Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

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Riber, Ulla; Boesen, Henriette Toft; Jakobsen, Jeanne Toft

2011-01-01

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evalu......The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult...

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals.

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Mendiratta, Sanjay; Vajpayee, Madhu; Malhotra, Uma; Kaushik, Shweta; Dar, Lalit; Mojumdar, Kamalika; Chauhan, Neeraj Kumar; Sreenivas, Vishnubhatla

2009-02-01

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

Development of a lion-specific interferon-gamma assay.

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Maas, M; van Kooten, P J S; Schreuder, J; Morar, D; Tijhaar, E; Michel, A L; Rutten, V P M G

2012-10-15

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species. Copyright © 2012 Elsevier B.V. All rights reserved.

Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

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Lyerly Herbert K

2008-03-01

Full Text Available Abstract Background Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC, as well as tetramer assays. Results Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen. Conclusion These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

TLR2-dependent inhibition of macrophage responses to IFN-gamma is mediated by distinct, gene-specific mechanisms.

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Sarah A Benson

2009-07-01

Full Text Available Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-gamma through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam(3CSK(4 and assayed responses to IFN-gamma. Pam(3CSK(4 stimulation prior to IFN-gamma inhibited transcription of the unrelated IFN-gamma-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-gamma-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-kappaB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

Short communication an interferon-γ ELISPOT assay with two cytotoxic T cell epitopes derived from HTLV-1 tax region 161-233 discriminates HTLV-1-associated myelopathy/tropical spastic paraparesis patients from asymptomatic HTLV-1 carriers in a Peruvian population.

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Best, Ivan; López, Giovanni; Talledo, Michael; MacNamara, Aidan; Verdonck, Kristien; González, Elsa; Tipismana, Martín; Asquith, Becca; Gotuzzo, Eduardo; Vanham, Guido; Clark, Daniel

2011-11-01

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic and progressive disorder caused by the human T-lymphotropic virus type 1 (HTLV-1). In HTLV-1 infection, a strong cytotoxic T cell (CTL) response is mounted against the immunodominant protein Tax. Previous studies carried out by our group reported that increased IFN-γ enzyme-linked immunospot (ELISPOT) responses against the region spanning amino acids 161 to 233 of the Tax protein were associated with HAM/TSP and increased HTLV-1 proviral load (PVL). An exploratory study was conducted on 16 subjects with HAM/TSP, 13 asymptomatic carriers (AC), and 10 HTLV-1-seronegative controls (SC) to map the HAM/TSP-associated CTL epitopes within Tax region 161-233. The PVL of the infected subjects was determined and the specific CTL response was evaluated with a 6-h incubation IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs) stimulated with 16 individual overlapping peptides covering the Tax region 161-233. Other proinflammatory and Th1/Th2 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. In addition, a set of human leukocyte antigen (HLA) class I alleles that bind with high affinity to the CTL epitopes of interest was determined using computational tools. Univariate analyses identified an association between ELISPOT responses to two new CTL epitopes, Tax 173-185 and Tax 181-193, and the presence of HAM/TSP as well as an increased PVL. The HLA-A*6801 allele, which is predicted to bind to the Tax 181-193 peptide, was overpresented in the HAM/TSP patients tested.

Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction

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Huang Jun

2012-09-01

Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. Results We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A, lysine (K or aspartic acid (D, respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. Conclusions We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.

High Reproducibility of ELISPOT Counts from Nine Different Laboratories

DEFF Research Database (Denmark)

Sundararaman, Srividya; Karulin, Alexey Y; Ansari, Tameem

2015-01-01

The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable...

A molecular assay for sensitive detection of pathogen-specific T-cells.

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Victoria O Kasprowicz

Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

Critical role of IFN-gamma in CFA-mediated protection of NOD mice from diabetes development.

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Mori, Yoshiko; Kodaka, Tetsuro; Kato, Takako; Kanagawa, Edith M; Kanagawa, Osami

2009-11-01

IFN-gamma signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN-gamma signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN-gamma receptor-deficient (IFN-gammaR(-/-)) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice. These data clearly show that the pro-inflammatory cytokine IFN-gamma is necessary for the CFA-mediated protection of NOD mice from diabetes. There is no difference in the T(h)1/T(h)17 balance between IFN-gammaR(-/-) NOD and wild-type NOD mice. There is also no difference in the total numbers and percentages of regulatory T (Treg) cells in the lymph node CD4(+) T-cell populations between IFN-gammaR(-/-) NOD and wild-type NOD mice. However, pathogenic T cells lacking IFN-gammaR are resistant to the suppressive effect of Treg cells, both in vivo and in vitro. Therefore, it is likely that CFA-mediated protection against diabetes development depends on a change in the balance between Treg cells and pathogenic T cells, and IFN-gamma signaling seems to control the susceptibility of pathogenic T cells to the inhibitory activity of Treg cells.

S100A7 (Psoriasin), highly expressed in Ductal Carcinoma In Situ (DCIS), is regulated by IFN-gamma in mammary epithelial cells

International Nuclear Information System (INIS)

Petersson, Stina; Bylander, Anna; Yhr, Maria; Enerbäck, Charlotta

2007-01-01

The aim of the present work was to explore signal transduction pathways used in the regulation of S100A7 (psoriasin). Members of the S100 gene family participate in many important cellular functions. Psoriasin, S100A8 (calgranulin A) and S100A9 (calgranulin B) are expressed in ductal carcinoma in situ (DCIS), as well as in the hyperproliferative skin disease, psoriasis. In the latter condition, a disturbance in the STAT pathway has recently been reported. This pathway is implicated in the regulation of IFN-gamma, widely recognized as a key cytokine in psoriasis. IFN-gamma also exerts anti-tumor action in a number of tumor cell types, including breast cancer. We therefore examined the effect of IFN-gamma and STAT-signaling on the psoriasin expression. We established a TAC2 mouse mammary epithelial cell line with tetracycline-inducible psoriasin expression (Tet-Off). Viability in cell culture was estimated using MTS assay. Protein and gene expression were evaluated by Western blotting and quantitative real-time PCR. Statistical analyses were assessed using a one-tailed, paired t-test. We report the downregulation of psoriasin by IFN-gamma in the MDA-MB-468 breast cancer cell line, as well as the downregulation of psoriasin induced by anoikis in cell lines derived from different epithelial tissues. In contrast, IFN-gamma had no suppressive effect on calgranulin A or calgranulin B. IFN-gamma is an important activator of the STAT1 pathway and we confirmed an active signaling pathway in the cell lines that responded to IFN-gamma treatment. In contrast, in the SUM190 breast carcinoma cell line, IFN-gamma did not suppress the expression of endogenous psoriasin. Moreover, a reduced phosphorylation of the STAT1 protein was observed. We showed that IFN-gamma treatment and the inhibition of the transcription factor NFkappaB had a synergistic effect on psoriasin levels. Finally, in TAC2 cells with tetracycline-induced psoriasin expression, we observed the increased viability of

Lion (Panthera leo) and cheetah (Acinonyx jubatus) IFN-gamma sequences.

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Maas, Miriam; Van Rhijn, Ildiko; Allsopp, Maria T E P; Rutten, Victor P M G

2010-04-15

Cloning and sequencing of the full length lion and cheetah interferon-gamma (IFN-gamma) transcript will enable the expression of the recombinant cytokine, to be used for production of monoclonal antibodies and to set up lion and cheetah-specific IFN-gamma ELISAs. These are relevant in blood-based diagnosis of bovine tuberculosis, an important threat to lions in the Kruger National Park. Alignment of nucleotide and amino acid sequences of lion and cheetah and that of domestic cats showed homologies of 97-100%. Copyright 2009 Elsevier B.V. All rights reserved.

Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis▿ †

Science.gov (United States)

Feske, Marsha; Nudelman, Rodolfo J.; Medina, Miguel; Lew, Justin; Singh, Manisha; Couturier, Jacob; Graviss, Edward A.; Lewis, Dorothy E.

2008-01-01

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB. PMID:18753334

Targeted Recombinant Fusion Proteins of IFN gamma and Mimetic IFN gamma with PDGF beta R Bicyclic Peptide Inhibits Liver Fibrogenesis In Vivo

NARCIS (Netherlands)

Bansal, Ruchi; Prakash, Jai; De Ruiter, Marieke; Poelstra, Klaas

2014-01-01

Hepatic stellate cells (HSCs), following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFN gamma) is a potent anti-fibrotic cytokine, but its

Use of the johnin PPD interferon-gamma assay in control of bovine paratuberculosis

DEFF Research Database (Denmark)

Jungersen, Gregers; Mikkelsen, Heidi; Grell, Susanne N.

2012-01-01

Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other...

IFN-gamma-induced chemokines synergize with pertussis toxin to promote T cell entry to the central nervous system

DEFF Research Database (Denmark)

Millward, Jason M; Caruso, Maria; Campbell, Iain L

2007-01-01

Inflammation of the CNS, which occurs during multiple sclerosis and experimental autoimmune encephalomyelitis, is characterized by increased levels of IFN-gamma, a cytokine not normally expressed in the CNS. To investigate the role of IFN-gamma in CNS, we used intrathecal injection of a replication......-defective adenovirus encoding murine IFN-gamma (AdIFNgamma) to IFN-gamma-deficient (GKO) mice. This method resulted in stable, long-lived expression of IFN-gamma that could be detected in cerebrospinal fluid using ELISA and Luminex bead immunoassay. IFN-gamma induced expression in the CNS of message and protein...... was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-gamma-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-gamma vector, there was a dramatic increase in the number of T...

Thyroid epithelial cell hyperplasia in IFN-gamma deficient NOD.H-2h4 mice.

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Yu, Shiguang; Sharp, Gordon C; Braley-Mullen, Helen

2006-01-01

The role of inflammatory cells in thyroid epithelial cell (thyrocyte) hyperplasia is unknown. Here, we demonstrate that thyrocyte hyperplasia in IFN-gamma-/- NOD.H-2h4 mice has an autoimmune basis. After chronic exposure to increased dietary iodine, 60% of IFN-gamma-/- mice had severe thyrocyte hyperplasia with minimal or moderate lymphocyte infiltration, and thyroid dysfunction with reduced serum T4. All mice produced anti-thyroglobulin autoantibody. Some wild-type NOD.H-2h4 mice had isolated areas of thyrocyte hyperplasia with predominantly lymphocytic infiltration, whereas IL-4-/- and 50% of wild-type NOD.H-2h4 mice developed lymphocytic thyroiditis but no thyrocyte hyperplasia. Both thyroid infiltrating inflammatory cells and environmental factors (iodine) were required to induce thyrocyte hyperplasia. Splenocytes from IFN-gamma-/- mice with thyrocyte hyperplasia, but not splenocytes from naïve IFN-gamma-/- mice, induced hyperplasia in IFN-gamma-/- NOD.H-2h4.SCID mice. These results may provide clues for understanding the mechanisms underlying development of epithelial cell hyperplasia not only in thyroids but also in other tissues and organs.

Immunological tolerance induced by galectin-1 in rat allogeneic renal transplantation.

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Xu, Gaosi; Tu, Weiping; Xu, Chengyun

2010-06-01

The existed literatures indicated that galectin-1 has anti-inflammatory effects and plays a pivotal role in autoimmune diseases. Present study was to identify the roles of galectin-1 in acute animal renal allograft rejection. Rat acute rejection models were erected by allogeneic renal transplantation. Galectin-1 injection was performed in different concentrations in renal recipients post-transplantation. Recipient survivals, CD8+ T cell proliferation, production of IFN-gamma, levels of serum CD30, enzyme-linked immunoabsorbent spot assay (ELISPOT) and immunohistochemistry were observed or tested 7days after renal transplantation. Galectin-1 injection can prolong the recipient animal survival, reduce the serum levels of IFN-gamma, soluble CD30, percentage of CD8+ T cell subset, CD8+ T cell-mediated cytotoxicity, and IFN-gamma ELISPOT frequency for allograft recipients. The therapeutic effects of galectin-1 injection on recipient rats were dose-dependent. Galectin-1 plays an important role in CD8+ T cell-mediated renal rejection by inducing immunological tolerance. Copyright 2010 Elsevier B.V. All rights reserved.

IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines

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Tran, E H; Prince, E N; Owens, T

2000-01-01

Dynamic interplay between cytokines and chemokines directs trafficking of leukocyte subpopulations to tissues in autoimmune inflammation. We have examined the role of IFN-gamma in directing chemokine production and leukocyte infiltration to the CNS in experimental autoimmune encephalomyelitis (EA......-gamma in EAE, acting on T cell proliferation and directing chemokine production, with profound implications for the onset and progression of disease.......). BALB/c and C57BL/6 mice are resistant to induction of EAE by immunization with myelin basic protein. However, IFN-gamma-deficient (BALB/c) and IFN-gammaR-deficient (C57BL/6) mice developed rapidly progressing lethal disease. Widespread demyelination and disseminated leukocytic infiltration of spinal...

Lambda Interferon (IFN-gamma), a Type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

DEFF Research Database (Denmark)

Ank, Nina; West, Hans; Bartholdy, C.

2006-01-01

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN......-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN......-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral...

In vitro activated CD4+ T cells from interferon-gamma (IFN-gamma)-deficient mice induce intestinal inflammation in immunodeficient hosts

DEFF Research Database (Denmark)

Bregenholt, S; Brimnes, J; Nissen, Mogens Holst

1999-01-01

To investigate the role of IFN-gamma in the immunopathogenesis of inflammatory bowel disease (IBD), severe combined immunodeficient (SCID) mice were transplanted with in vitro activated CD4+ T cells from either wild-type (WT) or IFN-gamma-deficient (IFN-gammaKO) BALB/c mice. In vitro, the two types...... of T cells displayed comparable proliferation rates and production of tumour necrosis factor-alpha (TNF-alpha), IL-2, IL-4 and IL-10 after concanavalin A (Con A) stimulation. When transplanted into SCID mice, WT CD4+ blasts induced a lethal IBD, whereas IFN-gammaKO blasts induced a less severe...... intestinal inflammation with moderate weight loss. Intracellular cytokine staining of lamina propria lymphocytes (LPL) revealed comparable fractions of CD4+ T cells positive for TNF-alpha, IL-2 and IL-10 in the two groups of transplanted SCID mice, whereas a two-to-three-fold increase in the fraction of IL-4...

Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats

DEFF Research Database (Denmark)

Johansen, H K; Hougen, H P; Rygaard, J

1996-01-01

In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon-gamma (rrIFN-gamma). Rats were treated either before or after intratracheal ch...

Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2012-01-01

of the study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The antigens used were 4 ESAT-6 family members, 4 latency proteins, 4 secreted proteins including Ag85B, 3 other antigens and PPDj......Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection can be detected by measuring antigen specific cell mediated immune responses by the interferon gamma (IFN-γ) assay. Available IFN-γ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives...... of the infected and non-infected herds were significantly (Passay using PPDj did not correlate with the results using the novel antigens since 5 of the 17 animals that were positive to PPDj were...

Interleukin-4 (IL-4) and Interferon-Gamma (IFN-gamma) in pregnant ...

African Journals Online (AJOL)

Background and Objective:- To assess if gestational factors affect the resistance of C57BL/6 mice to L. major infection, this study determined the levels of IL-4 and IFN-gamma in popliteal lymph node cells of pregnant C57BL/6 mice infected with L. major at 16 hours, 5 days-, 10 days- and 15 days- post plug by PCR, ELISA ...

IFN-{gamma} enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease

DEFF Research Database (Denmark)

Baron, Rona; Nemirovsky, Anna; Harpaz, Idan

2008-01-01

the spatial learning and memory performance of the animals. In older mice, the effect of IFN-gamma is more pronounced in both wild-type mice and mice with Alzheimer's-like disease and is associated with neuroprotection. In addition, IFN-gamma reverses the increase in oligodendrogenesis observed in a mouse...... mechanisms can generate immunity to such deficits in neuronal repair. We demonstrate that in contrast to primarily innate immunity cytokines, such as interleukin-6 and tumor necrosis factor-alpha, the adaptive immunity cytokine IFN-gamma enhances neurogenesis in the dentate gyrus of adult mice and improves...

Evaluating pleural ADA, ADA2, IFN-γ and IGRA for diagnosing tuberculous pleurisy.

Science.gov (United States)

Keng, Li-Ta; Shu, Chin-Chung; Chen, Jason Yao-Ping; Liang, Sheng-Kai; Lin, Ching-Kai; Chang, Lih-Yu; Chang, Chia-Hao; Wang, Jann-Yuan; Yu, Chong-Jen; Lee, Li-Na

2013-10-01

Conventional methods for diagnosing tuberculous pleurisy (TB pleurisy) are either invasive or have a long turn-around-time. Performances of pleural adenosine deaminase (ADA), ADA2, interferon-gamma (IFN-γ), and interferon-gamma release assays (IGRA) as diagnostic tools for TB pleurisy were evaluated. Eighty-eight patients with lymphocyte-predominant pleural exudates between June 2010 and March 2011, including 31 with clinically diagnosed TB pleurisy, were prospectively studied. Pleural ADA and ADA2 activity were measured by colorimetric method, IFN-γ levels by enzyme-linked immuno-sorbent assay, and IGRA by enzyme-linked immuno-spot (T-SPOT.TB) assay. Pleural ADA, ADA2, and IFN-γ levels, but not the proportion of positive T-SPOT.TB assay, were significantly higher in patients with TB pleurisy than in those without TB pleurisy. The area under the receiver-operating-characteristic (ROC) curve was 0.920, 0.893, 0.875, and 0.544 for IFN-γ, ADA2, ADA, and T-SPOT.TB assay, respectively. The combination of ADA ≥ 40 IU/L and IFN-γ ≥ 75 pg/mL yielded a specificity of 100%. Pleural ADA, ADA2 and IFN-γ, but not T-SPOT.TB assay, are all sensitive and specific for TB pleurisy. In patients with lymphocyte-predominant pleural exudates, ADA ≥ 40 IU/L and IFN-γ ≥ 75 pg/mL in pleural effusion imply a very high probability of TB pleurisy. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Preassembly and ligand-induced restructuring of the chains of the IFN-gamma receptor complex: the roles of Jak kinases, Stat1 and the receptor chains.

Science.gov (United States)

Krause, Christopher D; Lavnikova, Natasha; Xie, Junxia; Mei, Erwen; Mirochnitchenko, Olga V; Jia, Yiwei; Hochstrasser, Robin M; Pestka, Sidney

2006-01-01

We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-gamma) receptor complex is preassembled (1). In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-gammaR1 results in a conformational change localized to IFN-gammaR1. Jak1 but not Jak2 is required for the two chains of the IFN-gamma receptor complex (IFN-gammaR1 and IFN-gammaR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-gammaR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-gammaR2 with IFN-gammaR1. These results agree with a detailed model of the IFN-gamma receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Identification of IFN-gamma-producing CD4+ T cells following PMA stimulation

DEFF Research Database (Denmark)

Kemp, K; Bruunsgaard, H

2001-01-01

Treatment of T cells with phorbol esters, such as phorbol myristate acetate (PMA), induces downregulation of CD4, making unambiguous identification of this subset difficult. In this study, the kinetics of intracellular expression of interferon-gamma (IFN-gamma) and downmodulation of surface CD4...... were measured in peripheral blood mononuclear cells (PBMC) after PMA stimulation. The number of IFN-gamma-producing cells increased within a 4-h period while the fluorescence intensity of the CD4(+) cell population decreased, and the two phenomena were correlated (n = 9; p = 0.01). Our data suggest...... that intracellular staining of CD4 together with cytokine staining will make identification of CD4(+) cells possible and facilitate the procedure of intracellular staining of cytokines....

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

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Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade

2006-12-15

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT

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Morse Michael A

2005-07-01

Full Text Available Abstract Background Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC, and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. Results Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001. The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001. Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation and tetramer staining (p Conclusion We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

Successful Application of the Gamma-Interferon Assay in a Bovine Tuberculosis Eradication Program: The French Bullfighting Herd Experience.

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Keck, Nicolas; Boschiroli, Maria-Laura; Smyej, Florence; Vogler, Valérie; Moyen, Jean-Louis; Desvaux, Stéphanie

2018-01-01

In the French Camargue region, where bovine tuberculosis had been enzootic for several years in bullfighting cattle herds, the gamma-interferon (IFN) assay was used since 2003 in parallel with the intradermal test in order to increase overall disease detection sensitivity in infected herds. This study presents the results of a field-evaluation of the assay during a 10-year period (2004-2014) of disease control and surveillance program and explores the particular pattern of IFN assay results in bullfight herds in comparison to cattle from other regions of France. The low sensitivity [59.2% (50.6; 67.3)] of IFN assay using the tuberculin stimulation could be related to the poor gamma-IFN production from bullfight cattle blood cells which is significantly lower than in animals of conventional breeds. The characteristics of the assay were progressively adapted to the epidemiological situation and the desired strategic applications. Data analysis with a receiver operating characteristic curve based on a simple S/P value algorithm allowed for the determination of a new cutoff adapted for a global screening, giving a high specificity of 99.9% results and a high accuracy of the assay. Having regularly risen to above 5% since 2005, with a peak around 10% in 2010, the annual incidence dropped to under 1% in 2014. The positive predictive value relative to the bacteriological confirmation evolved during the years, from 33% in 2009 to 12% during the last screening period, a normal trend in a context of decreasing prevalence. The estimated rate of false-positive reactions during screening campaigns was 0.67%, confirming the high specificity of the test, measured in bTB negative herds, in this epidemiological context. The proportion of false-positive reactions decreased with the age and was higher in males than in females. Although these results indicate that the IFN assay is accurate in the field, it also emphasizes great differences between interferon quantities produced by

Regulatory function of a novel population of mouse autoantigen-specific Foxp3 regulatory T cells depends on IFN-gamma, NO, and contact with target cells.

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Cyndi Chen

Full Text Available BACKGROUND: Both naturally arising Foxp3(+ and antigen-induced Foxp3(- regulatory T cells (Treg play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel class II MHC tetramer, we have isolated a population of CD4(+ Foxp3(- T cells specific for the autoantigen glutamic acid decarboxylase p286-300 peptide (NR286 T cells from diabetes-resistant non-obese resistant (NOR mice. These Foxp3(- NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-gamma was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg. CONCLUSIONS/SIGNIFICANCE: These studies identified a unique population of autoantigen-specific Foxp3(- Treg that can exert their regulatory function dependent on not only IFN-gamma and NO but also cell contact with target cells.

Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

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Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

1985-03-18

Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

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Jönsson, K H; Daas, A; Buchheit, K H; Terao, E

2016-01-01

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable

Thermostability of IFN-γ and IP-10 release assays for latent infection with Mycobacterium tuberculosis

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Blauenfeldt, Thomas; Wagner, Dirk; Aabye, Martine Grosos

2016-01-01

accuracy of IP-10 release assay and IGRAs. RESULTS: We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90-1.36) fold and IFN-γ responses decreased 0.88 (IQR 0......INTRODUCTION: Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher...

Evaluation of accuracy and uncertainty of ELISA assays for the determination of interleukin-4, interleukin-5, interferon-gamma and tumor necrosis factor-alpha

DEFF Research Database (Denmark)

Borg, Lone; Kristiansen, Jesper; Christensen, Jytte M

2002-01-01

. However, models for establishing the traceability and uncertainty of immunoassay results are lacking. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for determination of the human cytokines interleukin-4 (IL-4), interleukin-5 (IL-5), interferon-y (IFN-gamma) and tumor necrosis factor-alpha...... (TNF-alpha). The accuracy of each of the assays was evaluated in the ranges of 1-15 microg/l (IL-4), 0.001-1 microg/l (IL-5), 0.5-2.5 microg/l (IFN-T) and 0.14-2.2 microg/l (TNF-alpha). Other evaluated performance characteristics were the limit of detection (LOD), immunological specificity......) of the assessed ELISAs was found to be in the range of 11-18%, except for IL-5 where RSDA increased at decreasing concentrations. The LOD was 0.12 microg/l, 0.0077 microg/l, 0.0069 microg/l and 0.0063 microg/l for IL-4, IL-5, IFN-gamma and TNF-alpha, respectively. Traceability to the WHO IS was established...

Local IFN-gamma therapy of HPV16-associated tumours

Czech Academy of Sciences Publication Activity Database

Mikyšková, Romana; Bieblová, Jana; Šímová, Jana; Indrová, Marie; Jandlová, Táňa; Vonka, V.; Šmahel, M.; Bubeník, Jan; Mendoza, Luis

2003-01-01

Roč. 49, č. 1 (2003), s. 26-32 ISSN 0015-5500 R&D Projects: GA MZd NC7148; GA MZd NC5900; GA ČR GA301/01/0985 Grant - others:GA CZ(CZ) Liga proti rakovině Institutional research plan: CEZ:AV0Z5052915 Keywords : MHC class I * IFN-gamma * HPV16 Subject RIV: FD - Oncology ; Hematology Impact factor: 0.527, year: 2003

Nocardia brasiliensis Modulates IFN-gamma, IL-10, and IL-12 cytokine production by macrophages from BALB/c Mice.

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Salinas-Carmona, Mario C; Zúñiga, Juan M; Pérez-Rivera, Luz I; Segoviano-Ramírez, Juan C; Vázquez-Marmolejo, Anna V

2009-05-01

Interferon-gamma (IFN-gamma) is a critical cytokine involved in control of different infections. Actinomycetoma is a chronic infectious disease mainly caused by the bacterium Nocardia brasiliensis, which destroys subcutaneous tissue, including bone. Currently, the mechanism of pathogenesis in N. brasiliensis infection is not known. Here, we demonstrate that N. brasiliensis induced an IFN-gamma response in serum after 24 h of infection, while, in infected tissue, positive cells to IFN-gamma appeared in 2 early peaks: the first was present only 3 h after infection, then transiently decreased; and the second peak appeared 12 h after infection and was independent of interleukin-10. Resident macrophages produced an immediate IFN-gamma response 1 h after in vitro infection, and spleen-positive cells began later. The phase of growth of N. brasiliensis affected cytokine production, and exposure of macrophages to Nocardia opsonized with either polyclonal anti-Nocardia antibodies or anti-P61 monoclonal antibody led to a suppression of cytokine production. Our report provides evidence that N. brasiliensis as an intracellular bacterium modulates macrophage cytokine production, which helps survival of the pathogen. Modulation of these cytokines may contribute to pathogenesis once this bacterium is inside the macrophage.

Alopecia of IFN-gamma knockout mouse as a model for disturbance of the hair cycle: a unique arrest of the hair cycle at the anagen phase accompanied by mitosis.

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Hirota, Ryuichiro; Tajima, Sadao; Yoneda, Yukio; Tamayama, Takumi; Watanabe, Masahito; Ueda, Kouichi; Kubota, Takahiro; Yoshida, Ryotaro

2002-09-01

Interferon-gamma(-/-) (IFN-gamma(-/-)) and IFN-gamma(+/+) C57BL/6 mice (3 weeks of age) completed the production of morphogenesis-derived hair. Around 6 weeks of age, however, most of the IFN-gamma(-/-) but none of the IFN-gamma(+/+) mice began to lose hairs in the dorsal and occipital areas in the absence of inflammatory reactions, and the alopecia was sustained for at least several 10-week periods of observation. A single subcutaneous injection of IFN-gamma to IFN-gamma(-/-) mice at 3, but not 4, 5, or 8 weeks of age could protect all the mice from alopecia, revealing that the lack of IFN-gamma around 3 weeks of age is directly responsible for the alopecia. Histologic features showed that the hair follicles of the IFN-gamma(+/+) mice passed through the anagen (4-5 weeks of age) and catagen/telogen ( approximately 6 weeks of age) phases, whereas those of IFN-gamma(-/-) mice (5 weeks of age or older) stayed in the anagen phase. TUNEL and bromodeoxyuridine experiments suggested that an arrest with unlimited DNA synthesis of the hair cycle in the anagen phase by the lack of IFN-gamma-dependent apoptosis in the midfollicle region and diffuse shedding of previously formed hair induced alopecia in IFN-gamma(-/-) mice.

Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

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Kuo-Ching Sheng

2013-01-01

Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection

International Nuclear Information System (INIS)

Maier, K.; Gabriel, P.; Koscielniak, E.; Stierhof, Y.D.; Wiedmann, K.H.; Flehmig, B.; Vallbracht, A.

1988-01-01

The production of interferon (IFN) during a chromium-51 release assay with hepatitis A virus (HAV)-infected fibroblasts and autologous peripheral blood lymphocytes from patients with acute HAV infection was studied to determine whether IFN plays a role in immunopathogenesis of hepatitis A infection in humans. Skin fibroblasts of eight patients after acute HAV infection and from two control persons without history of current of past HAV infection were infected with HAV. Peripheral blood lymphocytes were collected at different times after the onset of icterus and tested in a chromium-51 release assay against autologous HAV-infected skin fibroblasts for their cytolytic and IFN-producing activity. The IFN produced during the assay was characterized and found to have the properties of human gamma IFN. Cytotoxicity and gamma IFN release were virus specific. The cell types responsible for both functions were characterized and found to be in the HLA-dependent T8 + lymphocyte subset. Considering that gamma IFN has an antiviral effect on persistent HAV infection in vitro and that it probably accounts for stimulation of HLA class I antigen expression on hepatocytes, these experimental results presented here demonstrate that human gamma IFN produced by HAV-specific T cells may participate in pathogenesis of hepatitis A infection in humans

CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.

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Haas, Jan D; González, Frano H Malinarich; Schmitz, Susanne; Chennupati, Vijaykumar; Föhse, Lisa; Kremmer, Elisabeth; Förster, Reinhold; Prinz, Immo

2009-12-01

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine.

Science.gov (United States)

Firbas, Christa; Boehm, Thomas; Buerger, Vera; Schuller, Elisabeth; Sabarth, Nicolas; Jilma, Bernd; Klade, Christoph S

2010-03-11

An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41. In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [(3)H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-gamma) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays. More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-gamma CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema. Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.

Immune defence against HIV-1 infection in HIV-1-exposed seronegative persons.

