a secondary injury, which is ischemia-reperfusion (I/R) injury. During ischemia, the reactive oxygen species (ROS) is over-produced, mostly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Besides, miRNAs are also associated with neuronal death in ischemic stroke. MiR-124-5p is selectively expressed within central nervous system (CNS) and is predicted to bind to NOX2 directly. Herein, we successfully set up cerebral I/R injury model in rats through middle cerebral artery occlusion (MCAO) surgery. After 12 h or 24 h of refusion, the superoxide dismutase (SOD) activity was significantly inhibited, accompanied by NOX2 protein increase within MCAO rat infarct area. In vitro, oxygen-glucose deprivation/refusion (OGD/R) stimulation on PC-12 cells significantly increased NOX2 protein levels, ROS production, and the cell apoptosis, while a significant suppression on SOD activity; OGD/R stimulation-induced changes in PC-12 cells described above could be significantly attenuated by NOX2 silence. In vivo, miR-124 overexpression improved, whereas miR-124 inhibition aggravated I/R injury in MCAO rats. miR-124-5p directly bound to the CYBB 3'-untranslated region (UTR) to negatively regulate CYBB expression and NOX2 protein level. In vitro, miR-124 overexpression improved, while NOX2 overexpression aggravated OGD/R-induced cellular injuries; NOX2 overexpression significantly attenuated the effects of miR-124 overexpression. Besides, miR-124 overexpression significantly repressed NF-κB signaling activation and TNFα and IL-6 production through regulating NOX2. In conclusion, miR-124-5p/NOX2 axis modulates NOX-mediated ROS production, the inflammatory microenvironment, subsequently the apoptosis of neurons, finally affecting the cerebral I/R injury.

Chronic granulomatous disease is a primary immunodeficiency, characterized by a violation of the oxygen-dependent mechanisms of phagocytosis. Mutations in the genes encoding proteins of the NADPH-oxidase complex lead to a violation of the respiratory burst. Clinical manifestations are recurrent bacterial and fungal infections, and the development of granulomatous complications due to a defect in autophagy, accompanied by an increase in the level of interleukin-1 inthe blood. The treatment of this disease is continuous preventive antimicrobial therapy, and specific therapy for the treatment of granulomatous complications. Hematopoietic stem cell transplantation from an allogeneic or unrelated donor is currently considered to be only radical treatment for CGD. This article presents a clinical case of the manifestation of an X-linked form of chronic granulomatous disease with granulomatous manifestations in the absence of an infectious history. Parents patient agreed to use personal data in research and publications.

Resistance to chemotherapeutic drugs is a major cause of treatment failure in Acute Myeloid Leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin (DNR) or cytarabine (Ara-C), the main drugs used for the induction therapy. The genes found activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong up-regulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase activity, which is particularly strong in DNR-resistant cells, participates in the acquisition and/or maintenance of resistance to DNR. In addition, analyzing gp91phox (CYBB-encoded Nox2 catalytic sub-unit) expression at the surface of leukemic blasts from 74 patients at diagnosis showed that NOX2 is generally more expressed and active in leukemic cells from the FAB M4/M5 subtypes compared to FAB M0-M2 ones. Using a gene expression-based score we demonstrate that high NOX2 subunit genes expression is a marker of adverse prognosis, independent of the cytogenetic-based risk classification, in AML patients.

