scholarly journals Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ

2021 ◽  
Vol 9 (1) ◽  
pp. 1-10
Author(s):  
Seyedeh Elham Badiee Kheirabadi ◽  
◽  
Kazem Mashayekhi ◽  
Malihe Moghadam ◽  
Mohammad Javad Mousavi ◽  
...  
Keyword(s):  
Western Blotting ◽  
Large Scale ◽  
Target Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pcr Amplification ◽  
Interferon Γ ◽  
Bacterial Cells ◽  
Experimental Conditions ◽  
Recombinant Mouse ◽  
Ifn Γ

Background: Interferon-gamma [IFN-γ) is the most important cytokine in the immune system. This protein has been expressed in bacterial cells. However, bacterial cloning is not an easy task. We aimed to clone, express, and purify recombinant mouse IFN-γ and overcome problems in favor of commercial purposes. Materials and Methods: To amplify the gene product for cloning, we primarily designed two specific primers for the target gene. Following PCR amplification, the amplicon was inserted into the pET-21b[+) vector. The E. coli BL21 [DE3) CodonPlus strain was chosen for the expression of the target gene. Finally, the expressed recombinant mouse IFN-γ was assessed through the western blotting method. Results: We performed a cloning process and produced recombinant mouse IFN-γ in an optimal condition. We also noticed that monomeric protein could be transformed to a homodimeric structure which can be observed using the SDS PAGE [SDS-polyacrylamide gel electrophoresis) and western blotting. Conclusion: Experimental conditions strongly affect the large-scale cloning procedures required to be optimized in each laboratory. The expressed recombinant mouse IFN-γ described here is appropriate for commercial purposes.

scholarly journals MyD88 Primes Macrophages for Full-Scale Activation by Interferon-γ yet Mediates Few Responses to Mycobacterium tuberculosis

2003 ◽  
Vol 198 (7) ◽  
pp. 987-997 ◽  
Author(s):  
Shuangping Shi ◽  
Carl Nathan ◽  
Dirk Schnappinger ◽  
Jörg Drenkow ◽  
Michele Fuortes ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
Nuclear Factor Κb ◽  
Interferon Γ ◽  
Full Scale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ifn Γ ◽  
Microbial Products ◽  
Oxide Synthase

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88−/− macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-γ from 5- to 100-fold less extensively in MyD88−/− macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-γ requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor κB, which synergizes with IFN-γ for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-γ–dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

scholarly journals Impact of Prior Influenza and Pneumoccocal Vaccines on Humoral and Cellular Response to SARS-CoV-2 BNT162b2 Vaccination

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 615
Author(s):  
Vincenzo Puro ◽  
Concetta Castilletti ◽  
Chiara Agrati ◽  
Delia Goletti ◽  
Sara Leone ◽  
...  
Keyword(s):  
Immune Response ◽  
Cellular Response ◽  
Large Scale ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Pneumococcal Vaccination ◽  
Interferon Γ ◽  
Direct Care ◽  
Neutralization Titer ◽  
Ifn Γ

