Enhanced Cas12a editing in mammalian cells and zebrafish.

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

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.

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.

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.

Syzygium jambolanum treatment improves survival in lethal sepsis induced in mice.

BACKGROUND: The leaves and the fruits from Syzygium jambolanum DC.(Myrtaceae), a plant known in Brazil as sweet olive or 'jambolão', have been used by native people to treat infectious diseases, diabetes, and stomachache. Since the bactericidal activity of S. jambolanum has been confirmed in vitro, the aim of this work was to evaluate the effect of the prophylactic treatment with S. jambolanum on the in vivo polymicrobial infection induced by cecal ligation and puncture (CLP) in mice. METHODS: C57Bl/6 mice were treated by the subcutaneous route with a hydroalcoholic extract from fresh leaves of S. jambolanum (HCE). After 6 h, a bacterial infection was induced in the peritoneum using the lethal CLP model. The mice were killed 12 h after the CLP induction to evaluate the cellular influx and local and systemic inflammatory mediators' production. Some animals were maintained alive to evaluate the survival rate. RESULTS: The prophylactic HCE treatment increased the mice survival, the neutrophil migration to infectious site, the spreading ability and the hydrogen peroxide release, but decreased the serum TNF and nitrite. Despite the increased migration and activation of peritoneal cells the HCE treatment did not decrease the number of CFU. The HCE treatment induced a significant decrease on the bone marrow cells number but did not alter the cell number of the spleen and lymph node. CONCLUSION: We conclude that the treatment with S. jambolanum has a potent prophylactic anti-septic effect that is not associated to a direct microbicidal effect but it is associated to a recruitment of activated neutrophils to the infectious site and to a diminished systemic inflammatory response.

Aquisição passiva de anticorpos anti-dermatophagoides pteronyssinus através de passagem transplacentária e aleitamento materno / Passive acquisition of anti-dermatophagoides pteronyssinus antibodies through placental transfer and breastfeeding

Introdução e objetivos: Eventos nos primeiros anos de vida podem ser responsáveis pelo aumento da prevalência de doenças alérgicas. Tendo em vista que o sistema imunológico da criança recebe anticorpos específicos a alérgenos ainda no útero e através da amamentação, caracterizamos a transferência passiva de IgG e IgA anti-Dermatophagoides pteronyssinus e verificamos o efeito da sensibilização materna na resposta imune humoral no cordão umbilical e no colostro. Métodos: Colostro e amostras pareadas de soro materno e de cordão umbilical foram coletadas de treze mães sensibilizadas (RAST anti-Der p > classe 3) e 25 mães não sensibilizadas (RAST anti-Der p = O). Quantificamos os níveis totais de IgG por nefelometria e a IgA total e os anticorpos específicos anti¬Der p por ELISA. Para análise funcional, verificamos a avidez dos anticorpos ao extrato total do ácaro, também por ELISA. Resultados: Recém-nascidos de mães sensibilizadas apre¬sentaram níveis significativamente mais elevados de IgG anti¬Derp no cordão umbilical (p=O,Ol), no entanto, estes anticor¬pos foram detectados em todas as amostras, estando fortemente correlacionados aos níveis maternos (r=0.81 p

Introduction and objectives: The early life sensitization might be one of the primary causes of the increased prevalen¬ce of allergy. Considering that infants received in the uteri and by the breastfeed specific antibodies to allergens, we investigated the passive transference of IgG and IgA antibodies to Dermatophagoides pteronyssinus to verify if mother sensitization influences the humoral immune response in umbilical cord blood and in colostrum...

Efficacy of the intralesional treatment with Chenopodium ambrosioides in the murine infection by Leishmania amazonensis.

AIM OF THE STUDY: Leishmaniasis, caused by protozoan from Leishmania genus, is an endemic disease in the tropical and subtropical regions of the world. The chemotherapy to this disease is not always effective and can cause several side effects. Chenopodium ambrosioides L. (Chenopodiaceae) is used by the native people in the treatment of cutaneous ulcers caused by different species of Leishmania. The aim of this study was to investigate the effect of the treatment with a hydroalcoholic crude extract (HCE) from the leaves of Chenopodium ambrosioides on the murine infection with Leishmania amazonensis. MATERIAL AND METHODS: The mice were treated for 4-6 weeks post-infection (p.i.) with HCE (5 mg/kg) or meglumine antimoniate (Sb(v)) (28 mg/kg) either by the oral route, once a day, for 15 days or by five intralesional (IL) injections at intervals of 4 days. The thickness of the infected paws was determined weekly and the parasite load evaluated in the draining lymph nodes (LN), the spleen and in the footpad after 7 weeks of infection. The nitric oxide (NO) production was evaluated in cultures with cells from peritoneum or LN. RESULTS: The IL treatment increased the NO production in the LN and peritoneum cultures and reduced the parasite load from the footpad, spleen and LN. On the other hand, the oral treatment decreased did alter neither the NO production nor the parasite load. CONCLUSIONS: IL HCE treatment was more efficient than the oral HCE treatment since the former was able to control the dissemination of infection. This effect can be due to either a direct leishmanicidal effect of HCE or the improvement in the NO production by HCE-stimulated macrophages. The results could justify the topical use of the Chenopodium ambrosioides' leaves in the treatment of the ulcers caused by Leishmania.

Increase of cellular recruitment, phagocytosis ability and nitric oxide production induced by hydroalcoholic extract from Chenopodium ambrosioides leaves.

The leaves and the oil from the seeds of Chenopodium ambrosioides L. (Chenopodiaceae), a plant known in Brazil as 'mastruz', have been used by native people to treat parasitic diseases. Experimentally it was shown that Chenopodium ambrosioides inhibits the Ehrlich tumor growth, what could be due to an immunomodulatory effect of this product. The aim of this study was to investigate the effect of hydroalcoholic crude extract (HCE) from leaves of Chenopodium ambrosioides on macrophage activity and on lymphoid organs cellularity. C3H/HePas mice received the HCE (5mg/kg) by intraperitoneal via and were sacrificed 2 days later. HCE treatment did not alter the cell number in bone marrow, but it increased the cell number in peritoneal cavity, spleen and lymph node. The spreading and phagocytosis activity, the PMA-induced hydrogen peroxide (H(2)O(2)) release and the nitric oxide (NO) production were also increased when compared to control group. Similar results were obtained with concanavalin A (Con A), used as a positive control, with exception of the NO production that was only detected in HCE-derived macrophages. The in vitro treatment with HCE induced a dose-dependent NO production by resident macrophages, but did not enhance the NO production by HCE-derived macrophage, which however, was enhanced by Con A, suggesting that HCE and Con A induce NO production by different routes. In conclusion, HCE-treatment was able to increase the macrophages activity and also the cellular recruitment to secondary lymphoid organs, what could explain the previously related anti-tumor activity of Chenopodium ambrosioides.