Science.gov (United States)

Schmechel, S C; Russell, N; Hladik, F; Lang, J; Wilson, A; Ha, R; Desbien, A; McElrath, M J

2001-11-01

Rare individuals who are repeatedly exposed to HIV-1 through unprotected sexual contact fail to acquire HIV-1 infection. These persons represent a unique study population to evaluate mechanisms by which HIV-1 replication is either prevented or controlled. We followed longitudinally a group of healthy HIV-1 seronegative persons each reporting repeated high-risk sexual activities with their HIV-1-infected partner at enrollment. The volunteers were primarily (90%) male homosexuals, maintaining high risk activities with their known infected partner (45%) or multiple other partners (61%). We evaluated the quantity and specificity of HIV-1-specific T cells in 31 exposed seronegatives (ES) using a IFN-gamma ELISPOT assay to enumerate T cells recognizing epitopes within HIV-1 Env, Gag, Pol and Nef. PBMC from only three of the 31 volunteers demonstrated ex vivo HIV-1-specific IFN-gamma secretion, in contrast to nearly 30% exhibiting cytolytic responses in previous studies. These findings suggest that if T cell responses in ES are induced by HIV-1 exposure, the frequency is at low levels in most of them, and below the level of detection using the ELISPOT assay. Alternative approaches to improve the sensitivity of detection may include use of dendritic cells as antigen-presenting cells in the ex vivo assay and more careful definition of the risk behavior and extent of HIV-1 exposure in conjunction with the evaluation of T cell responses.

Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

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Guillaume Sarrabayrouse

Full Text Available BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298 stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL, we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i activation of CD8 T lymphocytes (CD8+/CD69+; ii proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+; iii secretion of hIFN-gamma; and iv anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

Science.gov (United States)

Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

2011-06-01

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Compromised virus control and augmented perforin-mediated immunopathology in IFN-gamma-deficient mice infected with lymphocytic choriomeningitis virus

DEFF Research Database (Denmark)

Nansen, A; Jensen, Teis; Christensen, Jan Pravsgaard

1999-01-01

To define the role of IFN-gamma in the control of acute infection with a noncytopathogenic virus, mice with targeted defects of the genes encoding IFN-gamma, perforin, or both were infected i.v. with two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread...... in wild-type mice. Our results reveal that IFN-gamma is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome...... mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus...

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

International Nuclear Information System (INIS)

Rodríguez, Teresa; Méndez, Rosa; Del Campo, Ana; Jiménez, Pilar; Aptsiauri, Natalia; Garrido, Federico; Ruiz-Cabello, Francisco

2007-01-01

The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-γ-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-γ treatment in human melanoma cell lines. Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan ® Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-γ-treated cells. Altered IFN-γ mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-α led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-γ treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-α treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-γ in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-γ signaling pathway. We observed two distinct mechanisms of loss of IFN-γ inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-γ signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

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Magdalena Tary-Lehmann

2012-05-01

Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

Science.gov (United States)

Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

2012-01-01

Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-γ secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. PMID:24710419

Specific Oral Tolerance Induction Using IFN-Gamma in 2 Cases of Food-Dependent Exercise-Induced Anaphylaxis

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Geunwoong Noh

2013-01-01

Full Text Available Anaphylaxis induced by exercise after the intake of certain foods is referred to as food-dependent exercise-induced anaphylaxis (FDEIA. Only the preventive medication such as oral sodium cromoglycate and oral combined cetirizine-montelukast was tried in FDEIA. Specific oral tolerance induction (SOTI using IFN-gamma was tried in 2 cases of FDEIA for wheat. Merely, exercise accompanied every treatment just after the intake of allergenic foods during treatment. Patients acquired tolerance for wheat in both cases successfully. After treatment, two patients take wheat in their food living freely. Conclusively, SOTI using IFN-gamma was effective as the causative treatment for allergenic foods in FDEIA.

Inflammation in the CNS and Th17 Responses Are Inhibited by IFN-{gamma}-Induced IL-18 Binding Protein

DEFF Research Database (Denmark)

Millward, Jason M; Pedersen, Morten Løbner; Wheeler, Rachel D

2010-01-01

Inflammatory responses are essential for immune protection but may also cause pathology and must be regulated. Both Th1 and Th17 cells are implicated in the pathogenesis of autoimmune inflammatory diseases, such as multiple sclerosis. We show in this study that IL-18-binding protein (IL-18bp......), the endogenous inhibitor of the Th1-promoting cytokine IL-18, is upregulated by IFN-gamma in resident microglial cells in the CNS during multiple sclerosis-like disease in mice. Test of function by overexpression of IL-18bp in the CNS using a viral vector led to marked reduction in Th17 responses and robust...... inhibition of incidence, severity, and histopathology of disease, independently of IFN-gamma. The disease-limiting action of IL-18bp included suppression of APC-derived Th17-polarizing cytokines. IL-18bp thus acts as a sensor for IFN-gamma and can regulate both Th1 and Th17 responses in the CNS....

Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma.

Science.gov (United States)

Day, Yuan-Ji; Huang, Liping; Ye, Hong; Li, Li; Linden, Joel; Okusa, Mark D

2006-03-01

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.

Role of 4-1BB receptor in the control played by CD8(+ T cells on IFN-gamma production by Mycobacterium tuberculosis antigen-specific CD4(+ T Cells.

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Carla Palma

Full Text Available BACKGROUND: Antigen-specific IFN-gamma producing CD4(+ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+ T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+ T cells which suppressed IFN-gamma-secreting CD4(+ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+ T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+ T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+ T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+ T cells. The selective

Mullerian Inhibiting Substances (MIS) Augments IFN-gamma Mediated Inhibition of Breast Cancer Cell Growth

National Research Council Canada - National Science Library

Gupta, Vandana

2006-01-01

MIS is a member of the TGF family. The purpose of this study is to test the hypothesis that MIS and IFN-gamma might be more effective in the inhibition of breast cancer cell growth than either agent alone...

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling

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Elisa Fanunza

2018-02-01

Full Text Available The interferon (IFN system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24 is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE. Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

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Rajiv L Gaur

Full Text Available Activation of innate immunity via pathogen recognition receptors (PRR modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ release assays (IGRAs are functional T cell assays used to diagnose latent tuberculosis infection (LTBI; however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube with Toll-like receptor agonists poly(I:C, LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

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Martins, Marcia Valéria B S; Lima, Mônica Cristina B S; Duppre, Nadia C; Matos, Haroldo J; Spencer, John S; Brennan, Patrick J; Sarno, Euzenir N; Fonseca, Leila; Pereira, Geraldo M B; Pessolani, Maria Cristina V

2007-05-01

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome

Peripheral blood CD161+ T cells from asthmatic patients are activated during asthma attack and predominantly produce IFN-gamma.

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González-Hernández, Y; Pedraza-Sánchez, S; Blandón-Vijil, V; del Río-Navarro, B E; Vaughan, G; Moreno-Lafont, M; Escobar-Gutiérrez, A

2007-04-01

In humans, T cells expressing the CD161 molecule NKR-P1A constitute around 20% of the circulating CD3(+) cells and are potentially immunoregulatory in several diseases. Their role in asthma is not well known, but they could participate in asthma attacks. To determinate whether activation of CD161(+) T cells and their cytokine production correlate with clinical status of asthma, we analysed blood samples from asthma attack patients (AAP) and stable asthma patients (SAP) in comparison with healthy non-atopic controls (HC). There was a significant higher baseline expression of CD69 on T cells from AAP and the difference was more notorious on CD161(+) T cells; upregulation of CD69 was observed on both CD161(-) and CD161(+) T cells driven by Dermatophagoides pteronyssinus crude extract, whereas polyclonal stimulation with phorbol 12-myristate 13-acetate plus ionomycin predominantly induced IFN-gamma but no IL-4, IL-5 and IL-13 by CD161(+) T cells in all groups; upon polyclonal stimulation, there were more CD161(+) T cells producing IFN-gamma and less CD161(-) T cells producing this cytokine, contrasting with the opposite results observed in SAP and HC groups. Our results indicate that, during asthma attack, CD161(+) T cells are activated and are able to produce predominantly IFN-gamma but no Th2 cytokines. We hypothesize that during an asthma attack, IFN-gamma produced by CD161(+) T cells could help to reestablish the Th1/Th2 equilibrium. These observations may contribute to the understanding of the immune mechanisms involved in asthma attacks.

CD4(+) T cell-mediated control of a gamma-herpesvirus in B cell-deficient mice is mediated by IFN-gamma

DEFF Research Database (Denmark)

Christensen, Jan Pravsgaard; Cardin, R D; Branum, K C

1999-01-01

The lack of B cells and antibody does not prevent mice from dealing effectively with a pathogenic gamma-herpesvirus. Both CD4(+) and CD8(+) T cells contribute to the control of virus replication in the respiratory tract, with the depletion of either lymphocyte subset leading to increased titers...... for direct interaction with virus-infected targets expressing MHC class II glycoproteins, suggesting that the IFN-gamma produced by these lymphocytes is functioning at short range. The numbers of latently infected cells in the spleens of carrier mice are also significantly increased by the concurrent...

T cell-based tracking of multidrug resistant tuberculosis infection after brief exposure.

Science.gov (United States)

Richeldi, Luca; Ewer, Katie; Losi, Monica; Bergamini, Barbara M; Roversi, Pietro; Deeks, Jonathan; Fabbri, Leonardo M; Lalvani, Ajit

2004-08-01

Molecular epidemiology indicates significant transmission of Mycobacterium tuberculosis after casual contact with infectious tuberculosis cases. We investigated M. tuberculosis transmission after brief exposure using a T cell-based assay, the enzyme-linked-immunospot (ELISPOT) for IFN-gamma. After childbirth, a mother was diagnosed with sputum smear-positive multidrug-resistant tuberculosis. Forty-one neonates and 47 adults were present during her admission on the maternity unit; 11 weeks later, all underwent tuberculin skin testing (TST) and ELISPOT. We correlated test results with markers of exposure to the index case. The participants, who were asymptomatic and predominantly had no prior tuberculosis exposure, had 6.05 hours mean exposure (range: 0-65 hours) to the index case. Seventeen individuals, including two newborns, were ELISPOT-positive, and ELISPOT results correlated significantly with three of four predefined measures of tuberculosis exposure. For each hour sharing room air with the index case, the odds of a positive ELISPOT result increased by 1.05 (95% CI: 1.02-1.09, p = 0.003). Only four adults were TST-positive and TST results did not correlate with exposure. Thus, ELISPOT, but not TST, suggested quite extensive nosocomial transmission of multidrug-resistant M. tuberculosis after brief exposure. These results help to explain the apparent importance of casual contact for tuberculosis transmission, and may have implications for prevention.

Enzyme-linked immunospot: an alternative method for the detection of interferon gamma in Johne's disease

DEFF Research Database (Denmark)

Begg, Douglas J.; de Silva, Kumudika; Bosward, Katrina

2009-01-01

To date, the sensitivity of the interferon gamma (IFN-) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN- detection in the early stages of infection, an alternate assay needs to be ...

Different patterns of cytokines and chemokines combined with IFN-γ production reflect Mycobacterium tuberculosis infection and disease.

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Yang Yu

Full Text Available BACKGROUND: IFN-γ is presently the only soluble immunological marker used to help diagnose latent Mycobacterium tuberculosis (M.tb infection. However, IFN-γ is not available to distinguish latent from active TB infection. Moreover, extrapulmonary tuberculosis, such as tuberculous pleurisy, cannot be properly diagnosed by IFN-γ release assay. As a result, other disease- or infection-related immunological biomarkers that would be more effective need to be screened and identified. METHODOLOGY: A panel of 41 soluble immunological molecules (17 cytokines and 24 chemokines was tested using Luminex liquid array-based multiplexed immunoassays. Samples, including plasma and pleural effusions, from healthy donors (HD, n = 12 or patients with latent tuberculosis infection (LTBI, n = 20, pulmonary tuberculosis (TB, n = 12, tuberculous pleurisy (TP, n = 15 or lung cancer (LC, n = 15 were collected and screened for soluble markers. Peripheral blood mononuclear cells (PBMCs and pleural fluid mononuclear cells (PFMCs were also isolated to investigate antigen-specific immune factors. PRINCIPAL FINDINGS: For the 41 examined factors, our results indicated that three patterns were closely associated with infection and disease. (1 Significantly elevated plasma levels of IL-2, IP-10, CXCL11 and CXCL12 were present in both patients with tuberculosis and in a sub-group participant with latent tuberculosis infection who showed a higher level of IFN-γ producing cells by ELISPOT assay compared with other latently infected individuals. (2 IL-6 and IL-9 were only significantly increased in plasma from active TB patients, and the two factors were consistently highly secreted after M.tb antigen stimulation. (3 When patients developed tuberculous pleurisy, CCL1, CCL21 and IL-6 were specifically increased in the pleural effusions. In particular, these three factors were consistently highly secreted by pleural fluid mononuclear cells following M

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

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Chih-Peng Chang

Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

Two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications

Energy Technology Data Exchange (ETDEWEB)

Meager, A; Parti, S; Leung, H; Woolley, J; Peil, E; Sidhu, S; Roberts, T

1987-11-23

Three monoclonal antibodies (MoAbs L49-15, L81-11 and L238-14) were raised against recombinant human lymphotoxin (rLT) derived from E. coli containing the cDNA sequence specifying LT. These MoAbs were ideal reagents for immunoassay of LT and a very sensitive, highly specific immunoradiometric assay (IRMA) was developed. This assay was rapid to perform and was capable of detecting as little as 10 pg/ml of LT. Application of the LT IRMA in combination with previously developed human gamma-interferon (IFN-..gamma..) and human tumour necrosis factor (TNF)-specific IRMA permitted independent estimations of these three substances to be carried out in parallel. By these means, it was found that RPMI 1788 lymphoblastoid cell line produced both LT and TNF, but not IFN-..gamma... Extensive analyses on cytokine (monokine and lymphokine) preparations derived from a variety of activated lymphocytes are also reported. Co-production of LT, TNF, and IFN-..gamma.. was a common finding, even occurring in alloantigen-specific T helper cell clones. 45 refs.; 3 figs.; 4 tabs.

Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-gamma production of T cells.

Science.gov (United States)

Pierkes, M; Bellinghausen, I; Hultsch, T; Metz, G; Knop, J; Saloga, J

1999-02-01

Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

Lol p I-induced IL-4 and IFN-gamma production by peripheral blood mononuclear cells of atopic and nonatopic subjects during and out of the pollen season.

Science.gov (United States)

Gagnon, R; Akoum, A; Hébert, J

1993-04-01

The reciprocal effects of IL-4 and IFN-gamma on IgE synthesis have been well established. It has also been shown that these two lymphokines are secreted by different subsets of CD4+ T cells (TH1 and TH2), and that TH2 helper T lymphocytes could be involved in the pathophysiology of allergic diseases. But little is known about the effects of an allergen on the profile of lymphokine synthesis by human peripheral blood mononuclear cells (PBMCs) of allergic and nonallergic subjects. We studied the production of IL-4 and IFN-gamma by PBMCs of atopic and nonatopic donors after in vitro stimulation by the group 1 allergen from Lolium perenne pollen (Lol p I), during and out of the grass pollen season. On natural exposure to pollen, Lol p I-induced IL-4 production was observed only with atopic donors (6 of 8), whereas the synthesis of IFN-gamma was observed for all nonatopic donors (7 of 7) and most allergic patients (5 of 7). At the time of the study, higher amounts of IFN-gamma were produced by PBMCs of nonatopic donors than by PBMCs of atopic patients. Out of the pollen season the production of IL-4 was not observed either by atopic (n = 11) or by nonatopic subjects (n = 5). On the other hand, IFN-gamma was produced by PBMCs of most subjects (atopic, 10 of 11; nonatopic, 5 of 5), but at the time of the study no difference was observed between the two groups. These results show that Lol p I induces different profiles of IL-4 and IFN-gamma production by PBMCs of atopic and nonatopic subjects. In atopic subjects this profile of lymphokine synthesis is influenced by the natural exposure to pollen, which is in keeping with the seasonal rise of IgE antibodies.

Using ELISPOT to expose false positive skin test conversion in tuberculosis contacts.

Directory of Open Access Journals (Sweden)

Philip C Hill

2007-01-01

Full Text Available Repeat tuberculin skin tests may be false positive due to boosting of waned immunity to past mycobacterial exposure. We evaluated whether an ELISPOT test could identify tuberculosis (TB contacts with boosting of immunity to non-tuberculous mycobacterial exposure.We conducted tuberculin and ELISPOT tests in 1665 TB contacts: 799 were tuberculin test negative and were offered a repeat test after three months. Those with tuberculin test conversion had an ELISPOT, chest X-ray and sputum analysis if appropriate. We compared converters with non-converters, assessed the probability of each of four combinations of ELISPOT results over the two time points and estimated boosting with adjustment for ELISPOT sensitivity and specificity. 704 (72% contacts had a repeat tuberculin test; 176 (25% had test conversion, which increased with exposure to a case (p = 0.002, increasing age (p = 0.0006 and BCG scar (p = 0.06. 114 tuberculin test converters had ELISPOT results: 16(14% were recruitment positive/follow-up positive, 9 (8% positive/negative, 34 (30% negative/positive, and 55 (48% were negative/negative. There was a significant non-linear effect of age for ELISPOT results in skin test converters (p = 0.038. Estimates of boosting ranged from 32%-41% of skin test converters with increasing age. Three converters were diagnosed with TB, two had ELISPOT results: both were positive, including one at recruitment.We estimate that approximately one third of tuberculin skin test conversion in Gambian TB case contacts is due to boosting of immunity to non-tuberculous mycobacterial exposure. Further longitudinal studies are required to confirm whether ELISPOT can reliably identify case contacts with tuberculin test conversion that would benefit most from prophylactic treatment.

Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

DEFF Research Database (Denmark)

Ank, Nina; West, Hans; Bartholdy, Christina

2006-01-01

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN......-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN......-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral...

Increase in neutrophil Fc gamma receptor I expression following interferon gamma treatment in rheumatoid arthritis.

Science.gov (United States)

Goulding, N J; Knight, S M; Godolphin, J L; Guyre, P M

1992-04-01

The therapeutic potential of interferon gamma (IFN gamma) in a number of disease states is still being explored, but progress is hampered by the lack of a suitable measure of in vivo biological activity. To assess the in vivo biological effects of recombinant human IFN gamma (rhIFN gamma), 14 patients were studied in a randomised, prospective, double blind, placebo controlled trial of this cytokine for the treatment of rheumatoid arthritis. The levels of Fc gamma receptors on peripheral blood neutrophils were measured at baseline and after 21 days of once daily, subcutaneous injections of rhIFN gamma or placebo. An induction of neutrophil Fc gamma receptor type I (Fc gamma RI) was seen in the group of patients receiving recombinant human rhIFN gamma but not in those receiving placebo. No change in the expression of Fc gamma RII or Fc gamma RIII was detected. The amount of induction of Fc gamma RI detected on the neutrophils of patients receiving rhIFN gamma did not correlate with clinical measures of response at either 21 days or at the end of the study (24 weeks). No significant clinical responses were observed in the rhIFN gamma group at these times. These data confirm that the reported in vitro effect of IFN gamma on human neutrophil Fc receptor expression can be reproduced in vivo.

Recombinant human growth-regulated oncogene-alpha induces T lymphocyte chemotaxis. A process regulated via IL-8 receptors by IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-13

DEFF Research Database (Denmark)

Jinquan, T; Frydenberg, Jane; Mukaida, N

1995-01-01

receptors on the cells. This process can be augmented by IFN-gamma and TNF-alpha, and inhibited by IL-4, IL-10, and IL-13. In addition, we also document that on T lymphocytes there exist IL-8 receptors that can be up-regulated by IFN-gamma, TNF-alpha, and IL-2. Our results demonstrate that rhGRO-alpha gene...

Detection of IFN-γ Secretion by T Cells Collected Before and After Successful Treatment of Early Lyme Disease.

Science.gov (United States)

Callister, Steven M; Jobe, Dean A; Stuparic-Stancic, Aleksandra; Miyamasu, Misato; Boyle, Jeff; Dattwyler, Raymond J; Arnaboldi, Paul M

2016-05-15

Current serodiagnostics for Lyme disease lack sensitivity during early disease, and cannot determine treatment response. We evaluated an assay based on QuantiFERON technology utilizing peptide antigens derived from Borrelia burgdorferi to stimulate interferon-gamma (IFN-γ) release as an alternative to serodiagnosis for the laboratory detection of Lyme disease. Blood was obtained from patients with erythema migrans before (n = 29) and 2 months after (n = 27) antibiotic therapy. IFN-γ release was measured by enzyme-linked immunosorbent assay (ELISA) following overnight stimulation of whole blood with the peptide antigens, and compared to the results of standard serological assays (C6, ELISA, and Western blot). IFN-γ release was observed in pretreatment blood of 20 of 29 (69%) patients with Lyme disease. Following antibiotic treatment, IFN-γ was significantly reduced (P = .0002), and was detectable in only 4 of 20 (20%) initially positive patients. By contrast, anti-C6 antibodies were detected in pretreatment sera from 17 of 29 (59%) subjects, whereas only 5 of 29 (17%) patients had positive Western blot seroreactivity. Antibody responses persisted and expanded following treatment. Our findings suggest that measurement of IFN-γ after incubating blood with Borrelia antigens could be useful in the laboratory diagnosis of early Lyme disease. Also, after antibiotic treatment, this response appears to be short lived. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Transient nature of long-term nonprogression and broad virus-specific proliferative T-cell responses with sustained thymic output in HIV-1 controllers.

Directory of Open Access Journals (Sweden)

Samantha J Westrop

Full Text Available HIV-1(+ individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4(+ T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-gamma ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1(+ individuals originally identified as "Long-term non-progressors" in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-gamma producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-gamma production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1(+ patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-gamma production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory.

Interferons and interferon (IFN)-inducible protein 10 during highly active anti-retroviral therapy (HAART)-possible immunosuppressive role of IFN-alpha in HIV infection

DEFF Research Database (Denmark)

Stylianou, E; Aukrust, P; Bendtzen, K

2000-01-01

-infected patients had raised levels of both IP-10 and IFN-alpha compared with healthy controls (n = 19), with particularly high levels in advanced disease. HAART induced a marked decrease in levels of both IFN-alpha, neopterin and IP-10, though not to normal concentrations. In contrast, IFN-gamma levels were low...

Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-gamma down-regulation in NC/Nga mice.

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Kim, Dong-Young; Jung, Jung-A; Kim, Tae-Ho; Seo, Sang-Wan; Jung, Sung-Ki; Park, Cheung-Seog

2009-04-21

Uncariae rhynchophylla (UR) is an herb which has blood pressure lowering and anti-inflammatory effects and has been prescribed traditionally to treat stroke and vascular dementia. In the present study, we examined whether UR suppress Atopic dermatitis (AD)-like skin lesions in NC/Nga mice treated with 2, 4-dinitrofluorobenzene (DNFB) under SPF conditions. The effect of UR in DNFB- treated NC/Nga mice was determined by measuring the skin symptom severity, levels of serum IgE, and of the amounts of IL-4 and IFN-gamma secreted by activated T cells in draining lymph nodes. Oral administration of UR to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. IFN-gamma production by CD4+ T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by UR treatment, although levels of IL-4 and total IgE in serum were not. UR may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing IFN-gamma production.

Induction of indolamine 2,3-dioxygenase and kynurenine 3-monooxygenase in rat brain following a systemic inflammatory challenge: a role for IFN-gamma?

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Connor, Thomas J; Starr, Neasa; O'Sullivan, Joan B; Harkin, Andrew

2008-08-15

Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,3-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating interferon (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-alpha and IL-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-3-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and 20-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection

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Denkinger, Claudia M.; Kik, Sandra V.; Rangaka, Molebogeng X.; Zwerling, Alice; Oxlade, Olivia; Metcalfe, John Z.; Cattamanchi, Adithya; Dowdy, David W.; Dheda, Keertan; Banaei, Niaz

2014-01-01

SUMMARY Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers. PMID:24396134

The effect of co-administration of DNA carrying chicken interferon-gamma gene on protection of chickens against infectious bursal disease by DNA-mediated vaccination.

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Hsieh, Ming Kun; Wu, Ching Ching; Lin, Tsang Long

2006-11-17

The purpose of the present study was to determine whether DNA vaccination by co-administration of DNA coding for chicken interferon-gamma (IFN-gamma) gene and DNA encoding for the VP243 gene of IBDV could enhance immune response and protection efficacy of chickens against challenge by IBDV. Plasmids carrying VP243 gene of IBDV strain variant E (VE) (P/VP243/E) and chicken IFN-gamma gene (P/cIFN-gamma) were constructed, respectively. One-day-old chickens were intramuscularly injected with P/VP243/E, or P/cIFN-gamma, or both once, twice, or three times into the thigh muscle of one leg or the thigh muscles of two separate legs at weekly intervals. Chickens were orally challenged with IBDV strain VE at 3 weeks of age and observed for 10 days. Chickens receiving two plasmids in the same site two times had significantly higher (Pprotection and those receiving two plasmids in the same sites did not have any protection against IBD. The enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers to IBDV of chickens in the groups with three doses of P/VP243/E were significantly higher (Pprotected by DNA vaccination did not have detectable IBDV antigen in the bursae as determined by immunofluorescent antibody assay (IFA). The results indicated that co-administration of plasmid encoding chicken IFN-gamma gene with plasmid encoding a large segment gene of the IBDV did not enhance immune response and protection against challenge by IBDV.

[Adenovirus-mediated canine interferon-gamma expression and its antiviral activity against canine parvovirus].

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Zhang, Kao; Jin, Huijun; Zhong, Fei; Li, Xiujin; Neng, Changai; Chen, Huihui; Li, Wenyan; Wen, Jiexia

2012-11-04

To construct recombinant adenovirus containing canine interferon-gamma (cIFN-gamma) gene and to investigate its antiviral activity against canine parvovirus in Madin-Darby canine kidney cells (MDCK). [Methods] The cIFN-gamma gene was inserted into adenovirus shuttle plasmid to construct pShuttle3-cIFN-gamma expression vector, from which the cIFN-gamma expression cassette was transferred into the adenovirus genomic plasmid pAdeno-X by specific restriction sites to generate recombinant adenovirus genomic plasmid pAd-cIFN-gamma. The pAd-cIFN-gamma plasmid was linearized by digestion and transfected into human embryonic kidney (HEK) 293T cells to generate the replication-defective cIFN-gamma recombinant adenovirus (Ad-cIFN-gamma). To analyze its anti-canine parvovirus activity, the MDCK cells were pre-infected by Ad-cIFN-gamma recombinant adenovirus, and then infected by canine parvovirus. The antiviral activity of the Ad-cIFN-gamma recombinant adenovirus against parvovirus was analyzed. The recombinant adenovirus containing cIFN-gamma gene was constructed by the ligation method. The recombinant adenovirus could mediates recombinant cIFN-gamma secretory expression in MDCK cells. The Ad-cIFN-gamma recombinant adenovirus could significantly inhibit canine parvovirus replication in MDCK cells pre-infected with the recombinant adenovirus. These results indicate that the Ad-cIFN-gamma recombinant adenovirus has the potent antiviral activity against canine parvovirus. The Ad-cIFN-gamma recombinant adenovirus was successfully constructed by the ligation method and possessed a powerful antiviral activity against canine parvovirus.

Gene expression in IFN-g-activated murine macrophages

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Pereira C.A.

2004-01-01

Full Text Available Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated or BALB/c (297 and 58 genes, respectively, up- and down-regulated mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis

NARCIS (Netherlands)

Chierakul, Wirongrong; de Fost, Maaike; Suputtamongkol, Yupin; Limpaiboon, Roongreung; Dondorp, Arjen; White, Nicholas J.; van der Poll, Tom

2004-01-01

Interferon (IFN)-gamma plays an important role in the induction of a type 1 immune response against intracellular pathogens. We compared the plasma levels of IFN-gamma and IFN-gamma-inducing cytokines in adult Thai patients with scrub typhus, caused by the obligate intracellular bacterium Orientia

Modulation of the allergen-induced human IgE response in Hu-SCID mice: inhibitory effect of human recombinant IFN-gamma and allergen-derived lipopeptide.

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Duez, C; Gras-Masse, H; Hammad, H; Akoum, H; Didierlaurent, A; André, C; Tonnel, A B; Pestel, J

2001-01-01

We have previously established a model to study the in vivo human IgE response using humanized SCID mice. Allergic SCID mice were obtained following intraperitoneal injection with mononuclear cells from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, and sensitization by Dpt allergen intraperitoneal injection (immunization) or Dpt aerosol (inhalation). Human serum IgE was measured in allergic SCID mice after administration of human recombinant IFN-gamma or the lipopeptide LP 52-71 (derived from peptide p52-71 from Der p 1, Dpt major allergen, coupled to a lipophilic moiety), during the immunization or the inhalation phase. IFN-gamma inhibited human IgE production when given at the time of immunization, but not during inhalation. This effect was long-lasting as Dpt aerosol, given one month after immunization and IFN-gamma administration, failed to increase IgE levels. Unlike Dpt or p52-71, LP 52-71 failed to induce human IgE production at day 14 and 21 after its injection, but did inhibit the development of the IgE response after a secondary Dpt-challenge. Moreover, LP 52-71 administration 14 days after Dpt inhalation decreased IgE levels, in contrast to peptide 52-71, which increased IgE levels. Thus, taken together these results indicate that the development of the human IgE response in allergic SCID mice can be modulated by modified allergen and a Th1 cytokine.

Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-gamma.

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Huang, T J; MacAry, P A; Eynott, P; Moussavi, A; Daniel, K C; Askenase, P W; Kemeny, D M; Chung, K F

2001-01-01

Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.

Abnormally high levels of virus-infected IFN-gamma+ CCR4+ CD4+ CD25+ T cells in a retrovirus-associated neuroinflammatory disorder.

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Yoshihisa Yamano

Full Text Available BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1 is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP, which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL. The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. PRINCIPAL FINDINGS: Here, we demonstrate that CD4(+CD25(+CCR4(+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2, Th17, and regulatory T (Treg cells in healthy individuals, we demonstrate that IFN-gamma production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4(+CD25(+CCR4(+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-gamma-producing CD4(+CD25(+CCR4(+Foxp3(- T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. CONCLUSIONS: We have defined a unique T cell subset--IFN-gamma(+CCR4(+CD4(+CD25(+ T cells--that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

Serial interferon-gamma release assays during treatment of active tuberculosis in young adults

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Lee Choon-Taek

2010-10-01

Full Text Available Abstract Background The role of interferon-γ release assay (IGRA in monitoring responses to anti-tuberculosis (TB treatment is not clear. We evaluated the results of the QuantiFERON-TB Gold In-tube (QFT-GIT assay over time during the anti-TB treatment of adults with no underlying disease. Methods We enrolled soldiers who were newly diagnosed with active TB and admitted to the central referral military hospital in South Korea between May 1, 2008 and September 30, 2009. For each participant, we preformed QFT-GIT assay before treatment (baseline and at 1, 3, and 6 months after initiating anti-TB medication. Results Of 67 eligible patients, 59 (88.1% completed the study protocol. All participants were males who were human immunodeficiency virus (HIV-negative and had no chronic diseases. Their median age was 21 years (range, 20-48. Initially, 57 (96.6% patients had positive QFT-GIT results, and 53 (89.8%, 42 (71.2%, and 39 (66.1% had positive QFT-GIT results at 1, 3, and 6 months, respectively. The IFN-γ level at baseline was 5.31 ± 5.34 IU/ml, and the levels at 1, 3, and 6 months were 3.95 ± 4.30, 1.82 ± 2.14, and 1.50 ± 2.12 IU/ml, respectively. All patients had clinical and radiologic improvements after treatment and were cured. A lower IFN-γ level, C-reactive protein ≥ 3 mg/dl, and the presence of fever (≥ 38.3°C at diagnosis were associated with negative reversion of the QFT-GIT assay. Conclusion Although the IFN-γ level measured by QFT-GIT assay decreased after successful anti-TB treatment in most participants, less than half of them exhibited QFT-GIT reversion. Thus, the reversion to negativity of the QFT-GIT assay may not be a good surrogate for treatment response in otherwise healthy young patients with TB.

Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes

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Ceretto Monica

2007-06-01

Full Text Available Abstract Background Enhanced production of nitric oxide (NO following upmodulation of the inducible isoform of NO synthase (iNOS by haemozoin (HZ, inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes. Methods Adherent immunopurified human monocytes (purity >85%, and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively. Results Phagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data. Conclusion Results indicate that human monocytes fed or not with HZ/sHZ were constantly

Immunological Roles of Elevated Plasma Levels of Matricellular Proteins in Japanese Patients with Pulmonary Tuberculosis

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Beata Shiratori

2016-12-01

Full Text Available Elevated matricellular proteins (MCPs, including osteopontin (OPN and galectin-9 (Gal-9, were observed in the plasma of patients with Manila-type tuberculosis (TB previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44 by enzyme-linked immunosorbent assay (ELISA, and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI, and 19 healthy controls (HCs from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs. Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-α, IFN-γ, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology.

Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts

NARCIS (Netherlands)

Arend, Sandra M.; Thijsen, Steven F. T.; Leyten, Eliane M. S.; Bouwman, John J. M.; Franken, Willeke P. J.; Koster, Ben F. P. J.; Cobelens, Frank G. J.; van Houte, Arend-Jan; Bossink, Ailko W. J.

2007-01-01

The tuberculin skin test (TST) has low specificity. QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB are based on interferon (IFN)-gamma responses to Mycobacterium tuberculosis-specific antigens. A novel in-tube format of QFT-G (QFT-GIT) offers logistical advantages. To compare TST, QFT-GIT, and T-SPOT.TB

Are 0.1%-accurate gamma-ray assays possible for 235U solutions

International Nuclear Information System (INIS)

Parker, J.L.

1983-01-01

The factors influencing the accuracy of passive gamma-ray assay of uniform, homogeneous solution samples have been studied in some detail, particularly for the assay of 235 U in uranium solutions. Factors considered are the overall long-term electronic stability, the information losses caused by the rate-related electronic processes of pulse pileup and dead-time, and the self-attenuation of gamma rays within the samples. Both experimental and computational studies indicate that gamma-ray assay procedures for solution samples of moderate size (from approx. 10 to perhaps a few hundred milliliters) are now capable of accuracies approaching 0.1% in many practical cases

T cell Ig domain and mucin domain 1 engagement on invariant NKT cells in the presence of TCR stimulation enhances IL-4 production but inhibits IFN-gamma production.

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Kim, Hye Sung; Kim, Hyun Soo; Lee, Chang Woo; Chung, Doo Hyun

2010-04-15

The T cell Ig domain and mucin domain (TIM)1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. However, the functional roles of TIM1 have not been examined in NKT cells. Therefore, we investigated the immunologic effects of TIM1 on NKT cells. We found that mouse NK1.1(+)TCR-beta(+), alpha-galactosyl ceramide/CD1d dimer(+) NKT, and NKT hybridoma (DN32.D3) cells constitutively express TIM1 and TIM4 on their surface. Engagement of TIM1 on NKT cells by any of several anti-TIM1 mAbs suppressed the production of IFN-gamma in the presence of TCR stimulation in vitro and in vivo, whereas the effects of such engagement on Th2 cytokine production by the NKT cells varied with the particular anti-TIM1 Ab clone. Moreover, in DN32.D3 TIM4-knockdown NKT hybridoma cells, TIM1 engagement by rTIM1 or TIM4 enhanced IL-4 production while inhibiting IFN-gamma production in the presence of alpha-galactosyl ceramide stimulation. TIM1 engagement increased GATA-3 expression but reduced T-bet expression in NKT cells in the presence of TCR engagement. The adoptive transfer of NKT cells preincubated with anti-TIM1 mAbs into Jalpha18(-/-) mice aggravated bleomycin-induced pulmonary fibrosis by suppressing IFN-gamma production. Taken together, these results suggest that TIM1 costimulation on NKT cells enhances the cellular production of IL-4 while inhibiting the production of IFN-gamma. Thus, as a differential regulator of the immune response, TIM1 on NKT cells may be a useful therapeutic target for immune diseases.

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

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Jeevan, A; McFarland, C T; Yoshimura, T; Skwor, T; Cho, H; Lasco, T; McMurray, D N

2006-01-01

Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

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Chungwon J Chung

Full Text Available Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI. However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates.An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983 and another strain (type-2 PRRSVVR2332 with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection.At 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate.These results demonstrated that T-lymphocytes recognizing antigenically and

Evaluation of gamma spectroscopy gauge for uranium-plutonium assay

International Nuclear Information System (INIS)

Notea, A.; Segal, Y.

1975-01-01

A procedure is presented for the characterization of a gamma passive method for nondestructive analysis of nuclear fuel. The approach provides an organized and systematic way for optimizing the assay system. The key function is the relative resolving power defined as the smallest relative change in the Quantity of radionuclide measured, that may be detected within a certain confidence level. This function is derived for nuclear fuel employing a model based on empirical parameters. The ability to detect changes in fuels of binary and trinary compositions with a 50 cc Ge(Li) at a 1 min counting period is discussed. As an example to a binary composition, an enriched uranium fuel was considered. The 185 keV and 1001 keV gamma lines are used for the assay of 235 U and 238 U respectively. As a trinary composition a plutonium-containing gamma line. The interference of the high energy lines is carefully analyzed, and numerical results are presented. For both cases the range of measurement under specific accuracy demands is determined. The approach described is suitable also for evaluation of other passive as well as active assay methods. (author)

Multi-isotopic gamma-ray assay system for alpha-contaminated waste

International Nuclear Information System (INIS)

Close, D.A.; Pratt, J.C.; Caldwell, J.T.; Kunz, W.E.; Schultz, F.J.; Haff, K.W.

1983-01-01

The capability of an existing segmented gamma-ray system is being expanded for the analysis of alpha-contaminated waste drums. A cursory assay of 114 transuranic waste drums of 208-l capacity has been made. Analysis of these data indicates a detection limit better than 100 nCi/g of waste for 237 Np/ 233 Pa, 239 Pu, 241 Am, 243 Am/ 239 Np, 60 Co, 125 Sb, 134 137 Cs, and 154 Eu. A pending Code of Federal Regulation (10CFR61) stipulates that the nuclear industry quantify not only its transuranic waste, but also certain beta- and gamma-ray-emitting fission products. An assay system based on gamma-ray spectroscopy is the only system that can meet this requirement for the fission products

EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

Science.gov (United States)

McCormack, Sheena; Stöhr, Wolfgang; Barber, Tristan; Bart, Pierre-Alexandre; Harari, Alexandre; Moog, Christiane; Ciuffreda, Donatella; Cellerai, Cristina; Cowen, Miranda; Gamboni, Romilda; Burnet, Séverine; Legg, Ken; Brodnicki, Elizabeth; Wolf, Hans; Wagner, Ralf; Heeney, Jonathan; Frachette, Marie-Joëlle; Tartaglia, Jim; Babiker, Abdel; Pantaleo, Giuseppe; Weber, Jonathan

2008-06-13

The aim of this randomised controlled trial was to see if the addition of 4 mg/ml DNA-C priming given by the intramuscular route at weeks 0 and 4 to NYVAC-C at weeks 20 and 24, safely increased the proportion of participants with HIV-specific T-cell responses measured by the interferon (IFN)-gamma ELISpot assay at weeks 26 and/or 28 compared to NYVAC-C alone. Although 2 individuals discontinued after the first DNA-C due to adverse events (1 vaso-vagal; 1 transient, asymptomatic elevation in alanine transaminase), the vaccines were well tolerated. Three others failed to complete the regimen (1 changed her mind; 2 lost to follow-up). Of the 35 that completed the regimen 90% (18/20) in the DNA-C group had ELISpot responses compared to 33% (5/15) that received NYVAC-C alone (p=0.001). Responses were to envelope in the majority (21/23). Of the 9 individuals with responses to envelope and other peptides, 8 were in the DNA-C group. These promising results suggest that DNA-C was an effective priming agent, that merits further investigation.

Activated umbilical cord blood cells from pre-term and term neonates express CD69 and synthesize IL-2 but are unable to produce IFN-gamma.

Science.gov (United States)

Cérbulo-Vázquez, Arturo; Valdés-Ramos, Roxana; Santos-Argumedo, Leopoldo

2003-01-01

The immune response exhibits quantitative and qualitative differences throughout human development. Both phenotypical and functional immaturity of newborn immune cellular components have been reported. We aimed to analyze possible differences in cellular activation assessed by expression of surface CD69 and cytokine production in mononuclear peripheral blood cells from premature (term (>37 weeks of gestation) neonates compared to adult donors. Ten persons from each group were selected; none was infected, immunodepressed, under medical treatment, or had any congenital abnormalities. Blood was obtained from umbilical cord of term and pre-term donors and vein punction of adults. All samples were collected in heparin and subsequently activated with PHA-L or PMA plus ionomycin at 37 degrees C for 4 h. After incubation, cells were labeled to determine CD69 expression on CD3+CD4+, CD3+CD8+, CD19+, and CD16+56+ subpopulations. Intracellular staining was performed to analyze IFN-gamma, IL-2, and CD69 in CD3+ cells. After staining, cells were analyzed by flow cytometry. We first found a substantially higher number of CD3+CD4+CD69+ cells in premature and term neonates than in adults. Secondly, percentage of CD3+CD8+, CD56+, and CD19+ cells expressing CD69 was similar among the three groups. Thirdly, expression of CD69 was higher in CD19+ cells than in CD16+56+ cells of all three groups. Regarding cytokine production, IFN-gamma was detected only in cells from adults and was consistent in all individuals analyzed. In sharp contrast, IL-2 and intracellular CD69 (iCD69) were detected in all three groups, with no significant differences among them. Induction of IL-2 and iCD69 showed that lack of response with IFN-gamma was restricted to pre-term and newborn populations. In summary, our results showed that a) CD69 is an early activation marker of both mononuclear umbilical cord and peripheral blood cells activated by a mitogenic stimulus, and b) newborn CD3+ cells probably lack

Standard test method for nondestructive assay of radioactive material by tomographic gamma scanning

CERN Document Server

American Society for Testing and Materials. Philadelphia

2010-01-01

1.1 This test method describes the nondestructive assay (NDA) of gamma ray emitting radionuclides inside containers using tomographic gamma scanning (TGS). High resolution gamma ray spectroscopy is used to detect and quantify the radionuclides of interest. The attenuation of an external gamma ray transmission source is used to correct the measurement of the emission gamma rays from radionuclides to arrive at a quantitative determination of the radionuclides present in the item. 1.2 The TGS technique covered by the test method may be used to assay scrap or waste material in cans or drums in the 1 to 500 litre volume range. Other items may be assayed as well. 1.3 The test method will cover two implementations of the TGS procedure: (1) Isotope Specific Calibration that uses standards of known radionuclide masses (or activities) to determine system response in a mass (or activity) versus corrected count rate calibration, that applies to only those specific radionuclides for which it is calibrated, and (2) Respo...

Whole blood assay for NK activity in splenectomized and non-splenectomized hairy cell leukemia patients during IFN-alpha-2b treatment

DEFF Research Database (Denmark)

Nielsen, B; Hokland, P; Ellegaard, J

1989-01-01

Natural killer cell (NK) activity in peripheral blood (PB) was followed longitudinally for up to 2 yr after initiation of low-dose IFN-alpha-2b therapy in nine hairy cell leukemia (HCL) patients. A whole blood NK (WB-NK) assay was employed in order to measure the NK activity per unit blood. The p...

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

Directory of Open Access Journals (Sweden)

Bosco Christiano Maciel da Silva

2014-12-01

Full Text Available The interferon (IFN-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT assays using peripheral blood mononuclear cells (PBMCs from 16 Mantoux tuberculin skin test (TST-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007. The response to GroEL2 (463-477 was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Interferon gamma increases survival in urine experimental cryptococcosis El Interferon gamma incrementa la sobrevida de un modelo experimental murino de criptococosis

Directory of Open Access Journals (Sweden)

Amadeo J. Bava

1995-10-01

Full Text Available Systemic disease by Cryptococcus neoformans (C. neoformans is a common opportunistic infection in immunodeficient patients. Cellular immunity seems to be the most important determinant of resistance. The aim of this study was to assess the effect of recombinant rat interferon gamma (IFN-gamma in murine cryptococcosis (Balb/c mice infected by IP route with the Rivas strain of C. neoformans, evaluating survival time, macroscopic and microscopic examination of the organs, and massive seeding of brain homogenate. IFN-gamma treatment, at a daily dose of 10,000 IU, did not modify significantly these variables when mice were challenged with a high inoculum (10(7 yeasts and treatment was delayed to 5 days after infection (median survival 21 days in control mice vs. 23 days in IFN-treated. Another set of experiments suggested that IFN-gamma treatment, at a dose of 10,000 IU/day, begun at the moment of infection could be useful (it prolonged survival from 20 to 28 days, although the difference did not achieve statistical signification. When used simultaneously with infection by 3.5 x 10(5 yeasts, IFN-gamma at 10,000 IU/day for 15 days significantly prolonged survival of mice (p = 0.004. These results suggest that, depending on the experimental conditions, IFN-gamma can improve survival of mice infected with a lethal dose of C. neoformans.Se evaluó la efectividad del interferon-gamma (IFN-gamma recombinante de rata en un modelo experimental de criptococosis desarollado en ratones Balb/C inoculados por vía intraperitoneal con la cepa Rivas de Cryptococcus neoformans (C. neoformans. Se tuvieron en cuenta el tiempo de sobrevida de los animales, el aspecto macroscópico de los órganos en la autopsia, la presencia de levaduras capsuladas en los tejidos y la siembra masiva de un homogenato de cerebro. El tratamiento con IFN-gamma, en dosis diarias de 10.000 UI, no modificó estos parámetros cuando la dosis infectante fue de 10(7 levaduras y el tratamiento se

A weighted least-squares lump correction algorithm for transmission-corrected gamma-ray nondestructive assay

International Nuclear Information System (INIS)

Prettyman, T.H.; Sprinkle, J.K. Jr.; Sheppard, G.A.

1993-01-01

With transmission-corrected gamma-ray nondestructive assay instruments such as the Segmented Gamma Scanner (SGS) and the Tomographic Gamma Scanner (TGS) that is currently under development at Los Alamos National Laboratory, the amount of gamma-ray emitting material can be underestimated for samples in which the emitting material consists of particles or lumps of highly attenuating material. This problem is encountered in the assay of uranium and plutonium-bearing samples. To correct for this source of bias, we have developed a least-squares algorithm that uses transmission-corrected assay results for several emitted energies and a weighting function to account for statistical uncertainties in the assay results. The variation of effective lump size in the fitted model is parameterized; this allows the correction to be performed for a wide range of lump-size distributions. It may be possible to use the reduced chi-squared value obtained in the fit to identify samples in which assay assumptions have been violated. We found that the algorithm significantly reduced bias in simulated assays and improved SGS assay results for plutonium-bearing samples. Further testing will be conducted with the TGS, which is expected to be less susceptible than the SGS to systematic source of bias

Quantitative radiological characterization of waste. Integration of gamma spectrometry and passive/active neutron assay

Energy Technology Data Exchange (ETDEWEB)

Simone, Gianluca; Mauro, Egidio; Gagliardi, Filippo; Gorello, Edoardo [Nucleco S.p.A., Rome (Italy)

2016-06-15

The radiological characterization of drums through Non-Destructive Assay (NDA) techniques commonly relies on gamma spectrometry. This paper introduces the procedure developed in Nucleco for the NDA radiological characterization of drums when the presence of Special Nuclear Material (SNM) is expected/observed. The procedure is based on the integration of a gamma spectrometry in SGS mode (Segmented Gamma Scanner) and a passive/active neutron assay. The application of this procedure is discussed on a real case of drums. The extension of the integration procedure to other gamma spectrometry systems is also discussed.

Interferon-gamma response to the treatment of active pulmonary and extra-pulmonary tuberculosis.

Science.gov (United States)

Liang, L; Shi, R; Liu, X; Yuan, X; Zheng, S; Zhang, G; Wang, W; Wang, J; England, K; Via, L E; Cai, Y; Goldfeder, L C; Dodd, L E; Barry, C E; Chen, R Y

2017-10-01

Interferon-gamma (IFN-γ) release assays (IGRAs) are used to diagnose tuberculosis (TB) but not to measure treatment response. To measure IFN-γ response to active anti-tuberculosis treatment. Patients from the Henan Provincial Chest Hospital, Henan, China, with TB symptoms and/or signs were enrolled into this prospective, observational cohort study and followed for 6 months of treatment, with blood and sputum samples collected at 0, 2, 4, 6, 8, 16 and 24 weeks. The QuantiFERON® TB-Gold assay was run on collected blood samples. Participants received a follow-up telephone call at 24 months to determine relapse status. Of the 152 TB patients enrolled, 135 were eligible for this analysis: 118 pulmonary (PTB) and 17 extra-pulmonary TB (EPTB) patients. IFN-γ levels declined significantly over time among all patients (P = 0.002), with this decline driven by PTB patients (P = 0.001), largely during the initial 8 weeks of treatment (P = 0.019). IFN-γ levels did not change among EPTB patients over time or against baseline culture or drug resistance status. After 6 months of effective anti-tuberculosis treatment, IFN-γ levels decreased significantly in PTB patients, largely over the initial 8 weeks of treatment. IFN-γ concentrations may offer some value for monitoring anti-tuberculosis treatment response among PTB patients.

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

Science.gov (United States)

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade

2006-01-01

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-gamma and IL-10.

Science.gov (United States)

Brandão, Geane Peroni; Melo, Maria Norma; Gazzinelli, Ricardo Tostes; Caetano, Braulia Costa; Ferreira, Adriana Melo; Silva, Letícia Azevedo; Vitor, Ricardo Wagner Almeida

2009-03-01

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-gamma and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-gamma were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

Intracellular fate of recombinant human interferon-gamma (rIFN) in U937 cells

International Nuclear Information System (INIS)

Finbloom, D.S.

1986-01-01

After IFN binds to specific receptors on macrophages, both modulation of surface molecules and induction of microbicidal and tumoricidal activity occurs 24-48 hr later. Since the intracellular events required to insure these responses are poorly defined, the fate of radiolabeled rIFN in U937 cells was examined. Endocytosis was determined by exposing cells to pH 2.5 to allow rIFN to dissociate leaving only intracellular ligand. Degradation was measured as trichloroacetic acid soluble radioactivity in the media. Of the 4-5000 molecules of rIFN that specifically and saturably (at 300 U/ml) bound at 4 0 C, 40% dissociated during 15-30 min after cells were warmed to 37 0 C. However, if cells were continuously exposed at 37 0 C to lower levels of rIFN (60-100 U/ml), 30-40% of those molecules capable of binding to the cell at that concentration were internalized. Furthermore, 60% of the molecules were degraded during 3-4 hr of additional culture. Since exposure of cells to chloroquine and monensin resulted in only partial inhibition of degradation (75% and 43%, respectively), there may also be degradation within endosomes or on the cell following binding to its receptor. Soon thereafter, degradation products are measurable. Since many biological responses require prolonged incubation with the molecule, intracellular processing of IFN may be important for expression of these effects

Evaluation of a gamma-spectroscopy gauge for uranium-plutonium assay

International Nuclear Information System (INIS)

Notea, A.; Segal, Y.

1976-01-01

A procedure is presented for the characterization of a gamma passive method for non-destructive analysis of nuclear fuel. The approachh provides an organized and systematic way for optimizing the assay system. The key function is the relative resolving power defined as the smallest relative change in the quantity of radionuclide measured that may be detected within a certain confidence level. This function is derived for nuclear fuel employing a model based on empirical parameters. The ability to detect changes in fuels of binary and trinary compositions with a 50-cm 3 Ge(Li) at a 1-min counting period is discussed. As an example to a binary composition, an enriched uranium fuel was considered. The 185-keV and 1001-keV gamma lines are used for the assay of 235 U and 238 U, respectively. As a trinary composition a plutonium-containing fuel was examined. The plutonium was identified by the 414-keV gamma line. The interference of the high-energy lines is carefully analysed, and numerical results are presented. For both cases the range of measurement under specific accuracy demands is determined. The approac described is suitable also for evaluation of other passive as well as active assay methods. (author)

[Effects of recombinant human alpha-2b and gamma interferons on bone marrow megakaryocyte progenitors (CFU-Meg) from patients with chronic myelocytic leukemia].

Science.gov (United States)

Tanabe, Y; Dan, K; Kuriya, S; Nomura, T

1989-10-01

The effects of recombinant human interferon (IFN) alpha-2b and gamma on the bone marrow megakaryocyte progenitors (CFU-Meg) were compared between eight patients in the chronic phase of Ph1-positive chronic myelocytic leukemia (CML) and five hematologically normal patients. CFU-Meg was assayed in plasma clot culture added with phytohemagglutinin-stimulated leukocyte-conditioned medium as a source of colony stimulating activity. The average count of CFU-Meg colonies formed from the bone marrow of CML patients was 5.5 times that of normal controls. Spontaneous CFU-Meg colonies were grown in seven of eight CML patients, but in none of five controls. Colony formation by CFU-Meg in CML as well as normal bone marrow was suppressed by the two preparations of IFN in a dose dependent fashion. Their suppressive influence on colonies from CFU-Meg was comparable between CML and normal bone marrow at lower concentrations, but was less marked for CML than normal bone marrow at higher concentrations. The formation of CFU-Meg colonies from CML bone marrow was more severely suppressed by IFN-gamma than IFN-alpha-2b. Depletion of either T lymphocytes or adherent cells from the CML bone marrow cells diminished the suppressive effects of IFN-gamma, but had no influence on the effects of IFN-alpha-2b.

IFN-γ and IP-10 in tracheal aspirates from premature infants: relationship with bronchopulmonary dysplasia.

Science.gov (United States)

Aghai, Zubair H; Saslow, Judy G; Mody, Kartik; Eydelman, Riva; Bhat, Vishwanath; Stahl, Gary; Pyon, Kee; Bhandari, Vineet

2013-01-01

Interferon-gamma (IFN-γ) and interferon-inducible protein of 10 kDa (IP-10) are potent inflammatory mediators and contribute to acute lung injury in adults. Recently, a potential role for IFN-γ and IP-10 in the pathogenesis of bronchopulmonary dysplasia (BPD) has been reported in animal models. To study the association between IFN-γ and IP-10 in tracheal aspirate (TA) and the development of BPD in premature infants. TA samples collected within 48 hr after birth from 79 mechanically ventilated premature neonates [gestational age (GA) IP-10 was determined using a commercially available ELISA kit. Total protein in TA was measured by Bradford assay to correct for sampling related dilution. BPD was defined as the need of supplemental oxygen at 36 weeks postmenstrual age (PMA). Twenty infants (GA 26.4 ± 1.9w, BW 860 ± 201 g) survived without BPD at 36 weeks PMA and 59 infants (GA 25.5 ± 1.5w, BW 751 ± 163 g) died before 36 weeks PMA or developed BPD. The mean IFN-γ level was higher in infants who died or developed BPD (9.7 ± 2.8 vs. 3.1 ± 1.1 pg/ml, P = 0.03). Similarly, the mean IP-10 level was higher in infants who died or developed BPD (63.4 ± 17.5 pg/ml) compared to those who survived without BPD (18.5 ± 7.5 pg/ml, P = 0.02). Higher IFN-γ and IP-10 levels in TA samples are associated with the development of BPD or death in premature infants. Copyright © 2012 Wiley Periodicals, Inc.

Incidence of tuberculosis and the predictive value of ELISPOT and Mantoux tests in Gambian case contacts.

Directory of Open Access Journals (Sweden)

Philip C Hill

2008-01-01

Full Text Available Studies of Tuberculosis (TB case contacts are increasingly being utilised for understanding the relationship between M. tuberculosis and the human host and for assessing new interventions and diagnostic tests. We aimed to identify the incidence rate of new TB cases among TB contacts and to relate this to their initial Mantoux and ELISPOT test results.After initial Mantoux and ELISPOT tests and exclusion of co-prevalent TB cases, we followed 2348 household contacts of sputum smear positive TB cases. We visited them at 3 months, 6 months, 12 months, 18 months and 24 months, and investigated those with symptoms consistent with TB. Those who were diagnosed separately at a government clinic had a chest x-ray. Twenty six contacts were diagnosed with definite TB over 4312 person years of follow-up (Incidence rate 603/100,000 person years; 95% Confidence Interval, 370-830. Nine index and secondary case pairs had cultured isolates available for genotyping. Of these, 6 pairs were concordant and 3 were discordant. 2.5% of non-progressors were HIV positive compared to 12% of progressors (HR 6.2; 95% CI 1.7-22.5; p = 0.010. 25 secondary cases had initial Mantoux results, 14 (56% were positive ; 21 had initial ELISPOT results, 11 (52% were positive; 15 (71% of 21 tested were positive by one or the other test. Of the 6 contacts who had concordant isolates with their respective index case, 4 (67% were Mantoux positive at recruitment, 3 (50% were ELISPOT positive; 5 (83% were positive by one or other of the two tests. ELISPOT positive contacts, and those with discordant results, had a similar rate of progression to those who were Mantoux positive. Those negative on either or both tests had the lowest rate of progression.The incidence rate of TB disease in Gambian TB case contacts, after screening for co-prevalent cases, was 603/100,000 person years. Since initial ELISPOT test and Mantoux tests were each positive in only just over half of cases, but 71% were

Interleukin 12 in part regulates gamma interferon release in human whole blood stimulated with Leptospira interrogans

NARCIS (Netherlands)

de Fost, Maaike; Hartskeerl, Rudy A.; Groenendijk, Martijn R.; van der Poll, Tom

2003-01-01

Heat-killed pathogenic Leptospira interrogans serovar rachmati induced the production of gamma interferon (IFN-gamma) and the IFN-gamma-inducing cytokines interleukin-12p40 (IL-12p40) and tumor necrosis factor alpha in human whole blood in vitro. The production of IFN-gamma was largely dependent on

Peripheral blood IFN-gamma-secreting V alpha 24(+)V beta 11(+) NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load

NARCIS (Netherlands)

Molling, JW; Kolgen, W; van der Vliet, HJJ; Boomsma, MF; Kruizenga, H; Smorenburg, CH; Molenkamp, BG; Langendijk, JA; Leemans, CR; von Blomberg, BME; Scheper, RJ; van den Eertwegh, AJM

2005-01-01

Natural killer T (NKT) cells are CDld-restricted lvmphoid cells and are characterized by an invariant T-cell receptor, which in humans consists of a V alpha 24 chain paired with a V beta 11 chain. These cells are known for their rapid production of large amounts of cytokines (e.g., IFN-gamma and

Ultraviolet-B irradiation decreases IFN-gamma and increases IL-4 expression in psoriatic lesional skin in situ and in cultured dermal T cells derived from these lesions

NARCIS (Netherlands)

Piskin, Gamze; Koomen, Cornelis W.; Picavet, Daisy; Bos, Jan D.; Teunissen, Marcel B. M.

2003-01-01

Type 1 cytokine producing T cells play an important role in the pathogenesis of psoriasis. Ultraviolet-B (UVB) irradiation is effective in the treatment of this disease. In normal skin, UVB causes a change in dermal microenvironment, leading to a decrease of IFN-gamma expressing type 1 T cells and a

Nondestructive assay of TRU waste using gamma-ray active and passive computed tomography

International Nuclear Information System (INIS)

Roberson, G.P.; Decman, D.; Martz, H.; Keto, E.R.; Johansson, E.M.

1995-01-01

The authors have developed an active and passive computed tomography (A and PCT) scanner for assaying radioactive waste drums. Here they describe the hardware components of their system and the software used for data acquisition, gamma-ray spectroscopy analysis, and image reconstruction. They have measured the performance of the system using ''mock'' waste drums and calibrated radioactive sources. They also describe the results of measurements using this system to assay a real TRU waste drum with relatively low Pu content. The results are compared with X-ray NDE studies of the same TRU waste drum as well as assay results from segmented gamma scanner (SGS) measurements

Immunomodulatory activities of some new synthesized compounds on serum IL-12 level and the production of IFN-gamma in irradiated female rats

International Nuclear Information System (INIS)

Noaman, E.; Elgawish, M.A.M.

2002-01-01

The immune responds to ionizing radiation with distinct characteristics depending on the dose and dose rate. The prominent suppressive effect of lethal and sublethal doses of ionizing radiation on immunity and hemopoiesis constitutes the basis of the chief clinical manifestations of acute radiation syndrome. The present of research was conducted to evaluate the effects of new compounds synthesized by adding histidine, glutathione or methionine to germanium element on the levels of interferon-gamma (IFN-γ)and interleukin-12 (IL-12) in serum of female rats exposure. The results revealed that exposure to γ-irradiation decreased significantly the levels of IL-12 and I FN-γ 1 and 3 days post-treatment. On the other hand, histidine-germanate could stimulate the production of IL-12 three days post-irradiation while glutathione-germanate and methionine-germanate may be considered as IFN-γ inducer during the investigated periods

Evaluation of Gamma Interferon and Antibody Tuberculosis Tests in Alpacas

Science.gov (United States)

Holder, Tom; Clifford, Derek; Dexter, Ian; Brewer, Jacky; Smith, Noel; Waring, Laura; Crawshaw, Tim; Gillgan, Steve; Lyashchenko, Konstantin; Lawrence, John; Clarke, John; de la Rua-Domenech, Ricardo; Vordermeier, Martin

2012-01-01

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a “test package,” although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations. PMID:22914362

Development of an aptamer beacon for detection of interferon-gamma.