Vaccination against SARS-CoV-2 is considered the most effective method of prevention to contain the pandemic. While highly effective SARS-CoV-2 vaccines are being applied on a large-scale, whether and to what extent the strength of the vaccine-induced immune response could be further potentiated is still an object of debate. Several reports studied the effect of different vaccines on the susceptibility and mortality of COVID-19, with conflicting results. We aimed to evaluate whether previous influenza and/or pneumococcal vaccination had an impact on the specific immune response to the SARS-CoV-2 BNT162b2 mRNA vaccine. The study population consists of 710 workers from our Institute who completed the BNT162b2 schedule and have been tested at least once after the second dose, from 27 December 2020 up to 15 April 2021. Of these, 152 (21.4%) had received an influenza and 215 (30.3%) a concomitant influenza and pneumococcal vaccination, a median of 102 days before the second dose of BNT162b2. Overall, 100% of workers were tested for anti-Spike receptor-binding domain (anti-S/RBD) antibodies, 224 workers for neutralization titer (Micro-neutralization assay, MNA), and 155 workers for a spike-specific T cell interferon-γ response (IFN-γ). The levels of anti-S/RBD, MNA and IFN-γ were evaluated and compared according to sex, age, involvement in direct care of COVID-19 patients, and previous influenza/pneumococcal vaccination. At the univariate analysis, no statistically significant association was observed with regard to a previous influenza and pneumococcal vaccination. A significant lower anti-S/RBD response was observed according to an older age and male sex, while MNA titers were significantly associated to sex but not to age. At the multivariable analysis, workers receiving a concomitant influenza and pneumococcal vaccination or only influenza showed a 58% (p 0.01) and 42% (p 0.07) increase in MNA titers, respectively, compared to those who did not receive an influenza/pneumococcal vaccination. Female workers showed an 81% MNA and a 44% anti-S/RBD increase compared to male workers (p < 0.001). Compared to workers aged 21 to 49 years, those aged 50 or older were associated with a reduction in the anti-S/RBD (16%; p 0.005), MNA (31%; p 0.019), and IFN.g (32%) immune response. Maintaining the influenza and pneumococcal immunization program for the coming season, in which COVID-19 could still be spreading, remains strongly recommended to protect those who are more vulnerable and to limit the potential burden of these infections on the healthcare system.

scholarly journals Lysozyme Aptamer-Functionalized Magnetic Nanoparticles for the Purification of Lysozyme from Chicken Egg White

Foods ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 67 ◽  
Author(s):  
Ruiping Luo ◽  
Xinrui Zhou ◽  
Yan Chen ◽  
Sicheng Tuo ◽  
Fulin Jiang ◽  
...  
Keyword(s):  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Superparamagnetic Nanoparticles ◽  
Maximum Adsorption Capacity ◽  
Amino Groups ◽  
Loop Analysis ◽  
Experimental Conditions ◽  
Fe3o4 Nps

Lysozyme is in high demand due to its many favorable characteristics such as being naturally occurring, non-toxic, and easy to digest and absorb. Recently, superparamagnetic nanoparticles with strong magnetic responsiveness have attracted significant interest for enzyme purification. The aptamer of the enzyme can be chemically synthesized rapidly at a large scale using simple and low-cost preparation methods. Therefore, Fe3O4 nanoparticles (Fe3O4 NPs) were prepared by chemical co-precipitation and were then functionalized with amino groups to produce NH2-Fe3O4 NPs. The specific reaction of aldehyde and amino groups was used to attach lysozyme aptamers with specific sequences to NH2-Fe3O4 NPs to produce Apt-NH2-Fe3O4 NPs. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), hysteresis loop analysis, and thermogravimetric analysis (TGA). The optimal experimental conditions for adsorption of lysozyme were investigated. The effects of initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength were determined. The maximum adsorption capacity and relevant activity of Apt-NH2-Fe3O4 NPs was 460 mg·g−1 and 16,412 ± 55 U·mg−1 in an aqueous lysozyme solution. In addition, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis analysis, lysozyme could be separated from crude fresh egg white using Apt-NH2-Fe3O4 NPs with an amount up to 113 ± 4.2 mg·g−1 and an activity up to 16,370 46 U·mg−1.

scholarly journals Natural History and Evolution of Anti-Interferon-γ Autoantibody-Associated Immunodeficiency Syndrome in Thailand and the United States

2019 ◽  
Vol 71 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Gloria H Hong ◽  
Ana M Ortega-Villa ◽  
Sally Hunsberger ◽  
Ploenchan Chetchotisakd ◽  
Siriluck Anunnatsiri ◽  
...  
Keyword(s):  
United States ◽  
Natural History ◽  
The United States ◽  
Interferon Γ ◽  
Mycobacterium Abscessus ◽  
Annual Data ◽  
Persistent Infections ◽  
Ifn Γ ◽  
Immunodeficiency Syndrome