Background Squamous cell carcinoma (SCC) is a disease with distinct management complexities as it displays a remarkably heterogeneous molecular subtype. However, the landscape of angiogenesis for SCC is not fully investigated. Method and materials The angiogenesis-related subtypes of SCC were established by using the ConsensusClusterPlus package based on angiogenesis-related genes and TCGA data. We analyzed the alteration of genes and miRNAs as well as pathways associated with angiogenesis subtypes. Next, the regulation network, the correlation with genomic characteristics, immune microenvironment, and clinical features of the angiogenesis subtypes were further investigated. Finally, the prognostic impact of the angiogenesis-related subtypes for SCC was also analyzed. Results A total of 1368 SCC samples were included in this study. Two angiogenesis subtypes were then identified based on the one hundred and sixty-three angiogenesis-related genes with subtype1 (angiogenesis subtype) of 951 SCC patients and subtype2 (non-angiogenesis subtype) of 417 SCC. GSEA revealed that angiogenesis and epithelial-mesenchymal transition, inflammatory response, and hypoxia were enriched in the angiogenesis subtype. Eight of the 15 immune checkpoints (ADORA2A, BTLA, CD276, CYBB, HAVCR2, SIGLEC7, SIGLEC9, and VTCN1) were significantly upregulated while C10orf54 were significantly downregulated in the angiogenesis subtype. The survival analysis revealed that the patients in the angiogenesis subtype have poorer survival outcomes than those in the non-angiogenesis subtype (P = 0.017 for disease-free interval and P = 0.00013 for overall survival). Conclusion Our analysis revealed a novel angiogenesis subtype classification in SCC and provides new insights into a hallmark of SCC progression.

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterized by impaired phagocyte function, recurrent fungal and bacterial infections and granuloma formation in multiple organs. Pediatric myelodysplastic Syndrome (MDS) is a rare hematological stem cell disease that leads to an ineffective hematopoiesis with variable risk of evolution to acute leukemias. Both disorders are rare and have distinct pathophysiologic mechanisms, with no known association. A 7-month-old boy presenting with recurrent infections and anemia at age 2 months underwent immunological, hematological and genetic investigation that culminated in the diagnosis of both CGD and MDS. Next generation sequencing was performed and identified a silent variant predicted as of Uncertain Significance, located in the splicing site at the end of exon 5 in CYBB. CYBB variants account for at least two thirds of CGD cases, but no previous descriptions of this variant were found in ClinVar or The Human Gene Mutation Database (HGMD) databases. We were able to demonstrate an exon 5 skipping on the proband’s cDNA, which strongly suggests the disruption of the NADPH oxidase complex, abrogating the formation of reactive oxygen species from neutrophils. Moreover, erythroid cell lineage could be also affected by NADPH oxidase complex damages. Further investigation is needed to evaluate the potential effect of CYBB gene alterations in hematopoiesis, as well as in MDS and CGD association.