Science.gov (United States)

Tuleuova, Nazgul; Jones, Caroline N; Yan, Jun; Ramanculov, Erlan; Yokobayashi, Yohei; Revzin, Alexander

2010-03-01

Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-gamma. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-gamma. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFN-gamma. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.

Interferon-gamma regulates oxidative stress during experimental autoimmune encephalomyelitis

DEFF Research Database (Denmark)

Espejo, Carmen; Penkowa, Milena; Sáez-Torres, Irene

2002-01-01

disease eliciting secretion of proinflammatory cytokines like IFN-gamma or TNF-alpha, and it has been suggested that cytokine-induced oxidative stress could have a role in EAE neuropathology. However, the individual roles of these and other cytokines in the pathogenesis of the disease are still uncertain....... Here we analyze the role of IFN-gamma during EAE by using both IFN-gamma receptor-knockout (IFN-gamma R(-/-)) and wild-type mice, both strains immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. The levels of oxidative stress were determined through the analysis...... of immunoreactivity for inducible NO synthase, nitrotyrosine, and malondialdehyde, as well as through the expression of the tissue-protective antioxidant factors metallothionein I+II (MT-I+II). We also examined the number of cells undergoing apoptosis as judged by using the TUNEL technique. The levels of oxidative...

Exogenous IFN-beta regulates the RANKL-c-Fos-IFN-beta signaling pathway in the collagen antibody-induced arthritis model.

Science.gov (United States)

Zhao, Rong; Chen, Ni-Nan; Zhou, Xiao-Wei; Miao, Ping; Hu, Chao-Ying; Qian, Liu; Yu, Qi-Wen; Zhang, Ji-Ying; Nie, Hong; Chen, Xue-hua; Li, Pu; Xu, Rong; Xiao, Lian-Bo; Zhang, Xin; Liu, Jian-Ren; Zhang, Dong-Qing

2014-12-10

Although a variety of drugs have been used to treat the symptoms of rheumatoid arthritis (RA), none of them are able to cure the disease. Interferon β (IFN-β) has pleiotropic effects on RA, but whether it can be used to treat RA remains globally controversial. Thus, in this study we tested the effects of IFN-β on RA patients and on collagen antibody-induced arthritis (CAIA) model mice. The cytokine and auto-antibody expression profiles in the serum and synovial fluid (SF) from RA patients were assessed using enzyme-linked immunosorbent assay (ELISA) and compared with the results from osteoarthritis (OA) patients. Exogenous IFN-β was administered to RA patients and CAIA model mice, and the therapeutic effects were evaluated. Endogenous IFN-β expression in the joint bones of CAIA model mice was evaluated by quantitative real-time PCR (qRT-PCR). The effects of exogenous IFN-β on CAIA model mice were assessed using a clinical scoring system, hematoxylin eosin and safranin-O with fast green counterstain histology, molybdenum target X-ray, and tartrate-resistant acid phosphatase (TRAP) staining. The RANKL-RANK signaling pathway was analyzed using qRT-PCR. The RAW 264.7 cell line was differentiated into osteoclasts with RANKL stimulation and then treated with exogenous IFN-β. The expression of inflammatory cytokines (IFN-γ, IL-17, MMP-3, and RANKL) and auto-antibodies (CII antibodies, RF-IgM, and anti-CCP/GPI) were significantly higher in RA compared with OA patients. After IFN-β intervention, some clinical symptoms in RA patients were partially alleviated, and the expression of IFN-γ, IL-17, MMP-3, and OPG) returned to normal levels. In the CAIA model, the expression of endogenous IFN-β in the joint bones was decreased. After IFN-β administration, the arthritis scores were decreased; synovial inflammation, cartilage, and bone destruction were clearly attenuated; and the expression of c-Fos and NFATc1 were reduced, while RANKL and TRAF6 expression was

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2012-01-01

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured...... to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10–21 months of age from a MAP infected herd with addition of either...... overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated...

MOLECULAR CLONING, SEQUENCING, EXPRESSION AND BIOLOGICAL ACTIVITY OF GIANT PANDA (AILUROPODA MELANOLEUCA) INTERFERON-GAMMA.

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Zhu, Hui; Wang, Wen-Xiu; Wang, Bao-Qin; Zhu, Xiao-Fu; Wu, Xu-Jin; Ma, Qing-Yi; Chen, De-Kun

2012-06-29

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. Interferon-gamma (IFN-γ) is the only member of type □ IFN and is vital for the regulation of host adapted immunity and inflammatory response. Little is known aboutthe FN-γ gene and its roles in giant panda.In this study, IFN-γ gene of Qinling giant panda was amplified from total blood RNA by RT-CPR, cloned, sequenced and analysed. The open reading frame (ORF) of Qinling giant panda IFN-γ encodes 152 amino acidsand is highly similar to Sichuan giant panda with an identity of 99.3% in cDNA sequence. The IFN-γ cDNA sequence was ligated to the pET32a vector and transformed into E. coli BL21 competent cells. Expression of recombinant IFN-γ protein of Qinling giant panda in E. coli was confirmed by SDS-PAGE and Western blot analysis. Biological activity assay indicated that the recombinant IFN-γ protein at the concentration of 4-10 µg/ml activated the giant panda peripheral blood lymphocytes,while at 12 µg/mlinhibited. the activation of the lymphocytes.These findings provide insights into the evolution of giant panda IFN-γ and information regarding amino acid residues essential for their biological activity.

The role of interferon gamma release assays in the monitoring of response to anti-tuberculosis treatment in children.

Science.gov (United States)

Shaik, Junaid; Pillay, Manormoney; Jeena, Prakash

2014-09-01

Successful control of childhood TB requires early diagnosis, effective chemotherapy and a method of evaluating the response to therapy. Identification of suitable biomarkers that predict the response to anti-TB therapy may allow the duration of treatment to be shortened. The majority of biomarker studies in paediatric TB have focused on the role of T cell-based interferon-gamma (IFN-γ) release assays (IGRAs) in the diagnosis of either latent or active disease. Little has been published on the role of IGRAs in the monitoring response to therapy in children. We reviewed the available literature to ascertain the value of IGRAs in the monitoring of response to anti-TB therapy in children. We explored the results of the few studies that have investigated the role of IGRAs as markers of response to anti-TB treatment in children. We conclude that the role of IGRAs as surrogate markers appears promising. Robust clinical trials are, however, needed to entrench the value of IGRAs as surrogate biomarkers of response to anti-TB therapy in children. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of IL-4, IL-17, and IFN-γ Levels in PatientsWith Breast Cancer

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Mina Rohani Borj

2017-03-01

Full Text Available Introduction: Tumor growth depends on intrinsic properties of malignant tumor and tumor microenvironment. Cytokines are secreted substances of the tumor microenvironment which are widely produced by tumor and immune cells. The aim of this research was to evaluate concentrations of interleukin 4 (IL-4, interleukin 7 (IL-17 and interferon gamma (IFN-γ in the breast cancer microenvironment. Methods: One hundred sixteen women between 18-73 years of age (61.15 ± 24.39 were enrolled in this study. Based on pathologic diagnostic assessment, patients were divided into 2 categories: those affected with benign breast tumor, and the subjects suffering from malignant breast tumors. Biopsy specimens were collected. Following homogenization, IFN-γ, IL-17, and IL-4 concentrations were determined in tumor tissues, adjacent tissues of the tumor, and blood serum samples of these 2 groups of patients by enzyme-linked immunosorbent assay (ELISA method. Results: Concentrations of IFN-γ, IL-17, and IL-4 were measured in tumor tissue samples, adjacent tissues of the tumor, and blood serum samples in both groups. Malignant breast tumor samples had significantly higher concentrations of IL-4 and IL-17 compared with benign breast tumor samples. And also the concentration of IFN-γ in adjacent tissues of the tumor and in blood serums in patients with malignant breast tumors was significantly higher than that in the benign breast tumor samples. However, there was no significant difference between the concentration of IFN-γ in neoplastic breast tumor tissues and that in the benign breast tumor tissues (P > 0. 05. Conclusion: Our data indicated that IL-17 and IL-4 cytokines but not IFN-γ had higher concentrations in the subjects with malignant tumor compared with those with benign tumor. The present findings indicated that the concentrations of IL-4 and IL-17 in tumor tissues may be associated with the severity of breast malignancy.

Role of IFN-gamma and LPS on Neuron/Glial Co-Cultures Infected by Neospora caninum

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Erica Etelvina Viana De Jesus

2014-10-01

Full Text Available Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 μg/mL of LPS on infected rat neuron/glial co-cultures. After 72 hours of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not, meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001, this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%. The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in CNS

Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

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Patcharaporn Tippayawat

Full Text Available Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined.Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry.B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+ (and CD8(+ T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum.Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

Tuberculin skin testing and treatment modulates interferon-gamma release assay results for latent tuberculosis in migrants.

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Matthew K O'Shea

Full Text Available Identifying latent tuberculosis infection (LTBI in people migrating from TB endemic regions to low incidence countries is an important control measure. However, no prospective longitudinal comparisons between diagnostic tests used in such migrant populations are available.To compare commercial interferon (IFN-gamma release assays (IGRAs and the tuberculin skin test (TST for diagnosing LTBI in a migrant population, and the influence of antecedent TST and LTBI treatment on IGRA performance.This cohort study, performed from February to September 2012, assessed longitudinal IGRA and TST responses in Nepalese military recruits recently arrived in the UK. Concomitant T-SPOT.TB, QFT-GIT and TST were performed on day 0, with IGRAs repeated 7 and 200 days later, following treatment for LTBI if necessary.166 Nepalese recruits were prospectively assessed. At entry, 21 individuals were positive by T-SPOT.TB and 8 individuals by QFT-GIT. There was substantial agreement between TST and T-SPOT.TB positives at baseline (71.4% agreement; κ = 0.62; 95% CI:0.44-0.79, but only moderate concordance between positive IGRAs (38.1% agreement; κ = 0.46; 95% CI:0.25-0.67. When reassessed 7 days following TST, numbers of IGRA-positive individuals changed from 8 to 23 for QFT-GIT (p = 0.0074 and from 21 to 23 for T-SPOT.TB (p = 0.87. This resulted in an increase in IGRA concordance to substantial (64.3% agreement; κ = 0.73; 95% CI:0.58-0.88. Thus, in total on day 0 and day 7 after testing, 29 out of 166 participants (17.5% provided a positive IGRA and of these 13 were TST negative. Two hundred days after the study commenced and three months after treatment for LTBI was completed by those who were given chemoprophylaxis, 23 and 21 participants were positive by T-SPOT.TB or QFT-GIT respectively. When individual responses were examined longitudinally within this population 35% of the day 7 QFT-GIT-positive, and 19% T-SPOT.TB-positive individuals, were

Use of the local lymph node assay in assessment of immune function.

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van den Berg, Femke A; Baken, Kirsten A; Vermeulen, Jolanda P; Gremmer, Eric R; van Steeg, Harry; van Loveren, Henk

2005-07-01

The murine local lymph node assay (LLNA) was originally developed as a predictive test method for the identification of chemicals with sensitizing potential. In this study we demonstrated that an adapted LLNA can also be used as an immune function assay by studying the effects of orally administered immunomodulating compounds on the T-cell-dependent immune response induced by the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). C57Bl/6 mice were treated with the immunotoxic compounds cyclosporin A (CsA), bis(tri-n-butyltin)oxide (TBTO) or benzo[a]pyrene, (B[a]P). Subsequently, cell proliferation and interferon-gamma (IFN-gamma) and interleukin (IL)-4 release were determined in the auricular lymph nodes (LNs) after DNCB application on both ears. Immunosuppression induced by CsA, TBTO and B[a]P was clearly detectable in this application of the LLNA. Cytokine release measurements proved valuable to confirm the results of the cell proliferation assay and to obtain an indication of the effect on Th1/Th2 balance. We believe to have demonstrated the applicability of an adapted LLNA as an immune function assay in the mouse.

Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence

DEFF Research Database (Denmark)

Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders

2016-01-01

light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell....... However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed...... concentration/population varied with whole blood, 10 × 106 cells/ml PBMC and 5 × 106 cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p

A Compton Suppressed Gamma Ray Counter For Radio Assay of Materials

Science.gov (United States)

Godfrey, Benjamin

2016-03-01

Rare event searches, such as direct dark matter experiments, require materials with ultra-low levels of natural radioactivity. We present a neutron activation analysis (NAA) technique for assaying metals, specifically titanium used for cryostat construction. Earlier attempts at NAA encountered limitations due to bulk activation via (n, p) reactions, which contributed to large continuum backgrounds due to Compton tails. Our method involves a heavy water shielded exposure to minimize (n,p) reactions and a sodium iodide shielded high purity germanium counter for the gamma ray assay. Preliminary results on assays for U/Th/K contamination in titaniumwill be presented.

The immunomodulatory effects of interferon-gamma on mature B-lymphocyte responses.

Science.gov (United States)

Jurado, A; Carballido, J; Griffel, H; Hochkeppel, H K; Wetzel, G D

1989-06-15

Interferon-gamma (IFN-gamma) exerts a broad spectrum of activities which affect the responses of mature B-cells. It strongly inhibits B-cell activation, acts as a B-cell growth factor (BCGF), and also induces final differentiation to immunoglobulin (Ig) production. IFN-gamma is deeply involved in the differential control of isotype expression, as it enhances IgG2a production and suppresses both IgG1 and IgE production. Although it is now possible to draw a general scheme of the effects of IFN-gamma on B-cells, a number of paradoxical results still exist in the field. In this manuscript, different experimental systems are analyzed in an attempt to explain these apparent paradoxes.

Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency.

Science.gov (United States)

Gul, Ersin; Sayar, Esra Hazar; Gungor, Bilgi; Eroglu, Fehime Kara; Surucu, Naz; Keles, Sevgi; Guner, Sukru Nail; Findik, Siddika; Alpdundar, Esin; Ayanoglu, Ihsan Cihan; Kayaoglu, Basak; Geckin, Busra Nur; Sanli, Hatice Asena; Kahraman, Tamer; Yakicier, Cengiz; Muftuoglu, Meltem; Oguz, Berna; Cagdas Ayvaz, Deniz Nazire; Gursel, Ihsan; Ozen, Seza; Reisli, Ismail; Gursel, Mayda

2017-11-16

Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

Science.gov (United States)

Xu, Lei; Zhou, Xinying; Wang, Wenshi; Wang, Yijin; Yin, Yuebang; Laan, Luc J W van der; Sprengers, Dave; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

2016-10-01

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes. © FASEB.

Diabetes is associated with lower tuberculosis antigen-specific interferon gamma release in Tanzanian tuberculosis patients and non-tuberculosis controls

DEFF Research Database (Denmark)

Faurholt-Jepsen, Daniel; Aabye, Martine Grosos; Jensen, Andreas Vestergaard

2014-01-01

in diabetes patients and therefore the validity of interferon gamma release assays (IGRA) may be compromised. The aim of the present study was to assess the association between diabetes and Mycobacterium tuberculosis (Mtb) antigen-specific interferon gamma (IFN-γ) release in a TB endemic area among culture......-confirmed TB patients and non-TB controls. Methods: Culture-confirmed pulmonary TB patients (n = 187) and healthy non-TB neighbourhood controls (n = 190) from Mwanza, Tanzania were tested for the presence of circulating T cells recognizing Mtb antigens using an IGRA. The diabetes status of all participants...

Delayed Gamma-Ray Spectroscopy for Non-Destructive Assay of Nuclear Materials

International Nuclear Information System (INIS)

Ludewigt, Bernhard; Mozin, Vladimir; Campbell, Luke; Favalli, Andrea; Hunt, Alan W.; Reedy, Edward T.E.; Seipel, Heather

2015-01-01

High-energy, beta-delayed gamma-ray spectroscopy is a potential, non-destructive assay techniques for the independent verification of declared quantities of special nuclear materials at key stages of the fuel cycle and for directly assaying nuclear material inventories for spent fuel handling, interim storage, reprocessing facilities, repository sites, and final disposal. Other potential applications include determination of MOX fuel composition, characterization of nuclear waste packages, and challenges in homeland security and arms control verification. Experimental measurements were performed to evaluate fission fragment yields, to test methods for determining isotopic fractions, and to benchmark the modeling code package. Experimental measurement campaigns were carried out at the IAC using a photo-neutron source and at OSU using a thermal neutron beam from the TRIGA reactor to characterize the emission of high-energy delayed gamma rays from 235 U, 239 Pu, and 241 Pu targets following neutron induced fission. Data were collected for pure and combined targets for several irradiation/spectroscopy cycle times ranging from 10/10 seconds to 15/30 minutes.The delayed gamma-ray signature of 241 Pu, a significant fissile constituent in spent fuel, was measured and compared to 239 Pu. The 241 Pu/ 239 Pu ratios varied between 0.5 and 1.2 for ten prominent lines in the 2700-3600 keV energy range. Such significant differences in relative peak intensities make it possible to determine relative fractions of these isotopes in a mixed sample. A method for determining fission product yields by fitting the energy and time dependence of the delayed gamma-ray emission was developed and demonstrated on a limited 235 U data set. De-convolution methods for determining fissile fractions were developed and tested on the experimental data. The use of high count-rate LaBr 3 detectors was investigated as a potential alternative to HPGe detectors. Modeling capabilities were added to an

Wholesomeness studies on gamma-irradiated smoked fish using short-term mutagenicity assays

International Nuclear Information System (INIS)

De la Rosa, A.M.; Banzon, R.B.

1985-12-01

The effect of gamma irradiation on the mutagenicity potential of wood-smoked mackerel (Rastrelliger sp.) was investigated. Smoked fish were irradiated with dose of 2.0, 4.0, 6.0 and 8.0 KGy, and tested for mutagenic activity using the Salmonella plate incorporation assay, host-mediated assay, and micronucleus test. The DMSO extract of unirradiated smoked fish was found to be mutagenic, without metabolic activation in Salmonella strains TA 100 and TA 104, both sensitive to base-pair substitution mutations. Strains TA 98 and TA 97 which are sensitive to frameshift mutations showed no mutagenic activity towards the same DMSO extract. The observed response towards the Salmonella strains was not affected by irradiation in the range of radiation doses studied. The presence of protamutagens in the DMSO extract of unirradiated smoked fish was not detected using the host-mediated assay. In another in-vivo test however, the same DMSO extract induced the formation of micronuclei in the bonemarrow cells of mice. Gamma irradiation up to a dose of 8.0 KGy did not affect the observed mutagenicity of wood-smoked fish. (author)

Increased TNF-alpha/IFN-gamma/IL-2 and decreased TNF-alpha/IFN-gamma production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination

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Mayara Fernanda Maggioli

2016-10-01

Full Text Available Central memory T cells (Tcm and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB; however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector / memory populations from bacille Calmette Guerin (BCG vaccinated and non-vaccinated calves prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves; only differing in magnitude (i.e., infected > vaccinated. BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (three weeks post-infection, non-vaccinates had greater antigen-specific IFN-γ/TNF-α and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains were detected in memory subsets, as well as in effector cells, as early as three weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB.

Cytokine mRNA profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: Association of sustained expression of IFN-gamma and IL-10 after viral clearance

DEFF Research Database (Denmark)

Johnsen, C. K.; Bøtner, Anette; Kamstrup, Søren

2002-01-01

An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALC's were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real......-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly...... elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV....

Type 1- and type 2-like lesional skin-derived Mycobacterium leprae-responsive T cell clones are characterized by coexpression of IFN-gamma/TNF-alpha and IL-4/IL-5/IL-13, respectively

NARCIS (Netherlands)

Verhagen, C. E.; van der Pouw Kraan, T. C.; Buffing, A. A.; Chand, M. A.; Faber, W. R.; Aarden, L. A.; Das, P. K.

1998-01-01

In an earlier study, we generated a large number of Mycobacterium leprae-responsive and M. leprae-nonresponsive T cell clones (TCC) from the lesional skin of immunologic unstable borderline leprosy patients. In that study, we divided TCC into type 1- and type 2-like on the basis of their IFN-gamma

Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas.

Science.gov (United States)

Zribi, Lilia; El-Goulli, Amel F; Ben-Abid, Meriem; Gharbi, Mohamed; Ben-Sghaier, Ines; Boufaden, Imed; Aoun, Karim; Bouratbine, Aïda

2017-11-15

Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood. The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs' status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls. Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age dogs (median age=72months, IQR: 45-84 months) that didn't respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn't respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2). The results of this study showed that IGRA is a useful new tool that can assess

Assay of plutonium contaminated waste by gamma spectrometry

International Nuclear Information System (INIS)

Adsley, I.; Bull, R.; Davies, M.; Green, M.

2011-01-01

The extreme toxicity of plutonium necessitates the segregation of plutonium contaminated materials (PCM) with extremely small (sub-μg) levels of contamination. The driver to measure accurately these small quantities of plutonium within (relatively) large volumes of waste is (in part) financial. In particular the cost of disposal (per unit volume) rises steeply with increasing waste-category. Within the UK, there has been a historical reluctance to use low energy gamma radiation to sentence PCM because of the potential for self attenuation by dense materials. This is unfortunate because the low-energy gamma radiation from PCM offers the only practicable technique for segregating PCM within the various Low Level Waste (LLW) (>0.4Bq/g) and sub-LLW categories. Whilst passive neutron counting techniques have proved successful for assay of waste well into the Intermediate Level Waste (ILW) (>100Bq/g) category, a cursory study reveals that these techniques are barely capable of detecting mg quantities of plutonium -- let alone the sub-μg quantities present in LLW. This paper considers the use of two types of gamma detector for assay of PCM: the thin sodium iodide FIDLER (Field Instrument for the Detection of Low Energy Radiation) and the HPGe (High Purity Germanium) detector. Systems utilising these two types of detector can provide complementary information. FIDLER measurements are conducted by careful, local, systematic monitoring of surfaces. By contrast a HPGe detector can be used to monitor entire walls, or even rooms, in one measurement. Thus, a HPGe detector placed in the centre of room (from which any radioactive hot-spots have previously been removed) could be used to demonstrate that the average activity remaining close to the surface of the walls/floor/ceiling is below a given limit. The Monte Carlo Code MCNP 1 has been used to model both FIDLER probe and HPGe detector in the measurement geometries described above. The MCNP simulations have been validated

Resolution of PMA-Induced Skin Inflammation Involves Interaction of IFN-γ and ALOX15

Directory of Open Access Journals (Sweden)

Guojun Zhang

2013-01-01

Full Text Available Background. Acute inflammation and its timely resolution play important roles in the body’s responses to the environmental stimulation. Although IFN-γ is well known for the induction of inflammation, its role in the inflammation resolution is still poorly understood. Methodology and Principal Findings. In this study, we investigated the function of interferon gamma (IFN-γ during the resolution of PMA-induced skin inflammation in vivo. The results revealed that the expression levels of IL-6, TNF-α, and monocyte chemoattractant protein 1 (MCP-1 in skin decreased during the resolution stage of PMA-induced inflammation, while IFN-γ is still maintained at a relatively high level. Neutralization of endogenous IFN-γ led to accelerated reduction of epidermal thickness and decreased epithelial cell proliferation. Similarly, decreased infiltration of inflammatory cells (Gr1+ or CD11b+ cells and a significant reduction of proinflammatory cytokines were also observed upon the blockade of IFN-γ. Furthermore, neutralization of IFN-γ boosted ALOX15 expression of the skin during inflammation resolution. In accordance, application of lipoxin A4 (LXA4, a product of ALOX15 obtained a proresolution effect similar to neutralization of IFN-γ. These results demonstrated that through upregulating ALOX15-LXA4 pathway, blockage of IFN-γ can promote the resolution of PMA-induced skin inflammation.

O desafio da tuberculose na faixa etária pediátrica frente a novas técnicas diagnósticas The challenge of pediatric tuberculosis in face of new diagnostic techniques

Directory of Open Access Journals (Sweden)

Flavio R. Sztajnbok

2009-06-01

Full Text Available OBJETIVOS: Apresentar uma revisão atualizada sobre os novos métodos para o diagnóstico da tuberculose baseados na produção in vitro de interferon-gama (IFN-γ por células T dos pacientes sob investigação, comparando-os com a tradicional prova tuberculínica. FONTES DOS DADOS: Revisão de literatura utilizando os bancos de dados MEDLINE e LILACS (2000-2008 utilizando as palavras-chave tuberculose, interferon-gama, quantiFERON, ELISPOT e T-SPOT.TB. SÍNTESE DOS DADOS: Esses novos testes mostraram-se, de um modo geral, tão ou mais sensíveis e específicos que a prova tuberculínica, tanto em adultos como em crianças e imunossuprimidos, para o diagnóstico da infecção latente e da doença ativa, apresentando vantagens como a menor interferência da vacinação prévia pelo BCG, menor influência de estados anérgicos e melhor acurácia em crianças menores. Nos Estados Unidos, já estão sendo utilizados em substituição à prova tuberculínica, e apesar dos custos ainda elevados, a Organização Mundial de Saúde vai priorizar a sua viabilidade econômica. CONCLUSÕES: Sempre levando em conta a importância da história clínica e epidemiológica, os novos testes baseados na produção de IFN-γ apresentam resultados promissores e deverão ser considerados na investigação de tuberculose em qualquer paciente, mas especialmente nos grupos de risco, como as crianças e os imunossuprimidos.OBJECTIVES: To present an updated review concerning new assays for diagnosing tuberculosis based on in vitro interferon-gamma production by host T cells, and compare them with tuberculin skin test. SOURCES: A literature review was carried out based on Medline and LILACS databases (2000-2008 searching for the following keywords: tuberculosis, interferon-gamma, quantiFERON, ELISPOT and T-SPOT.TB. SUMMARY OF THE FINDINGS: These new assays proved to have, in general, equal or superior sensitivity and specificity than the tuberculin skin test not only in

Delayed Gamma-Ray Spectroscopy for Non-Destructive Assay of Nuclear Materials

Energy Technology Data Exchange (ETDEWEB)

Ludewigt, Bernhard [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Mozin, Vladimir [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Campbell, Luke [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Favalli, Andrea [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Hunt, Alan W. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Reedy, Edward T.E. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Seipel, Heather [Idaho National Lab. (INL), Idaho Falls, ID (United States)

2015-06-01

High-­energy, beta-delayed gamma-­ray spectroscopy is a potential, non-­destructive assay techniques for the independent verification of declared quantities of special nuclear materials at key stages of the fuel cycle and for directly assaying nuclear material inventories for spent fuel handling, interim storage, reprocessing facilities, repository sites, and final disposal. Other potential applications include determination of MOX fuel composition, characterization of nuclear waste packages, and challenges in homeland security and arms control verification. Experimental measurements were performed to evaluate fission fragment yields, to test methods for determining isotopic fractions, and to benchmark the modeling code package. Experimental measurement campaigns were carried out at the IAC using a photo-­neutron source and at OSU using a thermal neutron beam from the TRIGA reactor to characterize the emission of high-­energy delayed gamma rays from ²³⁵U, ²³⁹Pu, and ²⁴¹Pu targets following neutron induced fission. Data were collected for pure and combined targets for several irradiation/spectroscopy cycle times ranging from 10/10 seconds to 15/30 minutes.The delayed gamma-ray signature of ²⁴¹Pu, a significant fissile constituent in spent fuel, was measured and compared to ²³⁹Pu. The ²⁴¹Pu/²³⁹Pu ratios varied between 0.5 and 1.2 for ten prominent lines in the 2700-­3600 keV energy range. Such significant differences in relative peak intensities make it possible to determine relative fractions of these isotopes in a mixed sample. A method for determining fission product yields by fitting the energy and time dependence of the delayed gamma-­ray emission was developed and demonstrated on a limited ²³⁵U data set. De-­convolution methods for determining fissile fractions were developed and tested on the experimental data. The use of high count-­rate LaBr₃ detectors

Production of interferon-gamma by in vivo tumor-sensitized T cells: Association with active antitumor immunity

International Nuclear Information System (INIS)

Bursuker, I.; Pearce, M.T.

1990-01-01

The state of active immunity to Meth A fibrosarcoma in mice immunized with an admixture of Meth A cells and Propionibacterium acnes is associated with possession by the host of spleen cells capable of producing interferon-gamma (IFN-gamma) upon in vitro restimulation with irradiated tumor cells. The ability of spleen cells from immunized mice to produce IFN-gamma in response to irradiated Meth A cells decays as active antitumor immunity is replaced by a state of immunological memory. The IFN-producing cells are L3T4+Ly2+, cyclophosphamide-sensitive and radiosensitive T cells, as determined by their sensitivity to corresponding monoclonal antibodies and complement. The induction of IFN-gamma production by in vivo tumor-sensitized T cells is tumor specific, in that spleen cells from mice immunized against Meth A fibrosarcoma can produce IFN in response to irradiated Meth A cells but not in response to another syngeneic tumor M109 lung carcinoma

Household food insecurity is associated with low interferon-gamma levels in pregnant Indian women.

Science.gov (United States)

Vaidya, A; Bhosale, R; Sambarey, P; Suryavanshi, N; Young, S; Mave, V; Kanade, S; Kulkarni, V; Deshpande, P; Balasubramanian, U; Elf, J; Gupte, N; Gupta, A; Mathad, J S

2017-07-01

Over 20% of tuberculosis (TB) cases during pregnancy occur in India. To determine the association between household food insecurity and interferon-gamma (IFN-γ) levels in pregnancy. Pregnant women in India were administered the Household Food Insecurity Access Scale (HFIAS) questionnaire and underwent an IFN-γ release assay. Logistic regression was used to identify factors associated with food insecurity. Of 538 women, 60 (11%) had household food insecurity, 47 (78%) of which were moderate or severe food insecure. After mitogen stimulation, moderate or severe food insecure women had a median IFN-γ concentration of 4.2 IU/ml (IQR 2.2-9.8) vs. 8.4 IU/ml (IQR 3.0-10) in women with no or mild food insecurity (P = 0.03). In multivariate analysis, higher IFN-γ concentrations were associated with human immunodeficiency virus infection (OR 1.3, 95%CI 0.51-2.1, P = 0.001), and inversely associated with moderate or severe food insecurity (OR -1.6, 95%CI -2.9 to -0.27, P = 0.02) and the number of adults in the household (OR -0.08, 95%CI -0.16 to -0.01, P = 0.03). There was no association between food insecurity and IFN-γ response to Mycobacterium tuberculosis antigen. Food insecurity in pregnancy is associated with low IFN-γ levels. There was no association between food insecurity and IFN-γ response to M. tuberculosis antigen, but our study was underpowered to detect this outcome.