Abstract Background The natural history of anti-interferon-γ (IFN-γ) autoantibody-associated immunodeficiency syndrome is not well understood. Methods Data of 74 patients with anti-IFN-γ autoantibodies at Srinagarind Hospital, Thailand, were collected annually (median follow-up duration, 7.5 years). Annual data for 19 patients and initial data for 4 patients with anti-IFN-γ autoantibodies at the US National Institutes of Health were collected (median follow-up duration, 4.5 years). Anti-IFN-γ autoantibody levels were measured in plasma samples. Results Ninety-one percent of US patients were of Southeast Asian descent; there was a stronger female predominance (91%) in US than Thai (64%) patients. Mycobacterium abscessus (34%) and Mycobacterium avium complex (83%) were the most common nontuberculous mycobacteria in Thailand and the United States, respectively. Skin infections were more common in Thailand (P = .001), whereas bone (P &lt; .0001), lung (P = .002), and central nervous system (P = .03) infections were more common in the United States. Twenty-four percent of Thai patients died, most from infections. None of the 19 US patients with follow-up data died. Anti-IFN-γ autoantibody levels decreased over time in Thailand (P &lt; .001) and the United States (P = .017), with either cyclophosphamide (P = .01) or rituximab therapy (P = .001). Conclusions Patients with anti-IFN-γ autoantibodies in Thailand and the United States had distinct demographic and clinical features. While titers generally decreased with time, anti-IFN-γ autoantibody disease had a chronic clinical course with persistent infections and death. Close long-term surveillance for new infections is recommended.

scholarly journals The Role of Interleukine-10 and Interferon-γ as Potential Markers of the Evolution of African Swine Fever Virus Infection in Wild Boar

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 757
Author(s):  
Sandra Barroso-Arévalo ◽  
Jose A. Barasona ◽  
Estefanía Cadenas-Fernández ◽  
José M. Sánchez-Vizcaíno
Keyword(s):  
Wild Boar ◽  
Vaccine Candidate ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Interferon Γ ◽  
Fever Virus ◽  
Control Group ◽  
Challenge Virus ◽  
Ifn Γ

African swine fever virus (ASFv) is one of the most challenging pathogens to affect both domestic and wild pigs. The disease has now spread to Europe and Asia, causing great damage to the pig industry. Although no commercial vaccine with which to control the disease is, as yet, available, some potential vaccine candidates have shown good results in terms of protection. However, little is known about the host immune mechanisms underlying that protection, especially in wild boar, which is the main reservoir of the disease in Europe. Here, we study the role played by two cytokines (IL-10 and IFN-γ) in wild boar orally inoculated with the attenuated vaccine candidate Lv17/WB/Rie1 and challenged with a virulent ASFv genotype II isolate. A group of naïve wild boar challenged with the latter isolate was also established as a control group. Our results showed that both cytokines play a key role in protecting the host against the challenge virus. While high levels of IL-10 in serum may trigger an immune system malfunctioning in challenged animals, the provision of stable levels of this cytokine over time may help to control the disease. This, together with high and timely induction of IFN-γ by the vaccine candidate, could help protect animals from fatal outcomes. Further studies should be conducted in order to support these preliminary results and confirm the role of these two cytokines as potential markers of the evolution of ASFV infection.

scholarly journals THU0052 RELATIONSHIP BETWEEN INTERFERON-Γ-PRODUCING IMMUNOCOMPETENT CELLS AND DISEASE ACTIVITY IN ADULT-ONSET STILL’S DISEASE

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 238.1-238
Author(s):  
Y. Shimojima ◽  
D. Kishida ◽  
T. Ichikawa ◽  
Y. Sekijima
Keyword(s):  
T Cells ◽  
Disease Activity ◽  
Nk Cells ◽  
Acute Phase ◽  
Serum Ferritin ◽  
Interferon Γ ◽  
Adult Onset Still’S Disease ◽  
Still’S Disease ◽  
Ifn Γ ◽  
Adult Onset Still's Disease