Orientador: Antonio Condino Neto Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: Doença Granulomatosa. Crônica (DOC) é uma imunodeficiência caracterizada por infecções recorrentes graves. Os defeitos moleculares que lavam a DGC são geralmente devidos a mau funcionamento, ausência o baixa expressão de um dos componentes do sistema NADPH oxidase. Este trabalho analisou o uso de RT-PCR para a triagem de defeitos moleculares responsáveis por DGC ligada ao X em 8 pacientes. RNA total foi preparado de linfócitos B transformados com vírus de Epstein-Barr e transcrição reversa com hexâmeros randomicos. O cDNA resultante foi amplificado por PCR com oligonucleotídeos específicos para 3 regiões exônicas abrangendo toda a extensão do gene. Com esta estratégia foi possível a detecção da expressão defeituosa de gp91-phox em 7 pacientes. Concluímos que a análise por meio de RT-PCR, um método alternativo menos complexo, rápido e econômico foi apropriado para detecção inicial de defeitos moleculares em 7 de 8 pacientes com DOC ligada ao X. Posteriormente investigamos em detalhe os defeitos genetico-moleculares de 7 crianças não relacionadas com DGC ligada ao X. Todos os pacientes foram procedentes do Chile e Brasil. Encontramos uma inserção c.1267_1268insA no paciente JY no exon 10 levando a uma mutação tipo "&ameshill:". Esta mutação é um novo registro na literatura. Detectamos duas substituições "nonsense", uma no paciente PT, c.95 G>A no exon 2 que leva a um códon de parada W28X e outra no paciente MF, c.229 C>T no exon 3 que leva a um códon de parada R73X. Em 4 casos, nos pacientes IC, Vin, RS, GO, diferentes erros de "splicing" foram encontrados. Dois pacientes apresentaram uma subtitução c.264 G> A ao final do exon 3. Os dois restantes apresentaram uma subtitução c.1326 + 1 G>A no intron 10 e outra subtitução c.1164 - 2 A>O no intron 9. Esta última mutação também é um novo registro na lit_ratura. As mutações identificadas na proteína gp91-phox confirmam um alto grau de heterogeneidade molecular como é relatado em outros grupos étnicos e a importância de investigar os defeitos moleculares em diferentes populações. Contrastando com esta heterogeneidade, a DGC associada com defeitos na proteína p47-phox, apresentam pouca variabilidade. Neste estudo, os pacientes analisados, dois irmãos, mostram uma deleção homozigota OT (L}.GT) no começo do exon 2. L Também é analisado o caso de um paciente com infecções recorrentes que inicaJmente recebeu o diagnóstico de deficiência de G6PD. Estudos moleculares mostraram que a deficiência de G6PD foi devida a uma mutação 202 G_A, variante Afi:icana. . O paciente também mostrou uma reduzida atividade da explosão respiratória como observado em DGC ligada ao X. A análise do gDNA mostrou uma subtitução 264 G_A na região do splicing do exon 3 da proteína gp91-phox. A seqüência do cDNA detectou uma deleção do exon 3, levando a uma mutante inestável ou não funcional da proteina gp91-phox e resultando no fenótipo de DGC ligada ao X. Propomos que ftente a um paciente com deficiência de G6PD com episódios de infecções graves considerem a possibilidade de um defeito na atividade fagocítica e uma eventual associação com DGC Abstract: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency characterized by ear1y onset recurrent severe infections. The molecular defects causing CGD are generally due to the absence, low expression or malfunctioning of one of the NADPH oxidase components. This work analyzed the potential use of reverse transcription (RT)PCR for screening molecular defects responsible for X-linked CGD in 8 Brazilian patients. Total RNA was prepared from EBV-transformed B-lymphocytes and reverse transcribed using random hexamers. The resultant cDNA was PCR-amplified by specific and overlapping pairs of primers regarding 3 exonic regions of gp91-phox gene. This strategy made possible the detection of defective gp91-phox expression in 7 patients. We conclude that RT -PCR analysis, a less complex, more economic and faster alternative method, was appropriate for screening molecular defects in 7 out 8 X-linked CGD patients. We further investigated the molecular genetic defects in 7 unrelated patients with X-linked CGD, from Chile and Brazil. We found an insertion c.1267_1268insA in exon 10 leading to a frameshift mutation. This mutation is a novel reporto we detected two single base-pair substitutions that lead to nonsense mutations. The first was a c.95 G>A substitution in the exon 2 which predicts a stop codon W28X and the second was a c.229 C> T substitution in the exon 3 which predicts a stop codon R73X. We also identified different splice site mutations in 4 cases. Two patients presented a c.264 G> A substitution at the end of exon 3. The remaining two patients presented either a c.1326 + 1 G>A substitution in intron 10 or a c.1164 - 2 A>G substitution in intron 9. This Iast mutation is also novel. The gp91-phox mutations identified in these patients show a high degree of molecular heterogeneity as reported in other ethnic groups and the importance to investigate molecular genetic defects in diferent populations. Contrasting with the heterogeneity of mutations observed in X-linked CGD, the disease associated with defect in p47-phox shows less variability. In this report, the patients with CGD, two sib1ings, show a homozygotous dinucleotide GT deletion (.6.GT) at the beginning of exon 2. We also reported a child with recurrent infections who initially received the diagnosis of G6PD deficiency. Molecular studies showed that the G6PD deficiency was due a 202 G-+A mutation, the A- variant common in African ethnic groups. The proband also exln'bited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-+A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of Xlinked CGD. We propose that clinicians in face of a patient with G6PD deficiency under a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes microbicidal activity, and the eventual association of G6PD deficiencyand chronic granulomatous disease Doutorado Saúde da Criança e do Adolescente Doutor em Saúde da Criança e do Adolescente