Consistency of Mycobacterium tuberculosis-Specific Interferon-Gamma Responses in HIV-1-Infected Women during Pregnancy and Postpartum

Directory of Open Access Journals (Sweden)

Sasi R. Jonnalagadda

2012-01-01

Full Text Available Background. We determined the consistency of positive interferon-gamma (IFN-γ release assays (IGRAs to detect latent TB infection (LTBI over one-year postpartum in HIV-1-infected women. Methods. Women with positive IGRAs during pregnancy had four 3-monthly postpartum IGRAs. Postpartum change in magnitude of IFN-γ response was determined using linear mixed models. Results. Among 18 women with positive pregnancy IGRA, 15 (83% had a subsequent positive IGRA; 9 (50% were always positive, 3 (17% were always negative, and 6 (33% fluctuated between positive and negative IGRAs. Women with pregnancy IGRA IFN-γ >8 spot forming cells (SFCs/well were more likely to have consistent postpartum IGRA response (odds ratio: 10.0; 95% confidence interval (CI: 0.9–117.0. Change in IFN-γ response over postpartum was 10.2 SFCs/well (95% CI: −1.5–21.8 SFCs/well. Conclusion. Pregnancy positive IGRAs were often maintained postpartum with increased consistency in women with higher baseline responses. There were modest increases in magnitude of IGRA responses postpartum.

Is There Any Difference between the In Situ and Systemic IL-10 and IFN-γ Production when Clinical Forms of Cutaneous Sporotrichosis Are Compared?

Directory of Open Access Journals (Sweden)

Fernanda N Morgado

Full Text Available Fungus of the Sporothrix schenckii complex can produce skin lesions in humans, commonly lymphocutaneous (LC and fixed (F forms of sporotrichosis. Some authors have suggested that clinical forms are influenced by differences in virulence and genetic profile of isolates. But little is known about the role of immune response in determining the clinical outcome of sporotrichosis. To verify the profile of systemic and in situ IFN-γ and IL-10 expression in sporotrichosis patients, and consequently to detect any difference between the two compartments and/or clinical presentation, we quantified the number of IFN-γ and IL-10 producer peripheral blood mononuclear cells stimulated with S. schenckii antigen (Ss-Ag by Elispot, and quantified cytokines expression by in situ immunohistochemistry in the same patient. Three groups were formed: 1- LC (n = 9; 2- F (n = 10; 3- healthy individuals (n = 14. All sporotrichosis patients produced high amounts of systemic IFN- γ when compared to uninfected individuals. No differences were observed between LC and F groups. Regarding in situ IL-10 expression, a difference between LC and F groups was observed: LC lesions presented higher amounts of IL-10 than F lesions differently from systemic IL-10 which showed similarities. Our data suggests that LC lesions present higher IL-10 expression which could be related to regulatory mechanisms for compensating the tissue injury, however favoring fungal persistence in the lesions. Surprisingly, there were no differences in systemic and in situ IFN- γ expression between CL and F patients, although it was significantly higher expressed in these patients than in healthy individuals.

Is There Any Difference between the In Situ and Systemic IL-10 and IFN-γ Production when Clinical Forms of Cutaneous Sporotrichosis Are Compared?

Science.gov (United States)

Morgado, Fernanda N; Schubach, Armando O; Pimentel, Maria Inês; Lyra, Marcelo R; Vasconcellos, Érica C F; Valete-Rosalino, Claudia M; Conceição-Silva, Fátima

2016-01-01

Fungus of the Sporothrix schenckii complex can produce skin lesions in humans, commonly lymphocutaneous (LC) and fixed (F) forms of sporotrichosis. Some authors have suggested that clinical forms are influenced by differences in virulence and genetic profile of isolates. But little is known about the role of immune response in determining the clinical outcome of sporotrichosis. To verify the profile of systemic and in situ IFN-γ and IL-10 expression in sporotrichosis patients, and consequently to detect any difference between the two compartments and/or clinical presentation, we quantified the number of IFN-γ and IL-10 producer peripheral blood mononuclear cells stimulated with S. schenckii antigen (Ss-Ag) by Elispot, and quantified cytokines expression by in situ immunohistochemistry in the same patient. Three groups were formed: 1- LC (n = 9); 2- F (n = 10); 3- healthy individuals (n = 14). All sporotrichosis patients produced high amounts of systemic IFN- γ when compared to uninfected individuals. No differences were observed between LC and F groups. Regarding in situ IL-10 expression, a difference between LC and F groups was observed: LC lesions presented higher amounts of IL-10 than F lesions differently from systemic IL-10 which showed similarities. Our data suggests that LC lesions present higher IL-10 expression which could be related to regulatory mechanisms for compensating the tissue injury, however favoring fungal persistence in the lesions. Surprisingly, there were no differences in systemic and in situ IFN- γ expression between CL and F patients, although it was significantly higher expressed in these patients than in healthy individuals.

Is There Any Difference between the In Situ and Systemic IL-10 and IFN-γ Production when Clinical Forms of Cutaneous Sporotrichosis Are Compared?

Science.gov (United States)

Morgado, Fernanda N.; Schubach, Armando O.; Pimentel, Maria Inês; Lyra, Marcelo R.; Vasconcellos, Érica C. F.; Valete-Rosalino, Claudia M.; Conceição-Silva, Fátima

2016-01-01

Fungus of the Sporothrix schenckii complex can produce skin lesions in humans, commonly lymphocutaneous (LC) and fixed (F) forms of sporotrichosis. Some authors have suggested that clinical forms are influenced by differences in virulence and genetic profile of isolates. But little is known about the role of immune response in determining the clinical outcome of sporotrichosis. To verify the profile of systemic and in situ IFN-γ and IL-10 expression in sporotrichosis patients, and consequently to detect any difference between the two compartments and/or clinical presentation, we quantified the number of IFN-γ and IL-10 producer peripheral blood mononuclear cells stimulated with S. schenckii antigen (Ss-Ag) by Elispot, and quantified cytokines expression by in situ immunohistochemistry in the same patient. Three groups were formed: 1- LC (n = 9); 2- F (n = 10); 3- healthy individuals (n = 14). All sporotrichosis patients produced high amounts of systemic IFN- γ when compared to uninfected individuals. No differences were observed between LC and F groups. Regarding in situ IL-10 expression, a difference between LC and F groups was observed: LC lesions presented higher amounts of IL-10 than F lesions differently from systemic IL-10 which showed similarities. Our data suggests that LC lesions present higher IL-10 expression which could be related to regulatory mechanisms for compensating the tissue injury, however favoring fungal persistence in the lesions. Surprisingly, there were no differences in systemic and in situ IFN- γ expression between CL and F patients, although it was significantly higher expressed in these patients than in healthy individuals. PMID:27622513

Total Measurement Uncertainty for the Plutonium Finishing Plant (PFP) Segmented Gamma Scan Assay System

CERN Document Server

Fazzari, D M

2001-01-01

This report presents the results of an evaluation of the Total Measurement Uncertainty (TMU) for the Canberra manufactured Segmented Gamma Scanner Assay System (SGSAS) as employed at the Hanford Plutonium Finishing Plant (PFP). In this document, TMU embodies the combined uncertainties due to all of the individual random and systematic sources of measurement uncertainty. It includes uncertainties arising from corrections and factors applied to the analysis of transuranic waste to compensate for inhomogeneities and interferences from the waste matrix and radioactive components. These include uncertainty components for any assumptions contained in the calibration of the system or computation of the data. Uncertainties are propagated at 1 sigma. The final total measurement uncertainty value is reported at the 95% confidence level. The SGSAS is a gamma assay system that is used to assay plutonium and uranium waste. The SGSAS system can be used in a stand-alone mode to perform the NDA characterization of a containe...

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

LENUS (Irish Health Repository)

O'Connell, J

2012-02-03

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell\\'s sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95\\/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

Relative mass resolution technique for optimum design of a gamma nondestructive assay system

International Nuclear Information System (INIS)

Koh, Duck Joon

1995-02-01

Nondestructive assay(NDA) is a widely used nuclear technology for quantitative elemental and isotopic assay. Nondestructive assay is performed by the detection of an identifying radiation emerging from the sample, which can be unambiguously related to the element or isotope of interest. In every assay we can identify two distinct factors that lead to measurement uncertainty. We refer to these as statistical and spatial uncertainties. If the spatial distribution of the analyte and the matrix material in the sample are known and fairly constant from sample to sample, then the major source of measurement uncertainty is the statistical uncertainty resulting from randomness in the counting process. The spatial uncertainty is independent of the measurement time and therefore sets a lower limit to the measurement uncertainty, which is inherent in the assay system in conjunction with the population of samples to be measured. The only way to minimize the spatial uncertainty is an optimized design of the assay system. Therefore we have to decide on the type and number of detectors to be used, their deployment around the sample, the type of radiation to be measured, the duration of each measurement, the size and shape of the sample drum. The design procedure leading to the optimal assay system should be based on a quantitative(RMR:Relative Mass Resolution) comparison of the performance of each proposed design. For NDA system design of low level radwaste, a specific purpose Monte Carlo code has been developed to simulate point-source responses for sources within an assayed radwaste drum and to analyze the effect of scattered gammas from higher energy gammas on the spectrum of a low energy gamma-ray. We could use the well-known Monte Carlo code, such as MCNP for the simulation of NDA in the case of low level radwaste. But, MCNP is a multi-purpose Monte Carlo transport code for several geometries which requires large memory and long CPU time. For some cases in nuclear

Viral infection causes rapid sensitization to lipopolysaccharide: central role of IFN-alpha beta

DEFF Research Database (Denmark)

Nansen, A; Randrup Thomsen, A

2001-01-01

LPS is the major active agent in the pathogenesis of Gram-negative septic shock. In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock. We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point...... following infection. Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect. This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo. The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab......-depleted and gene-targeted mice. Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice. Thus...

Ornithogalum virens as a plant assay for beta and gamma radiation effects

International Nuclear Information System (INIS)

Herron, V.J.

1979-01-01

The purpose of this study was to determine if the monocotyledonous angiosperm, Ornithogalum virens (Quintanilha and Cabral, 1947), could be used in such a biological assay system. After exposing O. virens plants to acute ( 60 Co) and chronic ( 137 Cs) gamma radiation and internal beta radiation ( 32 P), lethality (LD 50 , LD 100 ), growth inhibition, and chromosome aberrations were investigated. The LD 50 and LD 100 for acute gamma radiation were estimated to be between 0.91 to 1.8 krad and less than 3.6 krad, respectively. Though growth inhibition and abnormal growth were observed in the acute and chronic gamma radiation studies, the changes in the growth of the plants were so variable that these parameters were found to be unreliable measures of radiation effects. Chromosome aberrations were a more reliable measure of radiation damage because linear relationships between total aberrations and dose were found for both gamma and beta radiation

A technique for combining neutron and gamma-ray data into a single assay value

International Nuclear Information System (INIS)

Pickrell, M.M.; Mercer, D.; Sharpe, T.J.

1998-01-01

The authors explored the potentials of using both neutron and gamma-ray measurements on a single item and combining these data into a single assay value. The purpose was to improve assay capability for sample matrices that are difficult to measure. They chose an empirical approach because they wanted to address difficult-to-measure items for which the assay problem is complex. They used the tomographic gamma scanner; a passive, high-efficiency neutron counter with add-a-source and multiplicity; and an active neutron, californium shuffler to obtain measurements. Twenty-four 200-L drums were measured with various matrices using all three machines. The matrices were chosen specifically to spain the difficult-to-measure assay problems for some or all of the instruments. For example, the authors measured a drum filled with concrete and another filled with metal. The data from these measurements were analyzed using the alternating conditional expectation algorithm, which is one of a class of generalized additive models. Other data fusion algorithms are also possible and are being explored. The intent was to find ways to combine the data that would reduce the matrix-induced measurement error

Evaluation of gamma radiation induced genetic damage in the fish Cyprinus carpio using comet assay

International Nuclear Information System (INIS)

Praveen Kumar, M.K.; Shyama, S.K.; Bhagat, S.S.; Chaubey, R.C.

2013-01-01

Radionuclides released from various sources including the industries, as well as, accidental release during a nuclear disaster can contaminate inland water bodies. Suitable bio-monitoring methods/biomarkers are the need of the day to assess the impact of high/low levels of radiation exposure in aquatic environment. Fishes are very important as a group of ecologically and commercially important non-human biota and are often used as a bioindicators of aquatic pollution. Present work was carried out to assess the genotoxic effect of gamma radiation on fresh water fish Cyprinus carpio (common carp) in vivo using comet assay. Fishes were irradiated with 2, 4, 6, 8 and 10 Gy of gamma rays using a teletherapy machine and comet assay was performed on nucleated erythrocytes after 24, 48 and 72 h of irradiation . A significant increase in % tail DNA was observed at all the doses of gamma radiation as compared to controls indicating radiation induced DNA damage in a dose-dependent manner. Maximum % tail DNA was observed at 24 h which gradually declined till 72 h, in a time-dependent manner. This decrease in damage may indicate repair of the damaged DNA and or loss of heavily damaged cells, over a period of time. The study reveals that the comet assay may be used as a sensitive and rapid method to detect genotoxicity of gamma radiation and other environmental pollutants in sentinel species. (author)

Delayed Gamma-Ray Spectroscopy for Non-Destructive Assay of Nuclear Materials

Energy Technology Data Exchange (ETDEWEB)

Ludewigt, Bernhard [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Mozin, Vladimir [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Campbell, Luke [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Favalli, Andrea [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Hunt, Alan W. [Idaho State Univ., Pocatello, ID (United States); Reedy, Edward T. [Idaho State Univ., Pocatello, ID (United States); Seipel, Heather A. [Idaho State Univ., Pocatello, ID (United States)

2015-06-01

Modeling capabilities were added to an existing framework and codes were adapted as needed for analyzing experiments and assessing application-specific assay concepts including simulation of measurements over many short irradiation/spectroscopy cycles. The code package was benchmarked against the data collected at the IAC for small targets and assembly-scale data collected at LANL. A study of delayed gamma-ray spectroscopy for nuclear safeguards was performed for a variety of assemblies in the extensive NGSI spent fuel library. The modeling results indicate that delayed gamma-ray responses can be collected from spent fuel assemblies with statistical quality sufficient for analyzing their isotopic composition using a 10¹¹ n/s neutron generator and COTS detector instrumentation.

A Promising IFN-Deficient System to Manufacture IFN-Sensitive Influenza Vaccine Virus.

Science.gov (United States)

Chen, Can; Fan, Wenhui; Li, Jing; Zheng, Weinan; Zhang, Shuang; Yang, Limin; Liu, Di; Liu, Wenjun; Sun, Lei

2018-01-01

Interferon (IFN)-sensitive and replication-incompetent influenza viruses are likely to be the alternatives to inactivated and attenuated virus vaccines. Some IFN-sensitive influenza vaccine candidates with modified non-structural protein 1 (NS1) are highly attenuated in IFN-competent hosts but induce robust antiviral immune responses. However, little research has been done on the manufacturability of these IFN-sensitive vaccine viruses. Here, RIG-I-knockout 293T cells were used to package the IFN-sensitive influenza A/WSN/33 (H1N1) virus expressing the mutant NS1 R38A/K41A. We found that the packaging efficiency of the NS1 R38A/K41A virus in RIG-I-knockout 293T cells was much higher than that in 293T cells. Moreover, the NS1 R38A/K41A virus almost lost its IFN antagonist activity and could no longer replicate in A549, MDCK, and Vero cells after 3-6 passages. This indicated that the replication of NS1 R38A/K41A virus is limited in conventional cells. Therefore, we further established a stable Vero cell line expressing the wild-type (WT) NS1 of the WSN virus, based on the Tet-On 3G system. The NS1 R38A/K41A virus was able to steadily propagate in this IFN-deficient cell line for at least 20 passages. In a mouse model, the NS1 R38A/K41A virus showed more than a 4-log reduction in lung virus titers compared to the WT virus at 3 and 5 days post infection. Furthermore, we observed that the NS1 R38A/K41A virus triggered high-level of IFN-α/β production in lung tissues and was eliminated from the host in a relatively short period of time. Additionally, this virus induced high-titer neutralizing antibodies against the WT WSN, A/Puerto Rico/8/1934 (PR8), or A/California/04/2009 (CA04) viruses and provided 100% protection against the WT WSN virus. Thus, we found that the replication of the NS1 R38A/K41A virus was limited in IFN-competent cells and mice. We also presented a promising IFN-deficient system, involving a RIG-I-knockout 293T cell line to package the IFN

A maximum-likelihood reconstruction algorithm for tomographic gamma-ray nondestructive assay

International Nuclear Information System (INIS)

Prettyman, T.H.; Estep, R.J.; Cole, R.A.; Sheppard, G.A.

1994-01-01

A new tomographic reconstruction algorithm for nondestructive assay with high resolution gamma-ray spectroscopy (HRGS) is presented. The reconstruction problem is formulated using a maximum-likelihood approach in which the statistical structure of both the gross and continuum measurements used to determine the full-energy response in HRGS is precisely modeled. An accelerated expectation-maximization algorithm is used to determine the optimal solution. The algorithm is applied to safeguards and environmental assays of large samples (for example, 55-gal. drums) in which high continuum levels caused by Compton scattering are routinely encountered. Details of the implementation of the algorithm and a comparative study of the algorithm's performance are presented

Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Erik T Sakowski

2015-07-01

Full Text Available The success of Mycobacterium tuberculosis (Mtb as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1 promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

Association study of interferon gamma (IFN-γ) +874T/A gene polymorphism in patients with paranoid schizophrenia.

Science.gov (United States)

Paul-Samojedny, Monika; Owczarek, Aleksander; Suchanek, Renata; Kowalczyk, Malgorzata; Fila-Danilow, Anna; Borkowska, Paulina; Kucia, Krzysztof; Kowalski, Jan

2011-03-01

Schizophrenia is a multifactorial disease with changes affecting the immune system. Dysregulation of the cytokine network in schizophrenia has been well documented. Such changes may occur due to disturbances in cytokine levels that are linked to polymorphisms of cytokine genes. However, research in the role of cytokine gene polymorphisms in schizophrenia has been surprisingly scanty. The aim of this study was to identify, in a case control study, whether polymorphism of IFN-γ gene is a risk factor for the development of paranoid schizophrenia. To the best of our knowledge, this is the first study that examines the association between the IFN-γ gene polymorphism and psychopathological symptoms in patients with paranoid schizophrenia. Polymorphism of IFN-γ (+874T/A, rs 62559044) in schizophrenic patients (n=179), as well as healthy individuals (n=196), both Polish residents, was genotyped using AS-PCR method. Of note, when analyzing the results, we took into consideration the gender of studied individuals. Surprisingly, a single-nucleotide polymorphism in the first intron of the IFN-γ gene was found to be associated with paranoid schizophrenia in males, but not in females. The presence of allele A at position +874 in the IFN-γ gene correlates with 1.66-fold higher risk of paranoid schizophrenia development in males. Differences in the genotypes may have an important role in determining the level of I gene transcription. Because other polymorphisms have been demonstrated to influence IFN-γ transcription, further analysis is necessary to clarify the role of this gene in the pathogenesis of paranoid schizophrenia.

IFN-γ signaling to astrocytes protects from autoimmune mediated neurological disability.

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Claudia Hindinger

Full Text Available Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS. Cells resident within the central nervous system (CNS are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

Human umbilical cord-derived mesenchymal stem cells utilise Activin-A to suppress Interferon-gamma production by natural killer cells.

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Debanjana eChaterjee

2014-12-01

Full Text Available Following allogeneic hematopoietic stem cell transplantation (HSCT, interferon (IFN-gamma levels in the recipient’s body can strongly influence the clinical outcome. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs are lucrative as biological tolerance-inducers in HSCT settings. Hence, we studied the molecular mechanism of how UC-MSCs influence natural killer (NK cell-mediated IFN-gamma production. Allogeneic NK cells were cultured in direct contact with UC-MSCs or cell free supernatants from MSC cultures (MSC conditioned media. We found that soluble factors secreted by UC-MSCs strongly suppressed IL-12/IL-18-induced IFN-gamma production by NK cells by reducing phosphorylation of STAT4, NF-kB as well as T-bet activity. UC-MSCs secreted considerable amounts of Activin-A, which could suppress IFN-gamma production by NK cells. Neutralisation of Activin-A in MSC-conditioned media significantly abrogated their suppressive abilities. Till date, multiple groups have reported that prostaglandin (PG-E2 produced by MSCs can suppress NK cell functions. Indeed, we found that inhibition of PGE2 production by MSCs could also significantly restore IFN-gamma production. However, the effects of Activin-A and PGE2 were not cumulative. To the best of our knowledge, we are first to report the role of Activin-A in MSC-mediated suppression of IFN-gamma production by NK cells.

Advanced Laser-Compton Gamma-Ray Sources for Nuclear Materials Detection, Assay and Imaging

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Barty, C. P. J.

2015-10-01

Highly-collimated, polarized, mono-energetic beams of tunable gamma-rays may be created via the optimized Compton scattering of pulsed lasers off of ultra-bright, relativistic electron beams. Above 2 MeV, the peak brilliance of such sources can exceed that of the world's largest synchrotrons by more than 15 orders of magnitude and can enable for the first time the efficient pursuit of nuclear science and applications with photon beams, i.e. Nuclear Photonics. Potential applications are numerous and include isotope-specific nuclear materials management, element-specific medical radiography and radiology, non-destructive, isotope-specific, material assay and imaging, precision spectroscopy of nuclear resonances and photon-induced fission. This review covers activities at the Lawrence Livermore National Laboratory related to the design and optimization of mono-energetic, laser-Compton gamma-ray systems and introduces isotope-specific nuclear materials detection and assay applications enabled by them.

HCV-specific immune responses induced by CIGB-230 in combination with IFN-α plus ribavirin

Science.gov (United States)

Amador-Cañizares, Yalena; Martínez-Donato, Gillian; Álvarez-Lajonchere, Liz; Vasallo, Claudia; Dausá, Mariacarla; Aguilar-Noriega, Daylen; Valenzuela, Carmen; Raíces, Ivette; Dubuisson, Jean; Wychowski, Czeslaw; Cinza-Estévez, Zurina; Castellanos, Marlén; Núñez, Magdalys; Armas, Anny; González, Yaimé; Revé, Ismariley; Guerra, Ivis; Pérez Aguiar, Ángel; Dueñas-Carrera, Santiago

2014-01-01

AIM: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. METHODS: CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. RESULTS: From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CONCLUSION: CIGB-230, combined with IFN

Analysis and databasing software for integrated tomographic gamma scanner (TGS) and passive-active neutron (PAN) assay systems

International Nuclear Information System (INIS)

Estep, R.J.; Melton, S.G.; Buenafe, C.

2000-01-01

The CTEN-FIT program, written for Windows 9x/NT in C++,performs databasing and analysis of combined thermal/epithermal neutron (CTEN) passive and active neutron assay data and integrates that with isotopics results and gamma-ray data from methods such as tomographic gamma scanning (TGS). The binary database is reflected in a companion Excel database that allows extensive customization via Visual Basic for Applications macros. Automated analysis options make the analysis of the data transparent to the assay system operator. Various record browsers and information displays simplify record keeping tasks

Development and testing of species-specific ELISA assays to measure IFN-gamma and TNF-alpha in bottlenose dolphins (Tursiops truncatus)

Science.gov (United States)

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Thou...

1α,25(OH2 Vitamin D3 Modulates Avian T Lymphocyte Functions without Inducing CTL Unresponsiveness.

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Nitish Boodhoo

Full Text Available 1,25-Dihydroxyvitamin D3 (Vitamin D is a naturally synthesized fat soluble vitamin shown to have immunomodulatory, anti-inflammatory and cancer prevention properties in human and murine models. Here, we studied the effects of Vitamin D on the functional abilities of avian T lymphocytes using chicken Interferon (IFN-γ ELISPOT assay, BrdU proliferation assay, Annexin V apoptosis assay and PhosFlow for detecting phosphorylated signalling molecules. The results demonstrate that Vitamin D significantly inhibited the abilities of T lymphocytes to produce IFN-γ and proliferate in vitro (P≤0.05, but retained their ability to undergo degranulation, which is a maker for cytotoxicity of these cells. Similarly, Vitamin D did not inhibit Extracellular signal-Regulated Kinase (ERK 1/2 phosphorylation, a key mediator in T cell signalling, in the stimulated T lymphocytes population, while reduced ERK1/2 phosphorylation levels in the unstimulated cells. Our data provide evidence that Vitamin D has immuno-modulatory properties on chicken T lymphocytes without inducing unresponsiveness and by limiting immuno-pathology can promote protective immunity against infectious diseases of poultry.

Efecto de la maca roja (Lepidium meyenii sobre los niveles de IFN-γ en ratas ovariectomizadas

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Johanna Leiva-Revilla

Full Text Available Objetivos. Comparar el efecto de diferentes dosis de maca roja sobre los niveles de interferón gamma (IFN-γ en ratas ovariectomizadas (OVX. Materiales y métodos. Ratas hembras adultas fueron divididas al azar en los siguientes seis grupos: Grupo 1: ratas pesudo-ovariectomizadas (PO; Grupo 2: ratas OVX; Grupo 3: ratas OVX tratadas con 4 ug/kg de estradiol, y Grupo 4, 5 y 6: ratas OVX tratadas con extractos de maca con 2,15, 4,3 y 8,6 mg polifenoles/kilogramo de peso corporal, respectivamente. Resultados. Las ratas OVX mostraron niveles bajos de IFN-γ en comparación con las ratas PO. El estradiol y la maca roja revirtieron el efecto de la ovariectomía sobre los niveles de IFN-γ. La maca roja presenta un incremento dosis-respuesta de los niveles de IFN-γ (r=0,57, p<0,05. Conclusiones. La administración de la maca roja incrementa los niveles de IFN-γ en ratas ovariectomizadas.

Early-life gut bacteria associate with IL-4-, IL-10- and IFN-γ production at two years of age.

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Maria A Johansson

Full Text Available Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B. adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus as well as Staphylococcus (S. aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA and numbers of IL-4-, IL-10- and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4-, IL-10- and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4- (p = 0.022 and IL-10 (p = 0.016 producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4- (p = 0.004, IL-10- (p = 0.004 and IFN-γ (p = 0.034 secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.

IP-10 release assays in the diagnosis of tuberculosis infection

DEFF Research Database (Denmark)

Ruhwald, Morten; Aabye, Martine G; Ravn, Pernille

2012-01-01

The current state-of-the-art tests for infection with Mycobacterium tuberculosis - the IFN-γ release assays - rely on accurate measurement of the cytokine IFN-γ. Many other potential biomarkers are expressed in concert with IFN-γ, and IP-10 in particular has shown promising results. IP-10...

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

OpenAIRE

Kontny, U; Kurane, I; Ennis, F A

1988-01-01

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

IFN-γ regulates human dental pulp stem cells behavior via NF-κB and MAPK signaling

Science.gov (United States)

He, Xinyao; Jiang, Wenkai; Luo, Zhirong; Qu, Tiejun; Wang, Zhihua; Liu, Ningning; Zhang, Yaqing; Cooper, Paul R.; He, Wenxi

2017-01-01

During caries, dental pulp expresses a range of pro-inflammatory cytokines in response to the infectious challenge. Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is critical for immune responses. Previous study has demonstrated that IFN-γ at relative high concentration (100 ng/mL) treatment improved the impaired dentinogenic and immunosuppressive regulatory functions of disease-derived dental pulp stem cells (DPSCs). However, little is known about the regulatory effects of IFN-γ at relative low concentration on healthy DPSC behavior (including proliferation, migration, and multiple-potential differentiation). Here we demonstrate that IFN-γ at relatively low concentrations (0.5 ng/mL) promoted the proliferation and migration of DPSCs, but abrogated odonto/osteogenic differentiation. Additionally, we identified that NF-κB and MAPK signaling pathways are both involved in the process of IFN-γ-regulated odonto/osteogenic differentiation of DPSCs. DPSCs treated with IFN-γ and supplemented with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) or SB203580 (a MAPK inhibitor) showed significantly improved potential for odonto/osteogenic differentiation of DPSCs both in vivo and in vitro. These data provide important insight into the regulatory effects of IFN-γ on the biological behavior of DPSCs and indicate a promising therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment. PMID:28098169

IFN-γ regulates human dental pulp stem cells behavior via NF-κB and MAPK signaling.

Science.gov (United States)

He, Xinyao; Jiang, Wenkai; Luo, Zhirong; Qu, Tiejun; Wang, Zhihua; Liu, Ningning; Zhang, Yaqing; Cooper, Paul R; He, Wenxi

2017-01-18

During caries, dental pulp expresses a range of pro-inflammatory cytokines in response to the infectious challenge. Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is critical for immune responses. Previous study has demonstrated that IFN-γ at relative high concentration (100 ng/mL) treatment improved the impaired dentinogenic and immunosuppressive regulatory functions of disease-derived dental pulp stem cells (DPSCs). However, little is known about the regulatory effects of IFN-γ at relative low concentration on healthy DPSC behavior (including proliferation, migration, and multiple-potential differentiation). Here we demonstrate that IFN-γ at relatively low concentrations (0.5 ng/mL) promoted the proliferation and migration of DPSCs, but abrogated odonto/osteogenic differentiation. Additionally, we identified that NF-κB and MAPK signaling pathways are both involved in the process of IFN-γ-regulated odonto/osteogenic differentiation of DPSCs. DPSCs treated with IFN-γ and supplemented with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) or SB203580 (a MAPK inhibitor) showed significantly improved potential for odonto/osteogenic differentiation of DPSCs both in vivo and in vitro. These data provide important insight into the regulatory effects of IFN-γ on the biological behavior of DPSCs and indicate a promising therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

Tuberculin-purified protein derivative-, MPT-64-, and ESAT-6-stimulated gamma interferon responses in medical students before and after Mycobacterium bovis BCG vaccination and in patients with tuberculosis

DEFF Research Database (Denmark)

Johnson, P D; Stuart, R L; Grayson, M L

1999-01-01

QuantiFERON-TB (QIFN) (CSL Limited) is a whole-blood assay for the recognition of infection with Mycobacterium tuberculosis. QIFN measures gamma interferon (IFN-gamma) production when purified protein derivatives (PPDs) of mycobacteria are incubated with venous blood samples. The specificity...... of QIFN in medical students before and after BCG immunization was assessed, and sensitivity in patients with tuberculosis was assessed. Antigens were PPD derived from M. tuberculosis and two M. tuberculosis-specific proteins, ESAT-6 and MPT-64. Of 60 medical students, all of whom had 0-mm tuberculin skin...... tests (TSTs) at study entry, 58 (97%) were initially classified as negative for M. tuberculosis infection by PPD QIFN. Five months after BCG immunization, 7 of 54 students (13%) had a TST result of >/=10 mm and 11 of 54 students (20%) tested positive by PPD QIFN. ESAT-6- and MPT-64-stimulated IFN...

