Josias Brito Frazão

HOX antisense lincRNA HOXA-AS2 is an apoptosis repressor in all trans retinoic acid treated NB4 promyelocytic leukemia cells

HOXA cluster antisense RNA 2 (HOXA‐AS2) is a long non‐coding RNA located between the HOXA3 and HOXA4 genes in the HOXA cluster. Its transcript is expressed in NB4 promyelocytic leukemia cells and human peripheral blood neutrophils, and expression is increased in NB4 cells treated with all trans retinoic acid (ATRA). Knockdown of HOXA‐AS2 expression by transduced shRNA decreases the number of viable cells and increases the proportion of apoptotic cells, measured by annexin V binding and by activity and cleavage of caspases‐3, ‐8, and ‐9. The increase in death of HOXA‐AS2 knockdown cells was accompanied by an elevated TNF‐related apoptosis‐inducing ligand (TRAIL) levels, but ATRA‐induced NB4 cells treated with TRAIL did show an increase in HOXA‐AS2 expression. These results demonstrate that ATRA induction of HOXA‐AS2 suppresses ATRA‐induced apoptosis, possibly through a TRAIL‐mediated pathway. HOXA‐AS2‐mediated negative regulation thus contributes to the fine‐tuning of apoptosis during ATRA‐induced myeloid differentiation in NB4 cells. J. Cell. Biochem. 114: 2375–2383, 2013.

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Abstract Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.

The Use of Interferon-Gamma Therapy in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by early onset of recurrent and severe infections. The molecular defects causing CGD are heterogeneous and lead to absence, low expression, or malfunctioning of one of the phagocyte NADPH oxidase components. It is known that mutations leading to CGD reside within the genes encoding four essential components of the oxidase designated as gp91-phox (phagocyte oxidase), p22-phox, p47-phox and p67-phox. gp91- together with p22-phox form the membrane cytochrome b(558) and play an essential role in the transfer of electrons following assembly of the active oxidase with the cytoplasmic p47- and p67-phox components. In hematopoietic cells, CYBB expression (the gene encoding gp91-phox) is limited to the granulocyte and monocyte/macrophage lineages during the process of terminal differentiation. CYBB is responsive to a number of inflammatory cytokines, especially interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Cytokines have been also studied for activation of phagocytes respiratory burst. IFN-gamma stimulates superoxide release and is a prophylactic agent for CGD. It has been shown in vitro and in vivo to correct at least in part alterations of the oxidative metabolism, and to improve their microbicidal function. It has demonstrated clinical benefit in the majority of patients with CGD, reducing the relative risk of severe infections in 70%. In this study, we review mechanisms showing that IFN-gamma improves the splicing efficiency of CYBB gene transcripts in a particular group of CGD patients. The present article is an informative review of recent patents related to the use of interferon gamma therapy in chronic granulomatous disease.

Mother to child transfer of IgG and IgA antibodies against Dermatophagoides pteronyssinus.

There is strong evidence from animal models that placental and/or breast milk‐mediated transfer of maternal allergen‐specific IgG prevents allergic immune responses in the progeny. Both human and animal data also point to IgA as having an important regulatory role. In contrast, little is known about maternal transfer of IgG and IgA specific for respiratory allergens in humans. Dermatophagoides pteronyssinus (Der p) is an indoor allergen that is a major cause of asthma worldwide. We analysed maternal to child Der p‐specific IgG and IgA transfer in a cohort of 77 paired maternal and child samples. We found Der p‐specific IgG and its IgG1, IgG2 and IgG4 subclasses in all cord blood samples. Except for IgG1, cord levels were higher in newborns from atopic mothers (n = 29) compared to non‐atopic mothers (n = 48). Der p‐specific IgA was found in all colostrum samples and levels were independent of maternal atopic status. Notably, anti‐Der p IgG was also found in colostrum and levels were higher in atopic mothers. We believe that our work is a critical first step in the identification of early factors that may impact asthma development and should guide the development of clinical studies that assess whether Der p‐specific IgG and IgA protect children from allergy as demonstrated in animal models.