Background Multisystem Inflammatory Syndrome in Children (MIS-C) is a pediatric complication of SARS-CoV-2 infection characterized by multiorgan inflammation and frequently, cardiovascular dysfunction. It occurs predominantly in otherwise healthy children. We previously reported haploinsufficiency of Suppressor of Cytokine Signaling 1 (SOCS1), a negative regulator of Type I and II interferons, as a genetic risk factor for MIS-C. Objectives We aimed to identify additional genetic mechanisms underlying susceptibility to SARS-CoV-2-associated MIS-C. Methods In a single center, prospective cohort study, whole exome sequencing was performed on patients with MIS-C. The impact of candidate variants was tested using patients’ peripheral blood mononuclear cells obtained at least seven months after recovery. Results We enrolled 18 patients with MIS-C (median age: 8 years, IQR 5 – 12.25 years), of whom 89% had no conditions other than obesity. In two boys with no significant infection history, we identified and validated hemizygous, deleterious defects in XIAP, encoding X-linked inhibitor of apoptosis, and CYBB, encoding cytochrome b-245. Including the previously reported SOCS1 haploinsufficiency, a genetic diagnosis was identified in three (17%) of 18 patients. Even after recovery, patients with defects in SOCS1, XIAP, or CYBB exhibit an inflammatory immune cell transcriptome with enrichment of differentially expressed genes in pathways downstream of IL-18, oncostatin M, and NF-κB, compared to those with mild COVID-19. Conclusions Although inflammatory disorders are rare in the general population, our cohort of patients with MIS-C was enriched for monogenic susceptibility to inflammation. Our results support the use of next-generation sequencing in previously healthy children who develop MIS-C., Graphical abstract, Capsule Summary: Hypomorphic inborn errors of immunity may predispose children to SARS-CoV-2-associated Multisystem Inflammatory Syndrome in Children (MIS-C).

The tumor microenvironment plays an important role in tumor development and progression, but the role of immune and stromal cells in this environment has not been sufficiently studied. In this study, we aimed to identify key genes associated with the microenvironment of lung adenocarcinoma (LUAD). Raw data for stromal and immune cells in malignant tumors were downloaded from The Cancer Genome Atlas (TCGA). These expression data were used to identify the differentially expressed genes (DEGs) in tissue samples of LUAD with high and low immune scores. A protein-protein interaction (PPI) network based on genes with significant differential expression was constructed. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to functionally annotate putative hub genes. These genes were assessed via Kaplan Meier analysis to determine their correlation with overall survival. In total, we identified 216 DEGs which were correlated with immune and stromal scores, including 30 hub genes which were identified based on the PPI network. Further analysis suggested that the expression levels of 10 of these genes were significantly correlated with overall survival of LUAD patients. These key hub genes included CCR2, CCR5, CD53, CYBB, HCK, IRF8, LCP2, PLEK, PTPRC, and TLR7. Moreover, the expression level of CCR2 was found to have strong prognostic value for LUAD patients. Additionally, high expression of CYBB was also correlated with better survival of patients with LUAD. The results of this study open several new avenues to explore in the treatment of LUAD.

In the United Arab Emirates, BCG (Bacillus Calmette-Guérin) is administered to all newborns. We present here a young infant with an inborn error of immunity (IEI) who developed fatal adverse events to this live-attenuated vaccine. This male infant received BCG (Serum Institute of India Pvt., Ltd., India) on Day 11 of life. On Day 25, he developed fever, followed by cervical lymphadenitis and bilateral otitis media with fluid drainage. On Day 118, he was admitted with severe hemophagocytic lymphohistiocytosis (HLH), and passed away on Day 145. The diagnostic exome sequencing test identified a hemizygous nonsense variant, NM_000397.3(CYBB):c.676C>T, p.Arg226* (rs137854592). Pathogenic variants of CYBB [cytochrome b(-245), beta subunit; Mendelian Inheritance in Man [MIM] accession code, 300481] are known to cause “immunodeficiency 34, mycobacteriosis, X-linked” (IMD34, MIM#300645) and “chronic granulomatous disease, X-linked” (CGDX, MIM#306400). The natural history of his illness is consistent with “X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD).” This entity is responsible for his BCG disease and is a likely trigger of his HLH. This disastrous event underlines the importance of developing worldwide policies that target BCG disease prevention, especially in communities with high prevalence of IEI. Moreover, screening for genetic causes of MSMD in the community could pave the way, at least partially, for scale-up of tuberculosis (TB) prevention.