Background:In the acute phase of adult-onset Still’s disease (AOSD), elevated levels of proinflammatory cytokines including interferon-γ (IFN-γ) are shown. Moreover, IFN-γ impacts on activating macrophages which play a crucial role in the pathogenesis of AOSD. Natural killer (NK) cells and T helper cells are in charge of secreting IFN-γ in the innate and adaptive immune systems of disease, respectively. However, the features of their IFN-γ-producing variation depending on disease activity are still uncertain in AOSD.Objectives:We investigated characteristics of IFN-γ-producing CD4+T cells and NK cells in patients with AOSD.Methods:Twenty-four patients in the acute phase of AOSD (active AOSD), 8 of them after treatment (remission), and 12 healthy controls (HC) were recruited in this study. Peripheral blood mononuclear cells and serum samples were provided from them for the experimental analysis. Flow cytometry was used for analyzing CD4+T cells, CD4+regulatory T cells (Tregs), NK cells, and their intracellular IFN-γ expression levels as well as suppression assay of Tregs. The serum concentration of interleukin-18 (IL-18) was measured using commercially available ELISA kit. Relationship between the analyzed data and clinical findings related to disease activity were statistically evaluated.Results:IFN-γ expression in CD4+T cells was significantly higher in active AOSD than in HC (p < 0.05). Tregs also significantly indicated higher expression of IFN-γ in active AOSD than in HC (p < 0.0001); and moreover, Tregs were significantly impaired in their suppression ability (p < 0.05). In both CD4+T cells and Tregs, expression of IFN-γ was significantly correlated with serum ferritin levels in active AOSD (p < 0.05). IFN-γ expression in CD4+T cells was significantly higher in patients with splenomegaly than those without that (p < 0.05). The proportion of NK cells was significantly lower in active AOSD than in HC (p < 0.005), whereas IFN-γ expression in NK cells was significantly higher in active AOSD than in HC (p < 0.0005). The number of NK cells and IFN-γ-expressing NK cells had inverse relationship with serum ferritin levels in active AOSD (p < 0.05 and p < 0.005, respectively). Increased number of NK cells and their decreased expression of IFN-γ were significantly demonstrated in remission (p < 0.05). In the analyses of NK cell subsets, lower expression of IFN-γ in CD56brightNK cells and higher that in CD56dimNK cells were significantly indicated in active AOSD than HC (p < 0.05). In remission, IFN-γ expression was significantly decreased in CD56dimNK cells (p < 0.05) despite no significant recovery of that in CD56brightNK cells (p = 0.311). Meanwhile, increased expression of IFN-γ in CD56brightNK cells was demonstrated in only patients who were treated with biologics. Although serum levels of IL-18 were significantly higher in active AOSD than in remission and HC; however, they had no significant correlations with any analyzed data.Conclusion:CD4+T cells and NK cells promote IFN-γ expression in the acute phase of AOSD. Meanwhile, increased expression of IFN-γ in CD4+T cells and decreased number of NK cells were correlated with serum ferritin levels, suggesting that they are indicators of disease activity. Furthermore, high disease activity may impact on the alteration of IFN-γ-producing balance in two distinct population of NK cells, and the plasticity of Tregs leading to defect in suppression ability.Disclosure of Interests:None declared

scholarly journals Compartment and hub definitions tune metabolic networks for metabolomic interpretations

GigaScience ◽  
2020 ◽  
Vol 9 (1) ◽  
Author(s):  
T Cameron Waller ◽  
Jordan A Berg ◽  
Alexander Lex ◽  
Brian E Chapman ◽  
Jared Rutter
Keyword(s):  
Large Scale ◽  
Metabolic Networks ◽  
Shortest Paths ◽  
Large Data ◽  
Differential Regulation ◽  
Large Data Sets ◽  
Data Sets ◽  
Human Metabolism ◽  
Experimental Conditions ◽  
Systemic Model