High‐Performance Liquid Chromatography Under Partially Denaturing Conditions (dHPLC) is a Fast and Cost‐Effective Method for Screening Molecular Defects: Four Novel Mutations Found in X‐Linked Chronic Granulomatous Disease

Implementing precise techniques in routine diagnosis of chronic granulomatous disease (CGD), which expedite the screening of molecular defects, may be critical for a quick assumption of patient prognosis. This study compared the efficacy of single‐strand conformation polymorphism analysis (SSCP) and high‐performance liquid chromatography under partially denaturing conditions (dHPLC) for screening mutations in CGD patients. We selected 10 male CGD patients with a clinical history of severe recurrent infections and abnormal respiratory burst function. gDNA, mRNA and cDNA samples were prepared by standard methods. CYBB exons were amplified by PCR and screened by SSCP or dHPLC. Abnormal DNA fragments were sequenced to reveal the nature of the mutations. The SSCP and dHPLC methods showed DNA abnormalities, respectively, in 55% and 100% of the cases. Sequencing of the abnormal DNA samples confirmed mutations in all cases. Four novel mutations in CYBB were identified which were picked up only by the dHPLC screening (c.904 insC, c.141+5 g>t, c.553 T>C, and c.665 A>T). This work highlights the relevance of dHPLC, a sensitive, fast, reliable and cost‐effective method for screening mutations in CGD, which in combination with functional assays assessing the phagocyte respiratory burst will contribute to expedite the definitive diagnosis of X‐linked CGD, direct treatment, genetic counselling and to have a clear assumption of the prognosis. This strategy is especially suitable for developing countries.

Primary immunodeficiency association with systemic lupus erythematosus: review of literature and lessons learned by the Rheumatology Division of a tertiary university hospital at São Paulo, Brazil

Primary immunodeficiency disorders (PID) represent a heterogeneous group of diseases resulting from inherited defects in the development, maturation and normal function of immune cells; thus, turning individuals susceptible to recurrent infections, allergy, autoimmunity, and malignancies. In this retrospective study, autoimmune diseases (AIDs), in special systemic lupus erythematosus (SLE) which arose associated to the course of PID, are described. Classically, the literature describes three groups of PID associated with SLE: (1) deficiency of Complement pathway components, (2) defects in immunoglobulin synthesis, and (3) chronic granulomatous disease (CGD). Currently, other PID have been described with clinical manifestation of SLE, such as Wiskott-Aldrich syndrome (WAS), autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), autoimmune lymphoproliferative syndrome (ALPS) and idiopathic CD4(+) lymphocytopenia. Also we present findings from an adult cohort from the outpatient clinic of the Rheumatology Division of Universidade Federal de São Paulo. The PID manifestations found by our study group were considered mild in terms of severity of infections and mortality in early life. Thus, it is possible that some immunodeficiency states are compatible with survival regarding infectious susceptibility; however these states might represent a strong predisposing factor for the development of immune disorders like those observed in SLE.

Regulation of CYBB Gene Expression in Human Phagocytes by a Distant Upstream NF-κB Binding Site.

The human CYBB gene encodes the gp91‐phox component of the phagocyte oxidase enzyme complex, which is responsible for generating superoxide and other downstream reactive oxygen species essential to microbial killing. In the present study, we have identified by sequence analysis a putative NF‐κB binding site in a DNase I hypersensitive site, termed HS‐II, located in the distant 5′ flanking region of the CYBB gene. Electrophoretic mobility assays showed binding of the sequence element by recombinant NF‐κB protein p50 and by proteins in nuclear extract from the HL‐60 myeloid leukemia cell line corresponding to p50 and to p50/p65 heterodimers. Chromatin immunoprecipitation demonstrated NF‐κB binding to the site in intact HL‐60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF‐κB binding site and the CYBB promoter region. Inhibition of NF‐κB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of κB “super‐repressor” showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF‐κB site linked to the CYBB 5′ flanking promoter region revealed enhanced expression, augmented by treatment with interferon‐γ. These studies indicate a role for this distant, 15 kb upstream, binding site in NF‐κB regulation of the CYBB gene, an essential component of phagocyte‐mediated host defense. J. Cell. Biochem. 116: 2008–2017, 2015.

Gene expression in chronic granulomatous disease and interferon-γ receptor-deficient cells treated in vitro with interferon-γ.

Interferon‐γ (IFN‐γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN‐γ/interleukin (IL)‐12 axis. Using transcriptome profiling (RNA‐seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein‐Barr virus–transformed B lymphocytes (B‐EBV) cells from CGD patients, IFN‐γ receptor deficiency patients, and normal controls, treated in vitro with IFN‐γ for 48 hours. Our results show that IFN‐γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN‐γ‐induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome‐mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN‐γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre‐mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl‐transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN‐γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN‐γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN‐γ treatment. These data support the concept that IFN‐γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.