We sought to further investigate the efficacy and safety of pioglitazone for chronic granulomatous disease (CGD) patients with severe infection. CGD patients with severe infection were enrolled and treated with pioglitazone for 90 days. The degree of improvement in infection and the changes of dihydrorhodamine-123 (DHR) were used to evaluate the efficacy of pioglitazone. The adverse reaction of pioglitazone was also investigated. We planned to enroll 30 patients at first in the study. However, the study was terminated due to negative results from all 3 enrolled patients. The 3 patients were diagnosed with CGD by clinical characteristics, DHR analysis, and genetics analysis. Mutations were CYBB (c.177C>A; p.C59X) in P1, CYBB (c.1498G>T; p.D500Y) in P2, and NCF2 (c.137T>G; p.M46R) in P3, respectively. The age of onset of the 3 patients was within 2 years after birth. The most common sites of infection were lung, lymph node, skin, and soft tissue, which were experienced in all 3 patients. The age of administration with pioglitazone was 5.2 years, 16 years and 11.1 years, respectively. The 3 patients experienced no improvement in severity of infection and stimulation index of the DHR did not also improve after receiving pioglitazone 10, 45 and 90 days, respectively. No drug-related adverse reaction was found during the period of pioglitazone. Low dose of pioglitazone did not improve the severity of infection and production of ROS in CGD patients with severe infection.

Chronic granulomatous disease (CGD) is a primary immunodeficiency for which allogeneic hematopoietic stem cell transplant (HSCT) offers potential cure. Direct comparison of HSCT to non-HSCT management in the North American population was performed to identify clinical factors associated with overall survival (OS) and transplant-related survival (TRS). Retrospective review of CGD subjects enrolled in the United States Immunodeficiency Network. Survival was estimated by the Kaplan-Meier method and modeled by proportional hazards regression. We identified 507 patients (66% CYBB mutants) diagnosed in 1953–2016. Fifty underwent allogeneic HSCT. Median follow-up was 9.1 years after diagnosis (0–45.8 years). OS was negatively associated with CYBB mutation (HR = 6.25; p = 0.034) and not associated with HSCT (88% v. 85% ± HCT) (HR = 1.26; p = 0.65). Transplant at ≤ 14 years old was associated with improved TRS (93% v. 82% at T + 60 months) (HR = − 4.51; p = 0.035). Patients transplanted before 15 years old had fewer severe infections pre-HSCT (mean 0.95 v. 2.13; p = 0.047). No mortality was reported in patients receiving stem cells from matched siblings. Infection incidence declined post-HSCT in subjects with greater than or equal to four infections pre-HSCT (p = 0.0010). Compared to non-HSCT patients ≥ 15 years old, post-transplant survivors had higher mean performance score (93.2 v. 85.9; p = 0.0039) and lower frequency of disability (11% v. 52%; p = 0.014). Allogeneic HSCT was associated with reduced infection incidence and improved functional performance, but not with a change in overall survival. Transplant-related survival was elevated in patients undergoing HSCT before 15 years old. Consider HSCT prior to late adolescence in patients with severely diminished reactive oxygen intermediate synthesis, particularly if a matched sibling is available.

AIM To assess the level of hepatic oxidative damage and its correlation with clinical severity in biliary atresia (BA), and to understand BA mitochondrial gene sequencing. METHODS Forty-eight BA patients and 28 control subjects (20 hepatoblastoma and 8 cholestasis patients) were enrolled. Hepatic oxidative damage was assessed by the expression of oxidation and antioxidant genes, and the correlation between oxidative damage and BA incidence, liver inflammation, and fibrosis was evaluated. Moreover, 8-hydroxyguanine (8-OHdG), mitochondrial DNA (mtDNA) copy number, and mitochondrial gene sequences were determined to evaluate oxidative mtDNA damage in BA. RESULTS The expression of oxidation gene cytochrome b-245 beta chain (CYBB) in BA was significantly increased and patients with a higher CYBB expression had the higher risk of BA incidence, liver inflammation, and cirrhosis. However, the expression of antioxidant genes was significantly decreased, and glutathione S-transferase alpha 1 (GSTA1) negatively correlated with BA incidence and cirrhosis. When GSTA1 mRNA expression was