Abstract Background Metabolic networks represent all chemical reactions that occur between molecular metabolites in an organism’s cells. They offer biological context in which to integrate, analyze, and interpret omic measurements, but their large scale and extensive connectivity present unique challenges. While it is practical to simplify these networks by placing constraints on compartments and hubs, it is unclear how these simplifications alter the structure of metabolic networks and the interpretation of metabolomic experiments. Results We curated and adapted the latest systemic model of human metabolism and developed customizable tools to define metabolic networks with and without compartmentalization in subcellular organelles and with or without inclusion of prolific metabolite hubs. Compartmentalization made networks larger, less dense, and more modular, whereas hubs made networks larger, more dense, and less modular. When present, these hubs also dominated shortest paths in the network, yet their exclusion exposed the subtler prominence of other metabolites that are typically more relevant to metabolomic experiments. We applied the non-compartmental network without metabolite hubs in a retrospective, exploratory analysis of metabolomic measurements from 5 studies on human tissues. Network clusters identified individual reactions that might experience differential regulation between experimental conditions, several of which were not apparent in the original publications. Conclusions Exclusion of specific metabolite hubs exposes modularity in both compartmental and non-compartmental metabolic networks, improving detection of relevant clusters in omic measurements. Better computational detection of metabolic network clusters in large data sets has potential to identify differential regulation of individual genes, transcripts, and proteins.

Regulation of mouse natural killer (NK) activity by interferon-γ (IFN-γ)

1985 ◽  
Vol 7 (3) ◽  
pp. 327
Author(s):  
F. Velotti ◽  
A. Santoni ◽  
F. Cofano ◽  
S. Landolfo ◽  
M. Piccoli ◽  
...  
Keyword(s):  
Natural Killer ◽  
Interferon Γ ◽  
Nk Activity ◽  
Ifn Γ

A Summary of the Third Global Interferon-γ Release Assay Symposium

2013 ◽  
Vol 34 (6) ◽  
pp. 619-624 ◽  
Author(s):  
Antonino Catanzaro ◽  
Charles Daley
Keyword(s):  
Immune Response ◽  
Tuberculin Skin Test ◽  
Skin Test ◽  
Interferon Γ ◽  
Enzyme Linked Immunosorbent Assay ◽  
In Vitro Tests ◽  
The Third ◽  
Specific Antigens ◽  
Ifn Γ

Studies over the past several decades have dramatically increased our understanding of the immune response to Mycobacterium tuberculosis infection, and advances in proteomics and genomics have led to a new class of immune-diagnostic tests, termed interferon-γ (IFN-γ) release assays (IGRAs), which appear to obviate many of the problems encountered with the tuberculin skin test (TST). Worldwide, 2 IGRAs are currently commercially available. QuantiFERON-TB Gold In-Tube (Cellestis) is a third-generation product that uses an enzyme-linked immunosorbent assay to measure IFN-γ generated in whole blood stimulated with M. tuberculosis–specific antigens. T-Spot-TB (Oxford Immunotec) employs enzyme-linked immunosorbent spot technology to enumerate the number of purified lymphocytes that respond to M. tuberculosis–specific antigens by producing IFN-γ. These in vitro tests measure the host immune response to M. tuberculosis–specific antigens, which virtually eliminates false-positive cross reactions caused by bacillus Calmette-Guérin vaccination and/or exposure to environmental nontuberculous mycobacteria that plague the interpretation and accuracy of the tuberculin skin test (TST). The high specificity of IGRAs, together with sensitivity commensurate with or better than that of the TST, promises an accurate diagnosis and the ability to focus tuberculosis-control activities on those who are actually infected with M. tuberculosis. The Third Global Symposium was held over a 3-day period and was presented by the University of California, San Diego, Continuing Medical Education department; slides and sound recordings of each presentation are available at http://cme.ucsd.edu/igras/syllabus.html. A moderated discussion is also available at http://cme.ucsd.edu/igrasvideo. This document provides a summary of the key findings of the meeting, specifically focusing on the use of IGRAs in screening healthcare worker populations.

scholarly journals Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages

2003 ◽  
Vol 197 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Courtney Beers ◽  
Karen Honey ◽  
Susan Fink ◽  
Katherine Forbush ◽  
Alexander Rudensky
Keyword(s):  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Interferon Γ ◽  
Cathepsin S ◽  
Antigen Presenting Cell ◽  
Relative Contribution ◽  
Helper Cell Type ◽  
Ifn Γ ◽  
Antigen Presenting

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mϕs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mϕs. In addition, our studies show that the level of catL activity is significantly decreased in Mϕs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mϕs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mϕs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mϕs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.

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