Association between level of interferon gamma and acid-fast bacillipositivity in pulmonary tuberculosis

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Priwahyuningtyas, N. B.; Sinaga, B. Y. M.; Pandia, P.; Eyanoer, P. C.

2018-03-01

Tuberculosis is an infectious disease which caused by Mycobacterium tuberculosis (M. tuberculosis) that infected numerous organ especially the lung. A person’s immunity is very affecting for a person exposed to pulmonary tuberculosis. T-helper-1 cell (Th1) is very influential in the immune system especially in interfering intracellular bacterial infection. One of the cytokines known produced by Th1 cell is interferon gamma (IFN-γ) which is in eliminating M. tuberculosis. The study aims to identify the association between level of IFN-γ and AFB positivity in pulmonary tuberculosis patients in Medan. It is a case-control study. The subjects of the study were 60 new cases of pulmonary tuberculosis with AFB sputum smear- positive that never received ATT consisting 20 cases AFB (+1), 20 cases AFB (+2) and 20 cases AFB (+3).Samples were plasma collected from the venous blood of pulmonary tuberculosis patients. The plasma then underwent laboratory assay with ELISA techniques. Independent t-test was p<0.05 considered significant. Level of IFN-γ in TB AFB (+1) is higher than TB AFB (+2) and (+3), with thesignificant statistical result (p=0.001).

Adjuvant interferon gamma in patients with pulmonary atypical Mycobacteriosis: A randomized, double-blind, placebo-controlled study

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Sánchez-de la Osa Reinaldo B

2008-02-01

Full Text Available Abstract Background High antibiotic resistance is described in atypical Mycobacteriosis, mainly by Mycobacterium avium complex (MAC. Methods A randomized, double-blind, placebo-controlled clinical trial was carried out in two hospitals to evaluate the effect of interferon (IFN gamma as immunoadjuvant to chemotherapy on patients with atypical mycobacteria lung disease. Patients received placebo or 1 × 106 IU recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to daily oral azithromycin, ciprofloxacin, ethambutol and rifampin. Sputum samples collection for direct smear observation and culture as well as clinical and thorax radiography assessments were done during treatment and one year after. Cytokines and oxidative stress determinations were carried out in peripheral blood before and after treatment. Results Eighteen patients were included in the IFN group and 14 received placebo. Groups were homogeneous at entry; average age was 60 years, 75% men, 84% white; MAC infection prevailed (94%. At the end of treatment, 72% of patients treated with IFN gamma were evaluated as complete responders, but only 36% in the placebo group. The difference was maintained during follow-up. A more rapid complete response was obtained in the IFN group (5 months before, with a significantly earlier improvement in respiratory symptoms and pulmonary lesions reduction. Disease-related deaths were 35.7% of the patients in the placebo group and only 11.1% in the IFN group. Three patients in the IFN group normalized their globular sedimentation rate values. Although differences in bacteriology were not significant during the treatment period, some patients in the placebo group converted again to positive during follow-up. Significant increments in serum TGF-beta and advanced oxidation protein products were observed in the placebo group but not among IFN receiving patients. Treatments were well tolerated

[Polymorphism of TNF-alpha (308 A/G), IL-10 (1082 A/G, 819 C/T 592 A/C), IL-6 (174 G/C), and IFN-gamma (874 A/T); genetically conditioned cytokine synthesis level in children with diabetes type 1].

Science.gov (United States)

Siekiera, Urszula; Jarosz-Chobot, P; Janusz, J; Koehler, Brygida

2002-01-01

Type 1 diabetes is a genetically conditioned autoimmune disease. Genes that account for strong clustering of the disease susceptibility are located within the HLA region. There is also considerable individual variation in the immune response and role of cytokine genes in the disease predisposition. The aim of our research was identification of the genetically controlled TNF-alpha, IL-10, IL-6, IFN-gamma secretion profile in children with diabetes type 1. We have examined 36 children with diabetes type 1 and 36 healthy individuals. DNA was extracted from mononuclear peripheral blood cells. For identification of the cytokine polymorphism PCR-SSP method was used. Patients with diabetes type 1 differ from the group of healthy persons in the cytokine synthesis level and in the cytokine genotypes distribution. Genotype TNF-alpha (A/G) as well as IL-10 (ATA/ATA) was found only in group of children with diabetes but not in the control group. Genotypes IL-10 (GCC/GCC), IL-6 (C/C), IFN-gamma (T/T) were observed with decreased frequency in children with diabetes type 1. No differences between patients and control group in the frequency of IL-10 (GCC/ACC) (GCC/ATA), (ACC/ACC) (ACC/ATA) IL-6 (G/G), (G/C) and IFN-gamma (T/A), (A/A) genotypes were observed. Children with diabetes type 1 were more frequent "high producers" of TNF-alpha and IL-6. It is possible to us molecular method to estimate the genetically controlled immune reactivity. It is a very important immunogenetic factor of the disease predisposition.

Effects of interferon-gamma and tumor necrosis factor-alpha on macrophage enzyme levels

Science.gov (United States)

Pierangeli, Silvia S.; Sonnenfeld, Gerald

1989-01-01

Murine peritoneal macrophages were treated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF). Measurements of changes in acid phosphatase and beta-glucuronidase levels were made as an indication of activation by cytokine treatment. IFN-gamma or TNF-gamma treatment resulted in a significant increase in the activities of both enzymes measured in the cell lysates. This increase was observable after 6 h of incubation, but reached its maximum level after 24 h of incubation. The effect of the treatment of the cell with both cytokines together was additive. No synergistic effect of addition of both cytokines on the enzyme levels was observed.

Absence of autoreactive CD4+ T-cells targeting HLA-DQA1*01:02/DQB1*06:02 restricted hypocretin/orexin epitopes in narcolepsy type 1 when detected by EliSpot.

Science.gov (United States)

Kornum, Birgitte Rahbek; Burgdorf, Kristoffer Sølvsten; Holm, Anja; Ullum, Henrik; Jennum, Poul; Knudsen, Stine

2017-08-15

Narcolepsy type 1, a neurological sleep disorder strongly associated with Human Leukocyte Antigen (HLA-)DQB1*06:02, is caused by the loss of hypothalamic neurons producing the wake-promoting neuropeptide hypocretin (hcrt, also known as orexin). This loss is believed to be caused by an autoimmune reaction. To test whether hcrt itself could be a possible target in the autoimmune attack, CD4 + T-cell reactivity towards six different 15-mer peptides from prepro-hypocretin with high predicted affinity to the DQA1*01:02/DQB1*06:02 MHC class II dimer was tested using EliSpot in a cohort of 22 narcolepsy patients with low CSF hcrt levels, and 23 DQB1*06:02 positive healthy controls. Our ELISpot assay had a detection limit of 1:10,000 cells. We present data showing that autoreactive CD4 + T-cells targeting epitopes from the hcrt precursor in the context of MHC-DQA1*01:02/DQB1*06:02 are either not present or present in a frequency is <1:10,000 among peripheral CD4 + T-cells from narcolepsy type 1 patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Validation of Non-Invasive Waste Assay System (Gamma Box Counter) Performance at AECL Whiteshell Laboratories - 13136

International Nuclear Information System (INIS)

Attas, E.M.; Bialas, E.; Rhodes, M.J.

2013-01-01

Low-level radioactive waste (LLW) in solid form, resulting from decommissioning and operations activities at AECL's Whiteshell Laboratories (WL), is packaged in B-25 and B-1000 standard waste containers and characterized before it is shipped to an on-site interim storage facility, pending AECL decisions on long term management of its LLW. Assay of the waste packages before shipment contributes to an inventory of the interim storage facility and provides data to support acceptance at a future repository. A key characterization step is a gamma spectrometric measurement carried out under standard conditions using an automated, multi-detector Waste Assay System (WAS), purchased from Antech Corporation. A combination of ORTEC gamma acquisition software and custom software is used in this system to incorporate multiple measurements from two collimated high-resolution detectors. The software corrects the intensities of the gamma spectral lines for geometry and attenuation, and generates a table of calculated activities or limits of detection for a user-defined list of radioisotopes that may potentially be present. Validation of WAS performance was a prerequisite to routine operation. Documentation of the validation process provides assurance of the quality of the results produced, which may be needed one or two decades after they were generated. Aspects of the validation included setting up a quality control routine, measurements of standard point sources in reproducible positions, study of the gamma background, optimization of user-selectable software parameters, investigation of the effect of non-uniform distribution of materials and radionuclides, and comparison of results with measurements made using other gamma detector systems designed to assay bulk materials. The following key components of the validation process have been established. A daily quality control routine has been instituted, to verify stability of the gamma detector operation and the background levels

Gamma aminobutyric acid radioreceptor assay: a confirmatory quantitative assay for toxaphene in environmental and biological samples

International Nuclear Information System (INIS)

Saleh, M.A.; Blancato, J.N.

1993-01-01

Toxaphene is a complex mixture of polychlorinated monoterpenes, and was found to be acutely and chronically toxic to aquatic and wild life and posed a carcinogenic risk to humans before its ban in 1982. However, it is still found in the environment due to its relative persistence with an estimated half life time of about 10 years in soils. Toxaphenes neurotoxicity is attributed to a few isomers with a mode of action through binding to the chloride channel of the gamma-aminobutyric acid (GABA) receptor ionophore complex. [ 35 S] tertiary butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABA receptor for the development of radioreceptor assay technique. The GABA receptor was prepared by a sequence of ultra centrifugation and dialysis of mammalian (rats, cows, catfish and goats) brain homogenates. The receptor is then labeled with [ 35 S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with the sample containing the analytes. The assay is fast, sensitive and requires very little or no sample preparation prior to the analysis. (Author)

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease

DEFF Research Database (Denmark)

Andersen, Stig Henrik; Vervelde, Lonneke; Sutton, Kate

2017-01-01

controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine...... the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ− cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing...... cells were CD3− indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ−CD8α+ (CTL) population produced the highest NDV specific IFN...

Quantifying the passive gamma signal from spent nuclear fuel in support of determining the plutonium content in spent nuclear fuel with nondestructive assay

Energy Technology Data Exchange (ETDEWEB)

Fensin, Michael L [Los Alamos National Laboratory; Tobin, Steven J [Los Alamos National Laboratory; Menlove, Howard O [Los Alamos National Laboratory; Swinhoe, Martyn T [Los Alamos National Laboratory

2009-01-01

The objective of safeguarding nuclear material is to deter diversions of significant quantities of nuclear materials by timely monitoring and detection. There are a variety of motivations for quantifying plutonium in spent fuel (SF), by means of nondestructive assay (NDA), in order to meet this goal. These motivations include the following: strengthening the capabilities of the International Atomic Energy Agencies ability to safeguard nuclear facilities, shipper/receiver difference, input accountability at reprocessing facilities and burnup credit at repositories. Many NDA techniques exist for measuring signatures from SF; however, no single NDA technique can, in isolation, quantify elemental plutonium in SF. A study has been undertaken to determine the best integrated combination of 13 NDA techniques for characterizing Pu mass in spent fuel. This paper focuses on the development of a passive gamma measurement system in support the spent fuel assay system. Gamma ray detection for fresh nuclear fuel focuses on gamma ray emissions that directly coincide with the actinides of interest to the assay. For example, the 186-keV gamma ray is generally used for {sup 235}U assay and the 384-keV complex is generally used for assaying plutonium. In spent nuclear fuel, these signatures cannot be detected as the Compton continuum created from the fission products dominates the signal in this energy range. For SF, the measured gamma signatures from key fission products ({sup 134}Cs, {sup 137}Cs, {sup 154}Eu) are used to ascertain burnup, cooling time, and fissile content information. In this paper the Monte Carlo modeling set-up for a passive gamma spent fuel assay system will be described. The set-up of the system includes a germanium detector and an ion chamber and will be used to gain passive gamma information that will be integrated into a system for determining Pu in SF. The passive gamma signal will be determined from a library of {approx} 100 assemblies that have been

Novel methods of cytokine detection: Real-time PCR, ELISPOT, and intracellular cytokine staining

Directory of Open Access Journals (Sweden)

Eliza Turlej

2009-05-01

Full Text Available Cytokines are small hormone-like proteins that play important roles in immune system control. Cytokines regulate the proliferation and differentiation of cells and hematopoiesis and act as mediators in the inflammatory reaction. Changes in cytokine levels are found in many diseases, such as sepsis, bowel inflammatory disease, autoimmune diseases, as well as graft-versus-host disease. Cytokines levels can be detected using in vivo, in vitro, and ex vivo techniques. The level of cytokine produced can be measured by immunoenzymatic test (ELISA in supernatant after cell culture with the addition of stimulant and in plasma by techniques that measure the level of cytokine secretion in cells (e.g. immunohistochemical staining, ELISPOT, and intracellular cytokine staining, and by molecular biological methods (RPA, real-time PCR, in situ hybridization, and Northern blot. Detection of cytokine mRNA in tissues is useful in the direct determination of heterogenic populations of cytokine-producing cells. Nowadays the most frequently used methods for measuring cytokine level are ELISPOT, intracellular cytokine staining with flow cytometry detection, and real-time PCR. These methods have an important clinical role in vaccine efficacy, in viral, bacterial, and verminous diagnostics, and in determining the efficacy of cancer treatment.

Role of IFN-gamma and IL-6 in a protective immune response to Yersinia enterocolitica in mice

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Autenrieth Ingo B

2008-09-01

Full Text Available Abstract Background Yersinia outer protein (Yop H is a secreted virulence factor of Yersinia enterocolitica (Ye, which inhibits phagocytosis of Ye and contributes to the virulence of Ye in mice. The aim of this study was to address whether and how YopH affects the innate immune response to Ye in mice. Results For this purpose, mice were infected with wild type Ye (pYV+ or a YopH-deficient Ye mutant strain (ΔyopH. CD11b+ cells were isolated from the infected spleen and subjected to gene expression analysis using microarrays. Despite the attenuation of ΔyopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and ΔyopH infection, using either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes, including IFN-γ and IL-6, are high after pYV+ infection but low after sublethal ΔyopH infection. In line with these findings, infection of IFN-γR-/- and IL-6-/- mice with pYV+ or ΔyopH revealed that these cytokines are not necessarily required for control of ΔyopH, but are essential for defense against infection with the more virulent pYV+. Consistently, IFN-γ pretreatment of bone marrow derived macrophages (BMDM strongly enhanced their ability in killing intracellular Ye bacteria. Conclusion In conclusion, this data suggests that IFN-γ-mediated effector mechanisms can partially compensate virulence exerted by YopH. These results shed new light on the protective role of IFN-γ in Ye wild type infections.

Measurement of gamma-aminobutyric acid in human cerebrospinal fluid: radioreceptor assay using [3H]muscimol

International Nuclear Information System (INIS)

Baraczka, K.; Sperk, G.

1981-01-01

A method is described for the determination of gamma-aminobutyric acid (GABA) levels in human cerebrospinal fluid by modification of the radioreceptor assay utilizing [ 3 H]muscimol as labelled ligand. This method is compared with the radioreceptor assay using [ 3 H]GABA as labelled ligand. Although the [ 3 H]muscimol assay is less sensitive than the [ 3 H]GABA method, it offers the advantage of being more rapid due to the use of a filtration step instead of the usual, more time-consuming centrifugation of the samples. Samples of CSF of patients with various neurological or psychiatric disturbances were analysed. There was a satisfactory correlation between the GABA values obtained by the two assays. (Auth.)

Gamma ray scanner systems for nondestructive assay of heterogeneous waste barrels

International Nuclear Information System (INIS)

Martz, H.E.; Decman, B.J.; Roberson, G.P.; Levai, F.

1997-01-01

Traditional gamma safeguards measurements have usually been performed using a segmented gamma scanning (SGS) system. The accuracy of this technique relies on the assumption that the sample matrix and the activity are both uniform for a segment. Waste barrels are often highly heterogeneous, span a wide range of composition and matrix type. The primary sources of error are all directly or indirectly related to a non-uniform measurement response associated with unknown radioactive source spatial distribution and heterogeneity of the matrix. These errors can be significantly reduced by some imaging techniques that measure exact spatial locations of sources and attenuation maps. In this paper we describe a joint R ampersand D effort between the Lawrence Livermore National Laboratory (LLNL) and the Institute of Nuclear Techniques (INT) of the Technical University, Budapest, to compare results obtained by two different gamma-ray nondestructive assay (NDA) systems used for imaging waste barrels. The basic principles are the same, but the approaches are different. Key factors to judge the adequacy of a method are the detection limit and the accuracy. Test drums representing waste to be measured are used to determine basic parameters of these techniques

Roles of PU.1 in monocyte- and mast cell-specific gene regulation: PU.1 transactivates CIITA pIV in cooperation with IFN-gamma.

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Ito, Tomonobu; Nishiyama, Chiharu; Nakano, Nobuhiro; Nishiyama, Makoto; Usui, Yoshihiko; Takeda, Kazuyoshi; Kanada, Shunsuke; Fukuyama, Kanako; Akiba, Hisaya; Tokura, Tomoko; Hara, Mutsuko; Tsuboi, Ryoji; Ogawa, Hideoki; Okumura, Ko

2009-07-01

Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN-gamma was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr(-/-) BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN-gamma stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBPalpha in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.

Verification by the FISH translocation assay of historic doses to Mayak workers from external gamma radiation

Energy Technology Data Exchange (ETDEWEB)

Sotnik, Natalia V.; Azizova, Tamara V. [Southern Urals Biophysics Institute (SUBI), Ozyorsk, Chelyabinsk Region (Russian Federation); Darroudi, Firouz [Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands); College of North Atlantic, Department of Health Science, Centre for Human Safety and Environmental Research, Doha (Qatar); Ainsbury, Elizabeth A.; Moquet, Jayne E.; Lloyd, David C.; Hone, Pat A.; Edwards, Alan A. [Public Health England, Chilton, Oxfordshire (United Kingdom); Fomina, Janna [Leiden University Medical Center, Department of Toxicogenetics, Leiden (Netherlands)

2015-11-15

The aim of this study was to apply the fluorescence in situ hybridization (FISH) translocation assay in combination with chromosome painting of peripheral blood lymphocytes for retrospective biological dosimetry of Mayak nuclear power plant workers exposed chronically to external gamma radiation. These data were compared with physical dose estimates based on monitoring with badge dosimeters throughout each person's working life. Chromosome translocation yields for 94 workers of the Mayak production association were measured in three laboratories: Southern Urals Biophysics Institute, Leiden University Medical Center and the former Health Protection Agency of the UK (hereinafter Public Health England). The results of the study demonstrated that the FISH-based translocation assay in workers with prolonged (chronic) occupational gamma-ray exposure was a reliable biological dosimeter even many years after radiation exposure. Cytogenetic estimates of red bone marrow doses from external gamma rays were reasonably consistent with dose measurements based on film badge readings successfully validated in dosimetry system ''Doses-2005'' by FISH, within the bounds of the associated uncertainties. (orig.)

Gamma ray NDA assay system for total plutonium and isotopics in plutonium product solutions

International Nuclear Information System (INIS)

Cowder, L.R.; Hsue, S.T.; Johnson, S.S.; Parker, J.L.; Russo, P.A.; Sprinkle, J.K.; Asakura, Y.; Fukuda, T.; Kondo, I.

1979-01-01

A LASL-designed gamma-ray NDA instrument for assay of total plutonium and isotopics of product solutions at Tokai-Mura is currently installed and operating. The instrument is, optimally, a densitometer that uses radioisotopic sources for total plutonium measurements at the K absorption edge. The measured transmissions of additional gamma-ray lines from the same radioisotopic sources are used to correct for self-attenuation of passive gamma rays from plutonium. The corrected passive data give the plutonium isotopic content of freshly separated to moderately aged solutions. This off-line instrument is fully automated under computer control, with the exception of sample positioning, and operates routinely in a mode designed for measurement control. A one-half percent precision in total plutonium concentration is achieved with a 15-minute measurement

Lectin of Bacillus subtilis sp. as overinducer of gamma-interferonogenesis.

Science.gov (United States)

Kishko, Ia H; Vasylenko, M I; Pidhors'kyĭ, V S; Kovalenko, E O

1997-01-01

It has been demonstrated experimentally that lectin of Bacillus subtilis sp. in comparison with generally accepted Con A, PHA and lectin of "gold rain" grass--Laburnum anagyroides M e d i k in trials on white mice of CBA line gave in 4 hours of induction maximal titers of gamma-IFN in blood serum of animals--153.6 +/- 17.0 IU/ml. Practically identical titers had been obtained after induction by lectin "gold rain", some lower--after Con A and PHA. At swine gamma-IFN synthesis optimal density of cell suspension must contain 2.5 + 10(7) immunocytes in 1 ml, owing to which it is possible to obtain the titer equal 1 : 2150. Materials with using of bacterial lectins at various degree of purification had shown that maximal titers in blood serum of mongrel white mice were registered at administration to animals of non-purified lectin, 4 times lower--at using of half-purified and purified lectins. Data of these trials in vivo were confirmed by materials of gamma-IFN induction by immunocytes of swine, cattle and even man.

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays.

Science.gov (United States)

M K, Praveen Kumar; Soorambail K, Shyama; Bhagatsingh Harisingh, Sonaye; D'costa, Avelyno; Ramesh Chandra, Chaubey

2015-10-01

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2-10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution. Copyright © 2015 Elsevier B.V. All rights reserved.

Immunosenescent CD57+CD4+ T-cells Accumulate and Contribute to Interferon-γ Responses in HIV Patients Responding Stably to ART

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Sonia Fernandez

2011-01-01

Full Text Available HIV-infected individuals responding to antiretroviral therapy (ART after severe CD4+ T-cell depletion may retain low responses to recall antigens [eg: cytomegalovirus (CMV] and altered expression of T-cell co-stimulatory molecules consistent with immunosenescence. We investigated the capacity of phenotypically senescent cells to generate cytokines in HIV patients receiving long-term ART (n = 18 and in healthy controls (n = 10. Memory T-cells were assessed by interferon (IFN-γ ELISpot assay and flow cytometrically via IFN-γ or IL-2. Proportions of CD57brightCD28null CD4+ T-cells correlated with IFN-γ responses to CMV (p = 0.009 and anti-CD3 (p = 0.002 in HIV patients only. Proportions of CD57brightCD28null CD8+ T-cells and CD8+ T-cell IFN-γ responses to CMV peptides correlated in controls but not HIV patients. IL-2 was predominantly produced by CD28+T-cells from all donors, whereas IFN-γ was mostly produced by CD57+ T-cells. The findings provide evidence of an accumulation of immunosenescent T-cells able to make IFN-γ. This may influence the pathogenesis of secondary viral infections in HIV patients receiving ART.

Getting “Inside” Type I IFNs: Type I IFNs in Intracellular Bacterial Infections

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Deann T. Snyder

2017-01-01

Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

Delayed growth of EL4 lymphoma in SR-A-deficient mice is due to upregulation of nitric oxide and interferon-gamma production by tumor-associated macrophages.

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Komohara, Yoshihiro; Takemura, Kenichi; Lei, Xiao Feng; Sakashita, Naomi; Harada, Mamoru; Suzuki, Hiroshi; Kodama, Tatsuhiko; Takeya, Motohiro

2009-11-01

Class A scavenger receptors (SR-A, CD204) are highly expressed in tumor-associated macrophages (TAM). To investigate the function of SR-A in TAM, wild-type and SR-A-deficient (SR-A(-/-)) mice were injected with EL4 cells. Although these groups of mice did not differ in the numbers of infiltrating macrophages and lymphocytes and in neovascularization, SR-A(-/-) mice had delayed growth of EL4 tumors. Expression of inducible nitric oxide (NO) synthase and interferon (IFN)-gamma mRNA increased significantly in tumor tissues from SR-A(-/-) mice. Engulfment of necrotic EL4 cells induced upregulation of NO and IFN-gamma production by cultured macrophages, and production of NO and IFN-gamma increased in SR-A(-/-) macrophages in vitro. IFN-beta production by cultured macrophages was also elevated in SR-A(-/-) macrophages in vitro. These results suggested that the antitumor activity of macrophages increased in SR-A(-/-) mice because of upregulation of NO and IFN-gamma production. These data indicate an important role of SR-A in regulating TAM function by inhibiting toll-like receptor (TLR)4-IFN-beta signaling.

A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.

Science.gov (United States)

Breivik, Lars; Oftedal, Bergithe E V; Bøe Wolff, Anette S; Bratland, Eirik; Orlova, Elizaveta M; Husebye, Eystein S

2014-07-01

An important characteristic of autoimmune polyendocrine syndrome type 1 (APS 1) is the existence of neutralizing autoantibodies (nAbs) against the type I interferons (IFN) -α2 and -ω at frequencies close to 100%. Type 1 IFN autoantibodies are detected by antiviral neutralizing assays (AVA), binding assays with radiolabelled antigens (RLBA), enzyme-linked immunosorbent assay (ELISA), or by reporter-based cell assays. We here present a simple and reliable version of the latter utilizing a commercially available cell line (HEK-Blue IFN-α/β). All 67 APS 1 patients were positive for IFN-ω nAbs, while 90% were positive for IFN-α2 nAbs, a 100% and 96% correlation with RLBA, respectively. All blood donors and non-APS 1 patients were negative. The dilution titer required to reduce the effect of IFN-ω nAbs correlated with the RLBA index. This cell-based autoantibody assay (CBAA) is easy to perform, suitable for high throughput, while providing high specificity and sensitivity. Copyright © 2014 Elsevier Inc. All rights reserved.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

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Kathleen Ingenhoven

2017-07-01

Full Text Available ObjectiveTo develop and validate a method for the detection of binding anti-drug antibodies (ADAs against interferon beta (IFN-β in human serum as part of a European initiative (ABIRISK aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk.MethodA two-tiered bridging enzyme-linked immunosorbent assay (ELISA format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen “putative positive” samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-β and percentage of inhibition is calculated.ResultsThe assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control.ConclusionAn ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

IFN-γ release assay conversions and reversions. Challenges with serial testing in U.S. health care workers.

Science.gov (United States)

Joshi, Manish; Monson, Thomas P; Joshi, Anita; Woods, Gail L

2014-03-01

IFN-γ release assays (IGRAs) including the QuantiFERON-TB gold in-tube test (QFT-GIT) are increasingly used in place of the tuberculin skin test (TST) in surveillance programs for Mycobacterium tuberculosis infection in the United States. However, data on conversions, reversions, and predictive value of QFT in such programs for health care workers (HCWs) are limited. The purpose of this study is to assess long-term reproducibility and conversion and reversion rates of QFT-GIT among HCWs who underwent serial testing at a tertiary care center in the United States. Retrospective chart review of HCWs at the Central Arkansas Veterans Healthcare System (CAVHS) who underwent serial testing with QFT-GIT as a part of their employee screening between November 1, 2008 and January 31, 2011. A total of 2,303 HCWs had at least 2 QFT-GITs 1 year apart. The initial QFT-GIT was positive for 69 and 2 were indeterminate. Of these 69 HCWs, 31 (45%) reverted on repeat testing, and 25 of 31 (80.6%) HCWs who reverted had a negative look-back TST. Of the 2,232 HCWs with an initial negative QFT-GIT, 71 (3.2%) converted on repeat testing. A third QFT-GIT assay was performed in 41 of the 71 converters and 90% (37 of 41) reverted back to negative. Only two HCWs had TST and QFT-GIT conversion. Poor IGRA reproducibility and a low predictive value of QFT-GIT conversions indicate that QFT-GIT with current interpretation criteria should not be used for serial screening of U.S. HCWs. Negative TSTs have higher reproducibility than QFT-GIT for serial testing of HCWs in low tuberculosis incidence settings.

Evaluation of immune responses in HIV infected patients with pleural tuberculosis by the QuantiFERON® TB-Gold interferon-gamma assay

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Lekabe Jacob M

2008-03-01

Full Text Available Abstract Background Diagnosis of tuberculous (TB pleuritis is difficult and better diagnostic tools are needed. New blood based interferon-gamma (IFN-γ tests are promising, but sensitivity could be low in HIV positive patients. The IFN-γ tests have not yet been validated for use in pleural fluid, a compartment with higher level of immune activation than in blood. Methods The QuantiFERON TB®-Gold (QFT-TB test was analysed in blood and pleural fluid from 34 patients presenting with clinically suspected pleural TB. Clinical data, HIV status and CD4 cell counts were recorded. Adenosine deaminase activity (ADA analysis and TB culture were performed on pleural fluid. Results The patients were categorised as 'confirmed TB' (n = 12, 'probable TB' (n = 16 and 'non-TB' pleuritis (n = 6 based on TB culture results and clinical and biochemical criteria. The majority of the TB patients were HIV infected (82%. The QFT-TB in pleural fluid was positive in 27% and 56% of the 'confirmed TB' and 'probable TB' cases, respectively, whereas the corresponding sensitivities in blood were 58% and 83%. Indeterminate results in blood (25% were caused by low phytohemagglutinin (PHA = positive control IFN-γ responses, significantly lower in the TB patients as compared to the 'non-TB' cases (p = 0.02. Blood PHA responses correlated with CD4 cell count (r = 0.600, p = 0.028. In contrast, in pleural fluid indeterminate results (52% were caused by high Nil (negative control IFN-γ responses in both TB groups. Still, the Nil IFN-γ responses were lower than the TB antigen responses (p Conclusion The QFT-TB test in blood could contribute to the diagnosis of TB pleuritis in the HIV positive population. Still, the number of inconclusive results is too high to recommend the commercial QFT-TB test for routine use in pleural fluid in a TB/HIV endemic resource-limited setting.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

DEFF Research Database (Denmark)

Shaw, A.; Larsen, M.; Roepstorff, P.

1999-01-01

magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

OpenAIRE

Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

1992-01-01

We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

Severe respiratory syncytial virus bronchiolitis in infants is associated with reduced airway interferon gamma and substance P.