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type

Neutrophil trafficking is essential for a strong and productive immune response to infection and injury. During acute inflammation, signals from resident immune cells, fibroblasts, and the endothelium help to prime, attract, and activate circulating neutrophils at sites of inflammation. Due to current limitations with in vitro and animal models, our understanding of these events is incomplete. In this paper, we describe a microfluidic technology which incorporates a lumen-based vascular component with a high degree of spatiotemporal control to facilitate the study of neutrophil trafficking using primary human cells. The improved spatiotemporal control allows functional selection of neutrophils based on their migratory capacity. We use this technology to investigate neutrophil-endothelial interactions and find that these interactions are necessary for robust neutrophil chemotaxis to interleukin-8 (IL-8) and priming of the neutrophils. In agreement with previous studies, we observed that transendothelial migration (TEM) is required for neutrophils to enter a primed phenotypic state. TEM neutrophils not only produce a significantly higher amount of reactive oxygen species (ROS) when treated with PMA, but also upregulate genes involved in ROS production (CYBB, NCF1, NFKB1, NFKBIA), cell adhesion (CEACAM-8, ITGAM), and chemokine receptors (CXCR2, TNFRSF1A). These results suggest that neutrophil-endothelial interactions are crucial to neutrophil chemotaxis and ROS generation.

Background Although the incidence of tuberculosis (TB) has dropped substantially, it still is a serious threat to human health. And in recent years, the emergence of resistant bacilli and inadequate disease control and prevention has led to a significant rise in the global TB epidemic. It is known that the cause of TB is Mycobacterium tuberculosis infection. But it is not clear why some infected patients are active while others are latent. Methods We analyzed the blood gene expression profiles of 69 latent TB patients and 54 active pulmonary TB patients from GEO (Transcript Expression Omnibus) database. Results By applying minimal redundancy maximal relevance and incremental feature selection, we identified 24 signature genes which can predict the TB activation. The support vector machine predictor based on these 24 genes had a sensitivity of 0.907, specificity of 0.913, and accuracy of 0.911, respectively. Although they need to be validated in a large independent dataset, the biological analysis of these 24 genes showed great promise. Conclusion We found that cytokine production was a key process during TB activation and genes like CYBB, TSPO, CD36, and STAT1 worth further investigation.

BACKGROUND Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. MATERIALS AND METHODS Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. RESULTS All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. CONCLUSIONS Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

Background This study was aimed to discover more biomarkers associated with spinal cord injury (SCI) by constructing a competing endogenous RNA (ceRNA) network. Methods The transcriptome expression profile data related to SCI (GSE45006 GSE20907) were downloaded from GEO database. The differentially expressed RNAs (DERs), including lncRNAs, miRNAs, and mRNAs, between SCI and control groups were selected, which were then performed function enrichment analyses. Following that, a SCI-related ceRNA regulatory network was constructed. PCA analysis was performed on the genes constituting the ceRNA regulatory network directly related to SCI. Results In GSE45006 and GSE20907 datasets, there were respectively 3336 and 1453 DERs. Venn analysis showed that there were 429 DERs which had consistent differential expression direction. RGD1564534-miR-29b-5p relation pair and 103 miRNA-target regulatory pairs were integrated to construct the ceRNA regulatory network. Then a SCI-related ceRNA regulatory network including 8 mRNAs of IFNGR1, STAT2, CYBB, NFATC1, FCGR2B, HMOX1, TLR4, and HK2, a lncRNA of RGD1564534, and a miRNA of miR-29b-5p was constructed. Additionally, two pathways, osteoclast differentiation, and HIF-1 signaling pathway, were involved in this network. PCA indicated the samples before and after injury can be significantly distinguished based on the genes in the ceRNA network. Conclusion A total of 8 SCI-related mRNAs have been identified in the ceRNA network, including IFNGR1, STAT2, CYBB, NFATC1, FCGR2B, HMOX1, TLR4, and HK2. Moreover, RGD1564534 may serve as ceRNA by competitively binding to miR-29b-5p to regulate the expression of 8 SCI-related mRNAs. Therefore, these genes may serve as key biomarkers of SCI.