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Malcolm G Semple

2007-10-01

Full Text Available Severe human respiratory syncytial virus (hRSV bronchiolitis in previously well infants may be due to differences in the innate immune response to hRSV infection.to determine if factors mediating proposed mechanisms for severe bronchiolitis differ with severity of disease.197 infants admitted to hospital with hRSV bronchiolitis were recruited and grouped according to no oxygen requirement (n = 27, oxygen dependence (n = 114 or mechanical ventilation (n = 56. We collected clinical data, nasopharyngeal aspirate (NPA and if ventilated bronchoalveolar lavage (BAL. Interferon-gamma (IFN-gamma, substance P (SP, interleukin 9 (IL-9, urea and hRSV load, were measured in cell free supernatant from NPA and BAL. Multivariate analysis compared independent effects of clinical, virological and immunological variables upon disease severity. IFN-gamma and SP concentrations were lower in NPA from infants who required oxygen or mechanical ventilation. Viral load and IL-9 concentrations were high but did not vary with severity of disease. Independent predictors of severe disease (in diminishing size of effect were low weight on admission, low gestation at birth, low NPA IFN-gamma and NPA SP. Nasal airway sampling appears to be a useful surrogate for distal airway sampling since concentrations of IFN-gamma, SP, IL-9 and viral load in NPA correlate with the same in BAL.Our data support two proposed mechanisms for severe hRSV disease; reduced local IFN-gamma response and SP mediated inflammation. We found large amounts of hRSV and IL-9 in airways secretions from the upper and lower respiratory tract but could not associate these with disease severity.

Interferon Gamma in African Trypanosome Infections: Friends or Foes?

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Wu, Hui; Liu, Gongguan; Shi, Meiqing

2017-01-01

African trypanosomes cause fatal infections in both humans and livestock. Interferon gamma (IFN-γ) plays an essential role in resistance to African trypanosomes. However, increasing evidence suggests that IFN-γ, when excessively synthesized, also induces immunopathology, enhancing susceptibility to the infection. Thus, production of IFN-γ must be tightly regulated during infections with African trypanosomes to ensure that a robust immune response is elicited without tissue destruction. Early studies have shown that secretion of IFN-γ is downregulated by interleukin 10 (IL-10). More recently, IL-27 has been identified as a negative regulator of IFN-γ production during African trypanosome infections. In this review, we discuss the current state of our understanding of the role of IFN-γ in African trypanosome infections. We have focused on the cellular source of IFN-γ, its beneficial and detrimental effects, and mechanisms involved in regulation of its production, highlighting some recent advances and offering some perspectives on future directions.

Ursolic acid isolated from Uncaria rhynchophylla activates human dendritic cells via TLR2 and/or TLR4 and induces the production of IFN-gamma by CD4+ naïve T cells.

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Jung, Tae-Young; Pham, Thanh Nhan Nguyen; Umeyama, Akemi; Shoji, Noboru; Hashimoto, Toshihiro; Lee, Je-Jung; Takei, Masao

2010-09-25

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-gamma), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy. 2010 Elsevier B.V. All rights reserved.

Use of calibration standards and the correction for sample self-attenuation in gamma-ray nondestructive assay

International Nuclear Information System (INIS)

Parker, J.L.

1984-08-01

The efficient use of appropriate calibration standards and the correction for the attenuation of the gamma rays within an assay sample by the sample itself are two important and closely related subjects in gamma-ray nondestructive assay. Much research relating to those subjects has been done in the Nuclear Safeguards Research and Development program at the Los Alamos National Laboratory since 1970. This report brings together most of the significant results of that research. Also discussed are the nature of appropriate calibration standards and the necessary conditions on the composition, size, and shape of the samples to allow accurate assays. Procedures for determining the correction for the sample self-attenuation are described at length including both general principles and several specific useful cases. The most useful concept is that knowing the linear attenuation coefficient of the sample (which can usually be determined) and the size and shape of the sample and its position relative to the detector permits the computation of the correction factor for the self-attenuation. A major objective of the report is to explain how the procedures for determining the self-attenuation correction factor can be applied so that calibration standards can be entirely appropriate without being particularly similar, either physically or chemically, to the items to be assayed. This permits minimization of the number of standards required to assay items with a wide range of size, shape, and chemical composition. 17 references, 18 figures, 2 tables

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

Science.gov (United States)

Sowmithra, K; Shetty, N J; Jha, S K; Chaubey, R C

2015-12-01

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Whole blood interferon-gamma assay for baseline tuberculosis screening among Japanese healthcare students.

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Katsuyuki Hotta

Full Text Available BACKGROUND: The whole blood interferon-gamma assay (QuantiFERON-TB-2G; QFT has not been fully evaluated as a baseline tuberculosis screening test in Japanese healthcare students commencing clinical contact. The aim of this study was to compare the results from the QFT with those from the tuberculin skin test (TST in a population deemed to be at a low risk for infection with Mycobacterium tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Healthcare students recruited at Okayama University received both the TST and the QFT to assess the level of agreement between these two tests. The interleukin-10 levels before and after exposure to M tuberculosis-specific antigens (early-secreted antigenic target 6-kDa protein [ESAT-6] and culture filtrate protein 10 [CFP-10] were also measured. Of the 536 healthcare students, most of whom had been vaccinated with bacillus-Calmette-Guérin (BCG, 207 (56% were enrolled in this study. The agreement between the QFT and the TST results was poor, with positive result rates of 1.4% vs. 27.5%, respectively. A multivariate analysis also revealed that the induration diameter of the TST was not affected by the interferon-gamma concentration after exposure to either of the antigens but was influenced by the number of BCG needle scars (p = 0.046. The whole blood interleukin-10 assay revealed that after antigen exposure, the median increases in interleukin-10 concentration was higher in the subgroup with the small increase in interferon-gamma concentration than in the subgroup with the large increase in interferon-gamma concentration (0.3 vs. 0 pg/mL; p = 0.004. CONCLUSIONS/SIGNIFICANCE: As a baseline screening test for low-risk Japanese healthcare students at their course entry, QFT yielded quite discordant results, compared with the TST, probably because of the low specificity of the TST results in the BCG-vaccinated population. We also found, for the first time, that the change in the interleukin-10 level after exposure to

HIV control through a single nucleotide on the HLA-B locus

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Kløverpris, Henrik N; Harndahl, Mikkel; Leslie, Alasdair J

2012-01-01

Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells, and str......Genetic variation within the HLA-B locus has the strongest impact on HIV disease progression of any polymorphisms within the human genome. However, identifying the exact mechanism involved is complicated by several factors. HLA-Bw4 alleles provide ligands for NK cells and for CD8 T cells......:02, which differ by only a single amino acid. Crucially, they occur primarily on identical HLA class I haplotypes and, as Bw6 alleles, do not act as NK cell ligands and are therefore largely unconfounded by other genetic factors. We show that in an outbred cohort (n = 2,093) of HIV C......-clade-infected individuals, a single amino acid change at position 9 of the HLA-B molecule critically affects peptide binding and significantly alters the cytotoxic T lymphocyte (CTL) epitopes targeted, measured directly ex vivo by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay (P = 2 × 10...

Increased Interleukin-4 production by CD8 and gammadelta T cells in health-care workers is associated with the subsequent development of active tuberculosis.

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Ordway, Diane J; Costa, Leonor; Martins, Marta; Silveira, Henrique; Amaral, Leonard; Arroz, Maria J; Ventura, Fernando A; Dockrell, Hazel M

2004-08-15

We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and 10 non-HCWs and correlated their immune status with the development of active tuberculosis (TB). Twenty individuals were randomly recruited, tested, and monitored longitudinally for TB presentation. Peripheral blood mononuclear cells (PBMCs) from donors were stimulated with M. tuberculosis and tested for cell proliferation and the production of interferon (IFN)- gamma, interleukin (IL)-5, and IL-4, by use of enzyme-linked immunosorbent or flow-cytometric assays. HCWs had higher levels of cell proliferation (24,258 cpm) and IFN- gamma (6373 pg/mL) to M. tuberculosis than did non-HCWs (cell proliferation, 11,462 cpm; IFN- gamma, 3228 pg/mL). Six of 10 HCWs showed increased median percentages of CD8+IL-4+ (4.7%) and gammadelta +IL-4+ (2.3%) T cells and progressed to active TB. HCWs who remained healthy showed increased median percentages of CD8+IFN- gamma+ (25.0%) and gammadelta +IFN- gamma+ (8.0%) and lower percentages of CD8+IL-4+ (0.05%) and gammadelta +IL-4+ (0.03%) T cells.

CXCR3 expression and activation of eosinophils

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Jinquan, T; Jing, C; Jacobi, H H

2000-01-01

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce...... in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact...

Evaluation of an automated assay system to measure soil radionuclides by L x-ray and gamma-ray spectrometry

International Nuclear Information System (INIS)

Nyhan, J.W.; Drennon, B.J.; Crowell, J.M.

1982-08-01

An automated radionuclide assay system for conducting soil radioassays using L x-ray and gamma-ray spectrometry was evaluated. Wet chemistry assay procedures were shown to be considerably more time consuming than similar analyses of soil on this radionuclide assay system. The detection limits of 241 Am and plutonium were determined, as well as the reproducibility of radionuclide assay results. The L x-ray spectrometric measurements were compared with radiochemical analyses on several tuff samples. The assay system's intrinsic germanium detector was found to respond linearly to varying low concentrations of 241 Am and plutonium, both of which were easily detected in the presence of elevated concentrations of 137 Cs

Limitations of Using IL-17A and IFN-γ-Induced Protein 10 to Detect Bovine Tuberculosis

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Xin, Ting; Gao, Xintao; Yang, Hongjun; Li, Pingjun; Liang, Qianqian; Hou, Shaohua; Sui, Xiukun; Guo, Xiaoyu; Yuan, Weifeng; Zhu, Hongfei; Ding, Jiabo; Jia, Hong

2018-01-01

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

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Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Interferon-gamma (IFN-γ-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

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Shahid Husain

Full Text Available We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC death using this model. RGC function was measured by pattern electroretinograms (ERGs in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03 and RGC numbers (37%, p = 0.0001 were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174 ± 27% vs. HLA-A2: 100%; p = 0.04. The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189 ± 11% vs. HLA-A2: 100%; p = 0.023. Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer, which was substantially reduced in IFN-γ ((-/- knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

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Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru

2013-01-01

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4 + IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4 + IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4 + IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4 + IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4 + LPLs and primed splenic CD4 + T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4 + IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

The IFN Response in Bats Displays Distinctive IFN-Stimulated Gene Expression Kinetics with Atypical RNASEL Induction.

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De La Cruz-Rivera, Pamela C; Kanchwala, Mohammed; Liang, Hanquan; Kumar, Ashwani; Wang, Lin-Fa; Xing, Chao; Schoggins, John W

2018-01-01

Bats host a large number of zoonotic viruses, including several viruses that are highly pathogenic to other mammals. The mechanisms underlying this rich viral diversity are unknown, but they may be linked to unique immunological features that allow bats to act as asymptomatic viral reservoirs. Vertebrates respond to viral infection by inducing IFNs, which trigger antiviral defenses through IFN-stimulated gene (ISG) expression. Although the IFN system of several bats is characterized at the genomic level, less is known about bat IFN-mediated transcriptional responses. In this article, we show that IFN signaling in bat cells from the black flying fox ( Pteropus alecto ) consists of conserved and unique ISG expression profiles. In IFN-stimulated cells, bat ISGs comprise two unique temporal subclusters with similar early induction kinetics but distinct late-phase declines. In contrast, human ISGs lack this decline phase and remained elevated for longer periods. Notably, in unstimulated cells, bat ISGs were expressed more highly than their human counterparts. We also found that the antiviral effector 2-5A-dependent endoribonuclease, which is not an ISG in humans, is highly IFN inducible in black flying fox cells and contributes to cell-intrinsic control of viral infection. These studies reveal distinctive innate immune features that may underlie a unique virus-host relationship in bats. Copyright © 2017 by The American Association of Immunologists, Inc.

The use of calibration standards and the correction for sample self-attenuation in gamma-ray nondestructive assay

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Parker, J.L.

1986-11-01

The efficient use of appropriate calibration standards and the correction for the attenuation of the gamma rays within an assay sample by the sample itself are two important and closely related subjects in gamma-ray nondestructive assay. Much research relating to those subjects has been done in the Nuclear Safeguards Research and Development program at the Los Alamos National Laboratory since 1970. This report brings together most of the significant results of that research. Also discussed are the nature of appropriate calibration standards and the necessary conditions on the composition, size, and shape of the samples to allow accurate assays. Procedures for determining the correction for the sample self-attenuation are described at length including both general principles and several specific useful cases. The most useful concept is that knowing the linear attenuation coefficient of the sample (which can usually be determined) and the size and shape of the sample and its position relative to the detector permits the computation of the correction factor for the self-attenuation. A major objective of the report is to explain how the procedures for determining the self-attenuation correction factor can be applied so that calibration standards can be entirely appropriate without being particularly similar, either physically or chemically, to the items to be assayed. This permits minimization of the number of standards required to assay items with a wide range of size, shape, and chemical composition

In vivo IFN-γ secretion by NK cells in response to Salmonella typhimurium requires NLRC4 inflammasomes.

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Andreas Kupz

Full Text Available Natural killer (NK cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18, independently of Toll-like receptor (TLR-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line.

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Baker, Olga J; Camden, Jean M; Redman, Robert S; Jones, Jonathan E; Seye, Cheikh I; Erb, Laurie; Weisman, Gary A

2008-11-01

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Impaired virus control and severe CD8+ T-cell-mediated immunopathology in chimeric mice deficient in gamma interferon receptor expression on both parenchymal and hematopoietic cells

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Henrichsen, Pernille; Bartholdy, Christina; Christensen, Jan Pravsgaard

2005-01-01

be capable of responding to IFN-gamma, but expression of the relevant receptor on non-T cells could be experimentally controlled. Only when the IFN-gamma receptor is absent on both radioresistant parenchymal and bone marrow-derived cells will chimeric mice challenged with a highly invasive, noncytolytic...

Early Experience With CliniMACS Prodigy CCS (IFN-gamma) System in Selection of Virus-specific T Cells From Third-party Donors for Pediatric Patients With Severe Viral Infections After Hematopoietic Stem Cell Transplantation.

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Kállay, Krisztián; Kassa, Csaba; Réti, Marienn; Karászi, Éva; Sinkó, János; Goda, Vera; Stréhn, Anita; Csordás, Katalin; Horváth, Orsolya; Szederjesi, Attila; Tasnády, Szabolcs; Hardi, Apor; Kriván, Gergely

2018-04-01

Viral reactivation is a frequent complication of allogeneic hematopoietic stem cell transplantation especially in children. For refractory cases, rapid virus-specific T-cell therapy would be ideally implemented within a few days. Over the course of a year in our pediatric cohort of 43 allogeneic transplantation, 9 patients fulfilled criteria for virus-specific T-cell therapy. Viral infections were due to cytomegalovirus (CMV) in 3, Epstein-Barr virus (EBV) in 2, and adenovirus (AdV) in 1 case, whereas >1 virus was detected in 3 cases. Viral diseases necessitating a T-cell therapy were CMV pneumonitis and colitis, AdV enteritis and cystitis, and EBV-induced posttransplantation lymphoproliferative disease. Cells were produced by the CliniMACS Prodigy CCS (IFN-gamma) System within 24 hours after mononuclear leukapheresis. Eight patients became completely asymptomatic, whereas 7 also cleared the virus. Six patients are alive without viral illness or sequelae demonstrating viral DNA clearance in peripheral blood with a median follow-up of 535 (350-786) days. One patient with CMV pneumonitis died of respiratory insufficiency. In 2 cases the viral illness improved or cleared, however, the patients died of invasive aspergillosis. No cases of graft-versus-host disease, rejection, organ toxicity, or recurrent infection were noticed. Virus-specific T-cell therapy implemented by the CliniMACS Prodigy CCS (IFN-gamma) System is an automated, fast, safe, and probably effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation.

Impaired chromatin remodelling at STAT1-regulated promoters leads to global unresponsiveness of Toxoplasma gondii-infected macrophages to IFN-γ.

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Christine Lang

2012-01-01

Full Text Available Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ stimulated with interferon (IFN-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma

Efficiency and attenuation correction factors determination in gamma spectrometric assay of bulk samples using self radiation

International Nuclear Information System (INIS)

Haddad, Kh.

2009-02-01

Gamma spectrometry forms the most important and capable tool for measuring radioactive materials. Determination of the efficiency and attenuation correction factors is the most tedious problem in the gamma spectrometric assay of bulk samples. A new experimental and easy method for these correction factors determination using self radiation was proposed in this work. An experimental study of the correlation between self attenuation correction factor and sample thickness and its practical application was also introduced. The work was performed on NORM and uranyl nitrate bulk sample. The results of proposed methods agreed with those of traditional ones.(author)

Identifying Predictors of Interferon-γ Release Assay Results in Pediatric Latent Tuberculosis: A Protective Role of Bacillus Calmette-Guérin?

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Sotgiu, Giovanni; Altet-Gómez, Neus; Tsolia, Maria; Ruga, Ezia; Velizarova, Svetlana; Kampmann, Beate

2012-01-01

Rationale: Interferon-γ (IFN-γ) release assays are widely used to diagnose latent infection with Mycobacterium tuberculosis in adults, but their performance in children remains incompletely evaluated to date. Objectives: To investigate factors influencing results of IFN-γ release assays in children using a large European data set. Methods: The Pediatric Tuberculosis Network European Trials group pooled and analyzed data from five sites across Europe comprising 1,128 children who were all investigated for latent tuberculosis infection by tuberculin skin test and at least one IFN-γ release assay. Multivariate analyses examined age, bacillus Calmette-Guérin (BCG) vaccination status, and sex as predictor variables of results. Subgroup analyses included children who were household contacts. Measurements and Main Results: A total of 1,093 children had a QuantiFERON-TB Gold In-Tube assay and 382 had a T-SPOT.TB IFN-γ release assay. Age was positively correlated with a positive blood result (QuantiFERON-TB Gold In-Tube: odds ratio [OR], 1.08 per year increasing age [P 5 yr). Conclusions: Our data show that BCG vaccination may be effective in protecting children against Mycobacterium tuberculosis infection. To restrict use of IFN-γ release assays to children with positive skin tests risks underestimating latent infection. PMID:22700862

Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells.

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JoĂŤl Pestel

2008-06-01

Full Text Available Mycobacterium bovis bacillus Calmette-GuĂŠrin (BCG is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells.

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Kowalewicz-Kulbat, Magdalena; Kaźmierczak, Dominik; Donevski, Stefan; Biet, Franck; Pestel, Joël; Rudnicka, Wiesława

2008-01-01

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs) are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

IFN-gamma Impairs Release of IL-8 by IL-1beta-stimulated A549 Lung Carcinoma Cells

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Pfeilschifter Josef

2008-09-01

Full Text Available Abstract Background Production of interferon (IFN-γ is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNγ on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells. Methods A549 cells were cultured and stimulated with interleukin (IL-1β alone or in combination with IFNγ. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA. mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA, respectively. Expression of inhibitor-κ Bα, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis. Results Here we demonstrate that IFNγ efficiently reduced IL-8 secretion under the influence of IL-1β. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNγ on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNγ on IL-1β-induced NF-κB activation as assessed by cellular IκB levels. Moreover, analysis of intracellular IL-8 suggests that IFNγ modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1β-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNγ indicating that modulation of IL-1β action by this cytokine displays specificity. Conclusion Data presented herein agree with an angiostatic role of IFNγ as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1-like functions in lung cancer patients e.g. by local delivery of IFNγ may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

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Coppen, S R; Newsam, R; Bull, A T; Baines, A J

1995-04-20

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

Recombinant gamma interferon for the treatment of pulmonary and mycobacterial diseases

International Nuclear Information System (INIS)

Garcia, Idrian; Milanes, Maria T; Cayon, Isis; Santos, Yamilet et. al

2009-01-01

An increased antibiotic resistance is described for Mycobacterium tuberculosis and atypical mycobacterial species; therefore, new treatments are required. Immunocompromised patients have increased risk, as demonstrated by complications after BCG vaccination. On the other hand, idiopathic pulmonary fibrosis is a fatal disease, with no therapy available to modify course of the disease. Gamma interferon (IFN-γ) plays an essential role as main activator of cytokine secretion in macrophages, also showing a potent anti-fibrotic effects. To evaluate the adjuvant effect of IFN-γ on these three clinical scenarios, five clinical trials were carried out. Patients treated with IFN gamma had satisfactory response according to clinical, imaging and functional criteria since their first evaluations, significantly improving when compared to the control group receiving placebo in a study of pulmonary atypical mycobacteriosis. Fast sputum conversion was obtained in mycobacterial infections, including tuberculosis. In the idiopathic pulmonary fibrosis study, 75% of treated patients were considered as responders (improvement + stable). Here we report the cases of two nursing babies with suppurative regional lymphadenitis caused by BCG, who were successfully treated with recombinant human IFN-γ. Treatment was well tolerated, with most of the adverse reactions corresponding to classical flu-like symptoms produced by the cytokine. We can conclude that IFN-γ is useful and well tolerated as adjuvant therapy in patients with pulmonary mycobacterial diseases or idiopathic pulmonary fibrosis. (author)

IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as biomarkers for infection with M. tuberculosis in a whole blood based T-cell assay

DEFF Research Database (Denmark)

Ruhwald, Morten; Bjerregaard-Andersen, Morten; Rabna, Paulo

2009-01-01

and mitogen in the Quantiferon In Tube test tubes. Levels of biomarkers were measured using Luminex and ELISA (IFN-gamma). RESULTS: We found all five new biomarkers were expressed in significantly higher concentrations compared to IFN-gamma. IP-10 and MCP-3 levels in the un-stimulated samples were higher...

Gamma-ray imaging and holdup assays of 235-F PuFF cells 1 & 2

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Aucott, T. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

2017-12-20

Savannah River National Laboratory (SRNL) Nuclear Measurements (L4120) was tasked with performing enhanced characterization of the holdup in the PuFF shielded cells. Assays were performed in accordance with L16.1-ADS-2460 using two high-resolution gamma-ray detectors. The first detector, an In Situ Object Counting System (ISOCS)-characterized detector, was used in conjunction with the ISOCS Geometry Composer software to quantify grams of holdup. The second detector, a Germanium Gamma-ray Imager (GeGI), was used to visualize the location and relative intensity of the holdup in the cells. Carts and collimators were specially designed to perform optimum assays of the cells. Thick, pencil-beam tungsten collimators were fabricated to allow for extremely precise targeting of items of interest inside the cells. Carts were designed with a wide range of motion to position and align the detectors. A total of 24 measurements were made, each typically 24 hours or longer to provide sufficient statistical precision. This report presents the results of the enhanced characterization for cells 1 and 2. The measured gram values agree very well with results from the 2014 study. In addition, images were created using both the 2014 data and the new GeGI data. The GeGI images of the cells walls reveal significant Pu-238 holdup on the surface of the walls in cells 1 and 2. Additionally, holdup is visible in the two pass-throughs from cell 1 to the wing cabinets. This report documents the final element (exterior measurements coupled with gamma-ray imaging and modeling) of the enhanced characterization of cells 1-5 (East Cell Line).

IFN-alpha antibodies in patients with age-related macular degeneration treated with recombinant human IFN-alpha2a

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Ross, Christian; Engler, Claus Bødker; Sander, Birgit

2002-01-01

We tested for development of binding and neutralizing antibodies to interferon-alpha (IFN-alpha) during IFN-alpha2a therapy of patients with age-related macular degeneration (AMD) of the eyes. Antibodies were investigated retrospectively in sera of 34 patients treated with 3 x 10(6) IU IFN-alpha2...

IFN-alpha antibodies in patients with age-related macular degeneration treated with recombinant human IFN-alpha2a

DEFF Research Database (Denmark)

Ross, Christian; Engler, Claus Bødker; Sander, Birgit

2002-01-01

We tested for development of binding and neutralizing antibodies to interferon-alpha (IFN-alpha) during IFN-alpha2a therapy of patients with age-related macular degeneration (AMD) of the eyes. Antibodies were investigated retrospectively in sera of 34 patients treated with 3 x 10(6) IU IFN-alpha2a...

Leishmania-specific T cells expressing interferon-¿(IFN-¿) and IL-10 upon activation are expanded in individuals cured of visceral leishmaniasis

DEFF Research Database (Denmark)

Kemp, K; Kemp, M; Kharazmi, A

1999-01-01

Peripheral blood mononuclear cells (PBMC) from patients who have recovered from visceral leishmaniasis often respond to Leishmania antigens in vitro by production of both IL-4, IFN-gamma and IL-10. In order to establish the cellular sources of these cytokines, we activated cells from individuals...... with a history of visceral leishmaniasis with Leishmania antigen for 6 days in culture, and identified cytokine production at the single-cell level by flow cytometry. The cytokines were only found in CD3+ cells and among these mainly within the CD4+ subset. The percentage of cytokine-producing cells was compared...... in Leishmania-activated PBMC cultures from the previous patients and from individuals living in a village where leishmaniasis does not occur. The percentage of IL-10- and IFN-gamma-containing cells was significantly higher in the previous patients than in the controls, indicating that Leishmania-specific T...

Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

International Nuclear Information System (INIS)

Rojas, Olga Lucia; Gonzalez, Ana Maria; Gonzalez, Rosabel; Perez-Schael, Irene; Greenberg, Harry B.; Franco, Manuel A.; Angel, Juana

2003-01-01

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4 + and CD8 + T cells secreting INFγ, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFγ-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4 + and CD8 + T cell subsets, IFNγ-secreting RV-specific CD8 + but not CD4 + T cells were detected in recently infected children. Using the same approach, both CD4 + and CD8 + RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFγ-secreting CD4 + T cells from adult volunteers preferentially express the intestinal homing receptor α4β7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFγ-secreting CD4 + T cells preferentially express L-selectin but not α4β7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFγ is independent of the expression of these homing receptors

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

DEFF Research Database (Denmark)

Ingenhoven, Kathleen; Kramer, Daniel; Jensen, Poul Erik Hyldgaard

2017-01-01

to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-β in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-β-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-β with regards to its......OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-β) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization...... to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-β and biotinylated IFN-β, which are then detected using HRP labeled...

Association of the IFN-γ (+874A/T) Genetic Polymorphism with Paranoid Schizophrenia in Tunisian Population.

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Jemli, Achraf; Eshili, Awatef; Trifa, Fatma; Mechri, Anouar; Zaafrane, Ferid; Gaha, Lotfi; Juckel, George; Tensaout, Besma Bel Hadj Jrad

2017-02-01

Since growing evidence suggests a significant role of chronic low-grade inflammation in the physiopathology of schizophrenia, we have hypothesized that functional genetic variant of the IFN gamma (IFN-γ; +874A/T; rs2430561) gene may be involved in the predisposition to schizophrenia. This research is based on a case-control study which aims to identify whether polymorphism of the IFN-γ gene is a risk factor for the development of schizophrenia. The RFLP-PCR genotyping of the IFN-γ gene was conducted on a Tunisian population composed of 218 patients and 162 controls. The IFN-γ (+874A/T) polymorphism analysis showed higher frequencies of minor homozygous genotype (TT) and allele (T) in all patients compared with controls (11.5 vs. 4.9%; p = 0.03, OR = 2.64 and 30.7 vs. 24.1%, p = 0.04, OR = 1.4, respectively). This correlation was confirmed for male but not for female patients. Also, the T allele was significantly more common among patients with paranoid schizophrenia when compared with controls (25.8 vs. 4.9%, p = 0.0001; OR = 6.7). Using the binary regression analysis to eliminate confounding factors as age and sex, only this last association remained significant (p = 0.03; OR = 1.76, CI = 1.05-2.93). In conclusion, our results showed a significant association between +874A/T polymorphism of IFN-γ and paranoid schizophrenia, suggesting that this single nucleotide polymorphism (SNP) or another at proximity could predispose to paranoid schizophrenia. Since the minor allele of this polymorphism was correlated with an increased expression of their product, our study validates the hypothesis of excessive pro-inflammatory cytokine in the physiopathology of paranoid schizophrenia.

IFN-γ and TNF-α producing CD4+ T-cells in the blood after Mycoplasma hyosynoviae challenge of vaccinated pigs

DEFF Research Database (Denmark)

Riber, Ulla; Hansen, Mette Sif; Lauritsen, Klara Tølbøll

In a vaccine trial against Mycoplasma hyosynoviae infection, pigs were vaccinated with formalin fixed whole-cell-antigen formulated with adjuvant DDA/TDB (SSI). Placebo pigs received adjuvant with saline. Vaccinations were performed at five and eight weeks of age, followed by an intranasal M....... hyosynoviae challenge inoculation three weeks later. Vaccination induced both antibodies and a cell-mediated immune response (CMI) in vaccinated pigs compared to placebo pigs as shown by M. hyosynoviae antigen (Ag) specific IFN-γ response in an IL-18 potentiated whole-blood IFN-γ stimulation assay (mean IFN......-γ level 1936 pg/ml vs. 82 pg/ml (p=0.0001)). A central memory T cell phenotype with polyfunctional capacity to produce all three cytokines IFN-γ, TNF-α and IL-2 has recently been linked to development of vaccine induced protection in several infections. In a subset of seven vaccinated pigs and four...

Immune responses to mumps vaccine in adults who were vaccinated in childhood.

Science.gov (United States)

Hanna-Wakim, Rima; Yasukawa, Linda L; Sung, Phillip; Arvin, Ann M; Gans, Hayley A

2008-06-15

In a mumps outbreak in the United States, many infected individuals were adults who had received 2 doses of mumps vaccine. The persistence of cellular immunity to mumps vaccine has not been defined. This was an observational, nonrandomized cohort study evaluating cell-mediated and humoral immunity to mumps in 10 vaccinated and 10 naturally immune adults. Mumps-specific T cell activation and interferon (IFN)-gamma production were measured using lymphoproliferative and flow cytometry assays, and mumps immunoglobulin (Ig) G was measured using enzyme-linked immunosorbent assay. T cell immunity to mumps was high in both groups; 70% of vaccinated and 80% of naturally immune individuals had a positive (> or =3) stimulation index (SI) (P = 1.0). The mean percentages of mumps-specific CD4+ T cells that expressed CD69 and produced IFN-gamma were equivalent in the 2 groups: 0.06% and 0.12%, respectively (P = .11). The mean SIs in the groups were also equivalent, although IFN-gamma concentrations from cultures stimulated with mumps antigen were higher in naturally immune adults than in vaccinated adults (P < or = .01). All adults were positive for mumps IgG. T and B cell immunity to mumps was detected in adults at least 10 years after immunization. Except for IFN-gamma release, responses in vaccinated adults paralleled those observed in naturally immune individuals.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

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Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

2013-06-14

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

Induced expression of mRNA for IL-5, IL-6, TNF-alpha, MIP-2 and IFN-gamma in immunologically activated rat peritoneal mast cells: inhibition by dexamethasone and cyclosporin A.