Background Chronic granulomatous disease (CGD) presents with a myriad of clinical manifestations pertaining to both immunodeficiency and hyperinflammation. Although Candida infection is a signature organism for patients with CGD, C. lusitaniae pneumonia in CGD has rarely been reported. C. lusitaniae is a ubiquitous ascomycete predominantly infecting immunocompromised hosts and has the potential to rapidly develop multi-drug resistance during therapy. Additionally, C. lusitaniae is recognized for its variable resistance against amphotericin B. To date, C. lusitaniae infections in patients with CGD have not been reviewed in detail. False-positive HIV serology, resulting from polyclonal hypergammaglobulinemia, has been reported in association with several infections, auto-immune diseases, and malignancies. Although CGD is often associated with hypergammaglobulinemia, a false-positive HIV serology in CGD has not been reported previously. Procedure We report a combination of unique findings in a child with CGD – a false-positive HIV serology, Candida lusitaniae pneumonia, and a novel CYBB mutation. We also provide a detailed review of C. lusitaniae infections in patients with CGD. Results In patients with CGD, C. lusitaniae has been reported to cause lymphadenitis (cervical, abdominal), fungemia, meningoencephalitis, or abscesses in the liver and spleen. Many CGD patients with C. lusitaniae infection have associated inflammatory complications of the gut (inflammatory bowel disease, colitis). Additionally, almost all C. lusitaniae infections in CGD have been reported in young infants or in patients receiving long-term immunosuppressive therapy. This reflects that further immunocompromise (in addition to the underlying immune deficiency in CGD) may specifically predispose to C. lusitaniae infection (unlike other candidal infections). Most of the CGD patients with documented C. lusitaniae infection have X-linked form of the disease which generally has been postulated to have a more severe clinical phenotype than the autosomal recessive forms of the disease. Conclusions HIV serology may be positive in patients with CGD and other inborn errors of immunity as a result of hypergammaglobulinemia. C. lusitaniae, which may have peculiar and evolving antimicrobial sensitivity patterns, needs to be considered in patients with CGD and pneumonia. Lastly, to reiterate, CGD should to be considered in patients with proven C. lusitaniae infection.

Chronic granulomatous Disease (CGD) is a rare innate immunodeficiency disorder caused by mutations in one of the six genes (CYBA, CYBB, NCF1, NCF2, NCF4, and CYBC1/EROS) encoding the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH)—oxidase complex in phagocytes. In the Western population, the most prevalent form of CGD (about two-thirds of all cases) is the X-linked form (X-CGD) caused by mutations in CYBB. The autosomal recessive forms (AR-CGD), due to mutations in the other genes, collectively account for the remaining one-third of CGD cases. We investigated the clinical and molecular features of 22 Jordanian, 7 Libyan, and 2 Iraqi CGD patients from 21 different families. In addition, 11 sibling patients from these families were suspected to have been died from CGD as suggested by their familial and clinical history. All patients except 9 were children of consanguineous parents. Most of the patients suffered from AR-CGD, with mutations in CYBA, NCF1, and NCF2, encoding p22phox, p47phox, and p67phox proteins, respectively. AR-CGD was the most frequent form, in Jordan probably because consanguineous marriages are common in this country. Only one patient from non-consanguineous parents suffered from an X910 CGD subtype (0 indicates no protein expression). AR670 CGD and AR220 CGD appeared to be the most frequently found sub-types but also the most severe clinical forms compared to AR470 CGD. As a geographical clustering of 11 patients from eight Jordanian families exhibited the c.1171_1175delAAGCT mutation in NCF2, segregation analysis with nine polymorphic markers overlapping NCF2 indicates that a common ancestor has arisen ~1,075 years ago.