Science.gov (United States)

Williams, C M; Coleman, J W

1995-10-01

We examined the capacity of purified rat peritoneal connective tissue-type mast cells (PMC) to express mRNA for several cytokines. Stimulation of PMC with anti-IgE for 4 hr induced the expression of mRNA encoding interleukin-5 (IL-5), IL-6, tumour necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Unstimulated PMC expressed detectable mRNA for TNF-alpha but not for the other four cytokines. Incubation of PMC with cyclosporin A (CsA) or dexamethasone (DEX), each at 10(-6) M for 24 hr, significantly inhibited the induced expression of mRNA for each of the five cytokines, and also inhibited release of biologically active TNF-alpha. Throughout these experiments mRNA levels of the housekeeping gene G3PDH were not altered by stimulation with anti-IgE or incubation with CsA or DEX. We conclude that immunological activation of rat PMC induces gene expression of several cytokines and that expression of these genes can be inhibited by immunosuppressive drugs.

Effect of vaccine dose on the safety and immunogenicity of a candidate TB vaccine, MVA85A, in BCG vaccinated UK adults.

Science.gov (United States)

Pathan, Ansar A; Minassian, Angela M; Sander, Clare R; Rowland, Rosalind; Porter, David W; Poulton, Ian D; Hill, Adrian V S; Fletcher, Helen A; McShane, Helen

2012-08-17

A non-randomised, open-label, Phase I safety and immunogenicity dose-finding study to assess the safety and immunogenicity of the candidate TB vaccine Modified Vaccinia virus Ankara expressing Antigen 85A (MVA85A) from Mycobacterium tuberculosis (MTB) in healthy adult volunteers previously vaccinated with BCG. Healthy BCG-vaccinated volunteers were vaccinated with either 1×10(7) or 1×10(8)PFU of MVA85A. All adverse events were documented and antigen specific T cell responses were measured using an ex vivo IFN-γ ELISPOT assay. Safety and immunogenicity were compared between the 2 dose groups and with a previous trial in which a dose of 5×10(7)PFU MVA85A had been administered. There were no serious adverse events recorded following administration of either 1×10(7) or 1×10(8)PFU of MVA85A. Systemic adverse events were more frequently reported following administration of 1×10(8)PFU of MVA85A when compared to either 5×10(7) or 1×10(7)PFU of MVA85A but were mild or moderate in severity and resolved completely within 7 days of immunisation. Antigen specific T cell responses as measured by the IFN-γ ELISPOT were significantly higher following immunisation in adults receiving 1×10(8)PFU compared to the 5×10(7) and 1×10(7) doses. Additionally, a broader range of Ag85A epitopes are detected following 1×10(8)PFU of MVA85A. A higher dose of 1×10(8)PFU of MVA85A is well-tolerated, increases the frequency of IFN-γ secreting T cells detected following immunisation and broadens the range of Ag85A epitopes detected. Copyright © 2012 Elsevier Ltd. All rights reserved.

Melanoma NOS1 expression promotes dysfunctional IFN signaling.

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Liu, Qiuzhen; Tomei, Sara; Ascierto, Maria Libera; De Giorgi, Valeria; Bedognetti, Davide; Dai, Cuilian; Uccellini, Lorenzo; Spivey, Tara; Pos, Zoltan; Thomas, Jaime; Reinboth, Jennifer; Murtas, Daniela; Zhang, Qianbing; Chouchane, Lotfi; Weiss, Geoffrey R; Slingluff, Craig L; Lee, Peter P; Rosenberg, Steven A; Alter, Harvey; Yao, Kaitai; Wang, Ena; Marincola, Francesco M

2014-05-01

In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells.

Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

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Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

2010-10-15

The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

Cross-talk between IGF-1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells due to interferon-gamma exposure

Science.gov (United States)

Park, Sookyoung; Nozaki, Kenkichi; Smith, Joshua A.; Krause, James S.; Banik, Naren L.

2014-01-01

Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. In order to examine whether IGF-1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon-gamma (IFN-γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl-2 ratios, and expression of apoptosis related proteases (caspase-3 and −12) in motoneurons rendered by IFN-γ in a dose-dependent manner. Post-treatment with IGF-1 attenuated these changes. In addition, IGF-1 treatment of motoneurons exposed to IFN-γ decreased expression of inflammatory markers (cyclooxygenase-2 and nuclear factor-kappa B:inhibitor of kappa B ratio). Furthermore, IGF-1 attenuated the loss of expression of IGF-1 receptors (IGF-1Rα and IGF-1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN-γ. To determine whether the protective effects of IGF-1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ-silenced VSC4.1 motoneurons following IFN-γ and IGF-1 exposure. These results suggest that IGF-1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ. PMID:24188094

Cell Death of Gamma Interferon-Stimulated Human Fibroblasts upon Toxoplasma gondii Infection Induces Early Parasite Egress and Limits Parasite Replication

NARCIS (Netherlands)

Niedelman, Wendy; Sprokholt, Joris K.; Clough, Barbara; Frickel, Eva-Maria; Saeij, Jeroen P. J.

2013-01-01

The intracellular protozoan parasite Toxoplasma gondii is a major food-borne illness and opportunistic infection for the immunosuppressed. Resistance to Toxoplasma is dependent on gamma interferon (IFN-γ) activation of both hematopoietic and nonhematopoietic cells. Although IFN-γ-induced innate

Cell death of gamma interferon-stimulated human fibroblasts upon toxoplasma gondii infection induces early parasite egress and limits parasite replication

NARCIS (Netherlands)

Niedelman, W.; Sprokholt, J.K.; Clough, B.; Frickel, E.; Saeij, J.P.J.

2013-01-01

The intracellular protozoan parasite Toxoplasma gondii is a major food-borne illness and opportunistic infection for the immunosuppressed. Resistance to Toxoplasma is dependent on gamma interferon (IFN-¿) activation of both hematopoietic and nonhematopoietic cells. Although IFN-¿-induced innate

Analysis of copper-nickel ores by gamma-gamma method in ore enrichment works

International Nuclear Information System (INIS)

Bol'shakov, A.Yu.; Tovstenko, Yu.G.; Chinskij, E.B.; Eliseev, G.I.

1973-01-01

The paper presents experimental data on continuous gamma-gamma assay of copper-nickel ores on conveyor belts and of dry discrete samples of classifier overflow at the enrichment plants of the Pechenganikel' group. The relative errors are given of the results of comparison of two-hour rapid analyses and shift and 24-hour chemical analyses of classifier overflow samples with the figures for gamma-gamma assay. The factors affecting the accuracy of the latter technique are elucidated. Practical recommendations are given on the use of this technique at the above enrichment plants. (author)

Adjuvant interferon gamma in patients with drug – resistant pulmonary tuberculosis: a pilot study

Directory of Open Access Journals (Sweden)

Carbonell Dalia

2004-10-01

Full Text Available Abstract Background Tuberculosis (TB is increasing in the world and drug-resistant (DR disease beckons new treatments. Methods To evaluate the action of interferon (IFN gamma as immunoadjuvant to chemotherapy on pulmonary DR-TB patients, a pilot, open label clinical trial was carried out in the Cuban reference ward for the management of this disease. The eight subjects existing in the country at the moment received, as in-patients, 1 × 106 IU of recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to the indicated chemotherapy, according to their antibiograms and WHO guidelines. Sputum samples collection for direct smear observation and culture as well as routine clinical and thorax radiography assessments were done monthly. Results Sputum smears and cultures became negative for acid-fast-bacilli before three months of treatment in all patients. Lesion size was reduced at the end of 6 months treatment; the lesions disappeared in one case. Clinical improvement was also evident; body mass index increased in general. Interferon gamma was well tolerated. Few adverse events were registered, mostly mild; fever and arthralgias prevailed. Conclusions These data suggest that IFN gamma is useful and well tolerated as adjunctive therapy in patients with DR-TB. Further controlled clinical trials are encouraged.

Recombinant IFN-γ from the bank vole Myodes glareolus: a novel tool for research on rodent reservoirs of zoonotic pathogens.

Science.gov (United States)

Torelli, Francesca; Zander, Steffen; Ellerbrok, Heinz; Kochs, Georg; Ulrich, Rainer G; Klotz, Christian; Seeber, Frank

2018-02-12

Rodent species like Myodes glareolus and Microtus spp. are natural reservoirs for many zoonotic pathogens causing human diseases and are gaining increasing interest in the field of eco-immunology as candidate animal models. Despite their importance the lack of immunological reagents has hampered research in these animal species. Here we report the recombinant production and functional characterization of IFN-γ, a central mediator of host's innate and adaptive immune responses, from the bank vole M. glareolus. Soluble dimeric recMgIFN-γ was purified in high yield from Escherichia coli. Its activity on M. glareolus and Microtus arvalis kidney cell lines was assessed by immunofluorescent detection of nuclear translocation and phosphorylation of the transcription factor STAT1. RecMgIFN-γ also induced expression of an IFN-γ-regulated innate immunity gene. Inhibition of vesicular stomatitis virus replication in vole cells upon recMgIFN-γ treatment provided further evidence of its biological activity. Finally, we established a recMgIFN-γ-responsive bank vole reporter cell line that allows the sensitive titration of the cytokine activity via a bioluminescence reporter assay. Taken together, we report valuable tools for future investigations on the immune response against zoonotic pathogens in their natural animal hosts, which might foster the development of novel animal models.

Interferon gamma peptidomimetic targeted to hepatic stellate cells ameliorates acute and chronic liver fibrosis in vivo

NARCIS (Netherlands)

Bansal, Ruchi; Prakash, Jai; De Ruiter, Marieke; Poelstra, Klaas

2014-01-01

Hepatic stellate cells play a crucial role in the pathogenesis of hepatic fibrosis. Thus, pharmacological inhibition of pro-fibrotic activities of these cells might lead to an effective therapy for this disease. Among the potent antifibrotics, interferon gamma (IFN gamma), a proinflammatory

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

Directory of Open Access Journals (Sweden)

Kalyan C Nallaparaju

2011-03-01

Full Text Available Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918 of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida FTN_0444 (homolog of FTT0918 fopC mutant to study the virulence-associated mechanism(s of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO mice, including MHC I, MHC II, and µmT (B cell deficient, but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

Executive Summary of the Guidelines for the Use of interferon-gamma Release Assays in the Diagnosis of Tuberculosis Infection.

Science.gov (United States)

Santin, Miguel; García-García, José-María; Rigau, David; Altet, Neus; Anibarro, Luis; Casas, Irma; Díez, Nuria; García-Gasalla, Mercedes; Martínez-Lacasa, Xavier; Penas, Antón; Pérez-Escolano, Elvira; Sánchez, Francisca; Domínguez, José

2016-09-01

Interferon-gamma release assays are widely used for the diagnosis of tuberculosis infection in Spain. However, there is no consensus on their application in specific clinical scenarios. To develop a guide-line for their use, a panel of experts comprising specialists in infectious diseases, respiratory diseases, microbiology, pediatrics and preventive medicine, together with a methodologist, conducted a systematic literature search, summarized the findings, rated the quality of the evidence, and formulated recommendations following the Grading of Recommendations of Assessment Development and Evaluations methodology. This document provides evidence-based guidance on the use of interferon-gamma release assays for the diagnosis of tuberculosis infection in patients at risk of tuberculosis or suspected of having active disease. The guidelines will be applicable to specialist and primary care, and public health. Copyright © 2016 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.

Early IFN-gamma production after YF 17D vaccine virus immunization in mice and its association with adaptive immune responses.

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Patrícia C C Neves

Full Text Available Yellow Fever vaccine is one of the most efficacious human vaccines ever made. The vaccine (YF 17D virus induces polyvalent immune responses, with a mixed TH1/TH2 CD4(+ cell profile, which results in robust T CD8(+ responses and high titers of neutralizing antibody. In recent years, it has been suggested that early events after yellow fever vaccination are crucial to the development of adequate acquired immunity. We have previously shown that primary immunization of humans and monkeys with YF 17D virus vaccine resulted in the early synthesis of IFN-γ. Herein we have demonstrated, for the first time that early IFN-γ production after yellow fever vaccination is a feature also of murine infection and is much more pronounced in the C57BL/6 strain compared to the BALB/c strain. Likewise, in C57BL/6 strain, we have observed the highest CD8(+ T cells responses as well as higher titers of neutralizing antibodies and total anti-YF IgG. Regardless of this intense IFN-γ response in mice, it was not possible to see higher titers of IgG2a in relation to IgG1 in both mice lineages. However, IgG2a titers were positively correlated to neutralizing antibodies levels, pointing to an important role of IFN-γ in eliciting high quality responses against YF 17D, therefore influencing the immunogenicity of this vaccine.

Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

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Neelkamal Chaudhary

2010-02-01

Full Text Available Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H1-T(H17 and destructive allergic (T(H2 immunity. How Aspergillus allergens (Asp f proteins participate in the development of allergic sensitization is unknown.To determine whether Asp f proteins are strictly associated with T(H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17 to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H1-responses to Asp f3 (a putative peroxismal membrane protein, Asp f9/16 (cell wall glucanase, Asp f11 (cyclophilin type peptidyl-prolyl isomerase and Asp f22 (enolase. Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

[The clinical application of quantiferon TB-2G: its usefulness and limitations].

Science.gov (United States)

Sato, Shigeki; Nagai, Hideaki

2011-02-01

compromised hosts is essential for TB control, but T cell assay might be influenced by the degree of cell-mediated immunosuppression. The relationship between immunocompetence and specific interferon (IFN)-gamma response in whole blood QuantiFERON-TB Gold (QFT) is uncertain. Immune-related clinical indicators associated with the degree of antigen-specific IFN-gamma production were analysed using a large immunologically-unselected population with obvious TB infection. The absolute number of blood lymphocyte in TB patients was significantly associated with specific IFN-gamma production in a linear regression model. Sensitivity of 2 IFN-gamma Release Assays, QFT and ELISPOT, partly depends on peripheral lymphocyte counts. At low lymphocyte count conditions, ELISPOT assay is superior to whole blood QFT for detecting tuberculosis infection. (4) QuantiFERON TB-2G among staffs in the hospitals of Nationao Hospital Organization: Susumu OGURI, Chihiro NISHIO, Kensuke SUMI, Masayoshi MINAGUCHI, Tomomasa TSUBOI, Atuo SATOU, Osamu TOKUNAGA, Takeshi MIYAMOMAE, Takuya KURASAWA (National Hospital Organization Minami-Kyoto National Hospital) To investigate the infection rate of tuberculosis among staffs working in the hospitals of NHO. Questionnaires were sent to the hospitals and the responses were analyzed. Among the staffs working in the hospitals with tuberculosis wards, positive rate of QuantiFERON TB-2G was 6.9%, probable positive rate was 5.6%. On the other hand, among the staffs working in the hospitals without tuberculosis wards, positive rate was 4.4%, probable positive rate was 3.9%. It is necessary to monitor the infection rate among hospital staffs.

IL-18 potentiated whole blood IFN-γ assay can identify cell-mediated immune responses towards Lawsonia intracellularis in experimentally infected pigs

DEFF Research Database (Denmark)

Riber, Ulla; Jakobsen, Jeanne Toft; Hvass, Henriette Cordes

Lawsonia intracellularis is an obligate intracellular bacteria causing proliferative enteropathy (PE) in pigs. The infection causes diarrhoea, retarded growth and sudden death in pigs and is one of the most economically important diseases in the swine industry worldwide. The infection is one...... indications that cell-mediated immune responses (CMI) are important for the protection against infections with L. intracellularis and in mice models IFN-γ has been shown to play a key role in the host defence against experimental infections . In L. intracellularis infected pigs, IFN-γ is only sparsely...

Duck RIG-I CARD Domain Induces the Chicken IFN-β by Activating NF-κB

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Yang Chen

2015-01-01

Full Text Available Retinoic acid-inducible gene I- (RIG-I- like receptors (RLRs have recently been identified as cytoplasmic sensors for viral RNA. RIG-I, a member of RLRs family, plays an important role in innate immunity. Although previous investigations have proved that RIG-I is absent in chickens, it remains largely unknown whether the chicken can respond to RIG-I ligand. In this study, the eukaryotic expression vectors encoding duRIG-I full length (duck RIG-I, containing all domains, duRIG-I N-terminal (containing the two caspase activation and recruitment domain, CARDs, and duRIG-I C-terminal (containing helicase and regulatory domains labeled with 6*His tags were constructed successfully and detected by western blotting. Luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA detected the duRIG-I significantly activated NF-κB and induced the expression of IFN-β when polyinosinic-polycytidylic acid (poly[I:C], synthetic double-stranded RNA challenges chicken embryonic fibroblasts cells (DF1 cells, while the duRIG-I was inactive in the absence of poly[I:C]. Further analysis revealed that the CARDs (duRIG-I-N induced IFN-β production regardless of the presence of poly[I:C], while the CARD-lacking duRIG-I (duRIG-I-C was not capable of activating downstream signals. These results indicate that duRIG-I CARD domain plays an important role in the induction of IFN-β and provide a basis for further studying the function of RIG-I in avian innate immunity.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts.

Science.gov (United States)

Teixeira-Salum, Tatiana Beber; Rodrigues, Denise Bertulucci Rocha; Gervásio, Aurélia M; Souza, Cássio J A; Rodrigues, Virmondes; Loyola, Adriano Motta

2010-03-01

Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.

Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in Idiopathic Pulmonary Fibrosis

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Welsh Kenneth I

2003-06-01

Full Text Available Abstract Background Genes encoding cytokine mediators are prime candidates for genetic analysis in conditions with T-helper (Th cell disease driven imbalance. Idiopathic Pulmonary Fibrosis (IPF is a predominantly Th2 mediated disease associated with a paucity of interferon-gamma (IFN-γ. The paucity of IFN-γ may favor the development of progressive fibrosis in IPF. Interleukin-12 (IL-12 plays a key role in inducing IFN-γ production. The aim of the current study was to assess whether the 1188 (A/C 3'UTR single nucleotide polymorphism (SNP in the IL-12 p40 subunit gene which was recently found to be functional and the 5644 (G/A 3' UTR SNP of the IFN-γ gene were associated with susceptibility to IPF. Methods We investigated the allelic distribution in these loci in UK white Caucasoid subjects comprising 73 patients with IPF and 157 healthy controls. The SNPs were determined using the polymerase chain reaction in association with sequence-specific primers incorporating mismatches at the 3'-end. Results Our results showed that these polymorphisms were distributed similarly in the IPF and control groups Conclusion We conclude that these two potentially important candidate gene single nucleotide polymorphisms are not associated with susceptibility to IPF.

Cytokines affecting CD4+T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-γ) promotes survival of alloantigen-specific CD4+T regulatory cells.

Science.gov (United States)

Nomura, Masaru; Hodgkinson, Suzanne J; Tran, Giang T; Verma, Nirupama D; Robinson, Catherine; Plain, Karren M; Boyd, Rochelle; Hall, Bruce M

2017-06-01

CD4 + T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4 + T cells. Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4 + T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4 + T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4 + T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4 + T cells. Tolerant CD4 + CD25 + T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4 + CD25 + T cells to third-party Lewis. Tolerant CD4 + CD25 + T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4 + CD25 + T cells. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Verlauf der zellulären Immunantwort bei Lebendnierenempfängern - Messung von IFN-γ und IL-17 im Elispot-Assay

OpenAIRE

Grehn, Conrad

2015-01-01

Die Nierentransplantation ermöglicht Patienten die Wiederherstellung der Nierenfunktion. Aufgrund der begrenzten Verfügbarkeit an Organen nimmt dabei die Zahl der Transplantationen von einem lebenden Spender stetig zu. Zudem ermöglichen die präzisen und genauen Vorbereitungen und Abläufe bei Lebendnierenspenden eine bessere 5-Jahres-Überlebensrate als bei Kadaverspenden. Die genetische Verschiedenheit zwischen Spender und Empfänger bedingt jedoch eine lebenslange immunsuppressive Therapie, um...

Induction and assay of pure soyabean mutants obtained from gamma irradiation

International Nuclear Information System (INIS)

Nasseri Tafti, M.; Rezazadeh, M.; Yousefi, F.; Sabzi, H.

2002-01-01

Gamma ray is an electromagnetic type of radiation and produces ions when passing through biological matter. It can be applied in plant breeding to induce variation. The most important character of this ray is to produce changes in DNA structure existing in cell. Mutants induced by irradiation of soybean seeds were assayed for their agronomic traits. Two locations were used for this purpose, Alishtar and Karaj. There were significant differences between soybean mutant lines and their check cv. Williams at 1% level and cv.Clark at 5% level. Line No. 47 with 4782 kg/hect. Possessed the top of the list and next to it line No.38 with 4722 kg/hect. Some mutant lines reached maturity 10 to 12 days earlier than commercial cv s used as check cultivars

Assessment of gamma ray-induced DNA damage in Lasioderma serricorne using the comet assay

International Nuclear Information System (INIS)

Kameya, Hiromi; Miyanoshita, Akihiro; Imamura, Taro; Todoriki, Setsuko

2012-01-01

We attempted a DNA comet assay under alkaline conditions to verify the irradiation treatment of pests. Lasioderma serricorne (Fabricius) were chosen as test insects and irradiated with gamma rays from a 60 Co source at 1 kGy. We conducted the comet assay immediately after irradiation and over time for 7 day. Severe DNA fragmentation in L. serricorne cells was observed just after irradiation and the damage was repaired during the post-irradiation period in a time-dependent manner. The parameters of the comet image analysis were calculated, and the degree of DNA damage and repair were evaluated. Values for the Ratio (a percentage determined by fluorescence in the damaged area to overall luminance, including intact DNA and the damaged area of a comet image) of individual cells showed that no cells in the irradiated group were included in the Ratio<0.1 category, the lowest grade. This finding was observed consistently throughout the 7-day post-irradiation period. We suggest that the Ratio values of individual cells can be used as an index of irradiation history and conclude that the DNA comet assay under alkaline conditions, combined with comet image analysis, can be used to identify irradiation history. - Highlights: ► We investigated the DNA comet assay to verify the irradiation of pests. ► Ratio and Tail Moment were higher in irradiated groups than in the control group. ► The DNA comet assay can be used to identify irradiation history.

Use of IFN-γ and IP-10 detection in the diagnosis of latent tuberculosis infection in patients with inflammatory rheumatic diseases.

Science.gov (United States)

Villar-Hernández, Raquel; Latorre, Irene; Mínguez, Sonia; Díaz, Jéssica; García-García, Esther; Muriel-Moreno, Beatriz; Lacoma, Alicia; Prat, Cristina; Olivé, Alex; Ruhwald, Morten; Mateo, Lourdes; Domínguez, José

2017-10-01

Biologic agents are used against rheumatic diseases, however, they increase the risk of developing severe infections and diseases such as tuberculosis. We aimed to determine the benefits of IP-10 detection to diagnose latent tuberculosis infection (LTBI) in patients with inflammatory rheumatic diseases on different immunosuppressive drug regimens, and compare these results with IFN-γ detection. We included 64 patients with inflammatory rheumatic diseases. We used QuantiFERON Gold In-Tube (QFN-G-IT) and T-SPOT.TB to detect IFN-γ production, and an in-house ELISA for IP-10 detection from the previous QFN-G-IT stimulated samples. We assessed the combined use of IFN-γ release assays (IGRAs) and IP-10 test, and analyzed the influence of immunotherapy on the tests performance. We obtained 34.9% positive results by T-SPOT.TB, 25.0% by QFN-G-IT and 31.3% by IP-10 test. The combined use of IGRAs and IP-10 detection increased significantly the amount of positive results (p  0.05). IP-10 and IFN-γ detection is comparable and their combined use could increase the number of positive results in the diagnosis of LTBI in rheumatic patients. The tested assays were not influenced by rheumatoid immunosuppressive therapy. Thus, IP-10 could be of use in the development of new and improved LTBI diagnostic tools. Copyright © 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Passive nondestructive assay of nuclear materials

International Nuclear Information System (INIS)

Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

1991-03-01

The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

Standard test method for nondestructive assay of special nuclear material in low density scrap and waste by segmented passive gamma-Ray scanning

CERN Document Server

American Society for Testing and Materials. Philadelphia

2010-01-01

1.1 This test method covers the transmission-corrected nondestructive assay (NDA) of gamma-ray emitting special nuclear materials (SNMs), most commonly 235U, 239Pu, and 241Am, in low-density scrap or waste, packaged in cylindrical containers. The method can also be applied to NDA of other gamma-emitting nuclides including fission products. High-resolution gamma-ray spectroscopy is used to detect and measure the nuclides of interest and to measure and correct for gamma-ray attenuation in a series of horizontal segments (collimated gamma detector views) of the container. Corrections are also made for counting losses occasioned by signal processing limitations (1-3). 1.2 There are currently several systems in use or under development for determining the attenuation corrections for NDA of radioisotopic materials (4-8). A related technique, tomographic gamma-ray scanning (TGS), is not included in this test method (9, 10, 11). 1.2.1 This test method will cover two implementations of the Segmented Gamma Scanning ...

Cross-talk between IGF-1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells because of interferon-gamma exposure.

Science.gov (United States)

Park, Sookyoung; Nozaki, Kenkichi; Smith, Joshua A; Krause, James S; Banik, Naren L

2014-03-01

Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF-1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon-gamma (IFN-γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl-2 ratios, and expression of apoptosis-related proteases (caspase-3 and -12) in motoneurons rendered by IFN-γ in a dose-dependent manner. Post-treatment with IGF-1 attenuated these changes. In addition, IGF-1 treatment of motoneurons exposed to IFN-γ decreased expression of inflammatory markers (cyclooxygenase-2 and nuclear factor-kappa B:inhibitor of kappa B ratio). Furthermore, IGF-1 attenuated the loss of expression of IGF-1 receptors (IGF-1Rα and IGF-1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN-γ. To determine whether the protective effects of IGF-1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ-silenced VSC4.1 motoneurons following IFN-γ and IGF-1 exposure. These results suggest that IGF-1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ. © 2013 International Society for Neurochemistry.

Immunological role of CD4+CD28null T lymphocytes, natural killer cells, and interferon-gamma in pediatric patients with sickle cell disease: relation to disease severity and response to therapy.

Science.gov (United States)

ElAlfy, Mohsen Saleh; Adly, Amira Abdel Moneam; Ebeid, Fatma Soliman ElSayed; Eissa, Deena Samir; Ismail, Eman Abdel Rahman; Mohammed, Yasser Hassan; Ahmed, Manar Elsayed; Saad, Aya Sayed

2018-06-20

Sickle cell disease (SCD) is associated with alterations in immune phenotypes. CD4 + CD28 null T lymphocytes have pro-inflammatory functions and are linked to vascular diseases. To assess the percentage of CD4 + CD28 null T lymphocytes, natural killer cells (NK), and IFN-gamma levels, we compared 40 children and adolescents with SCD with 40 healthy controls and evaluated their relation to disease severity and response to therapy. Patients with SCD steady state were studied, focusing on history of frequent vaso-occlusive crisis, hydroxyurea therapy, and IFN-gamma levels. Analysis of CD4 + CD28 null T lymphocytes and NK cells was done by flow cytometry. Liver and cardiac iron overload were assessed. CD4 + CD28 null T lymphocytes, NK cells, and IFN-gamma levels were significantly higher in patients than controls. Patients with history of frequent vaso-occlusive crisis and those with vascular complications had higher percentage of CD4 + CD28 null T lymphocytes and IFN-gamma while levels were significantly lower among hydroxyurea-treated patients. CD4 + CD28 null T lymphocytes were positively correlated to transfusional iron input while these cells and IFN-gamma were negatively correlated to cardiac T2* and duration of hydroxyurea therapy. NK cells were correlated to HbS and indirect bilirubin. Increased expression of CD4 + CD28 null T lymphocytes highlights their role in immune dysfunction and pathophysiology of SCD complications.

Neutron and gamma-ray nondestructive examination of contact-handled transuranic waste at the ORNL TRU Waste Drum Assay Facility

International Nuclear Information System (INIS)

Schultz, F.J.; Coffey, D.E.; Norris, L.B.; Haff, K.W.

1985-03-01

A nondestructive assay system, which includes the Neutron Assay System (NAS) and the Segmented Gamma Scanner (SGS), for the quantification of contact-handled (<200 mrem/h total radiation dose rate at contact with container) transuranic elements (CH-TRU) in bulk solid waste contained in 208-L and 114-L drums has been in operation at the Oak Ridge National Laboratory since April 1982. The NAS has been developed and demonstrated by Los Alamos National Laboratory (LANL) and the Oak Ridge National Laboratory (ORNL) for use by most US Department of Energy Defense Plant (DOE-DP) sites. More research and development is required, however, before the NAS can provide complete assay results for other than routine defense waste. To date, 525 ORNL waste drums have been assayed, with varying degrees of success. The isotopic complexity of the ORNL waste creates a correspondingly complex assay problem. The NAS and SGS assay data are presented and discussed. Neutron matrix effects, the destructive examination facility, and enriched uranium fuel-element assays are also discussed

Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection.

Science.gov (United States)

Perry, Clarice L; Banasik, Brianne N; Gorder, Summer R; Xia, Jingya; Auclair, Sarah; Bourne, Nigel; Milligan, Gregg N

2016-12-01

Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency. Copyright © 2016 Elsevier B.V. All rights reserved.

Parasite-specific IL-4 responses in Ascaris suum and Trichuris suis-infected pigs evaluated by ELISPOT

DEFF Research Database (Denmark)

Steenhard, N. R.; Kringel, H.; Roepstorff, A.

2007-01-01

method is a valuable tool for future experimental settings as it enables repeated and parasite-specific measurement of IL-4 at protein level when investigating, for example, immunomodulatory properties of helminths. Furthermore, the method could be used to identify specific parasite antigens inducing IL......The objective of the present study was to develop an ELISPOT method to measure parasite-specific IL-4 producing cells during experimental Ascaris suum and Trichuris suis infections in pigs. In many experimental settings it is useful to be able to measure changes in specifically induced cytokines...

The Effect of Levothyroxine on Serum Levels of Interleukin 10 and Interferon-gamma in Rat Model of Multiple Sclerosis

Directory of Open Access Journals (Sweden)

Cobra Payghani

2017-01-01

Full Text Available Background: There is an increase in inflammatory and a reduction in anti-inflammatory cytokines in multiple sclerosis (MS. Considering the role of thyroid hormones in the development and regulation of both neural and immune systems, the aim of this study was to evaluate the effects of levothyroxine on serum concentrations of interleukin-10 (IL-10 and interferon gamma (IFN-γ in animal models of MS. Materials and Methods: To induce demyelination in male Wistar rats, lysolecithin was injected into the optic chiasm. Then levothyroxine was injected intraperitoneally (20, 50, and 100 μg/kg for 21 days. Serum levels of cytokines were measured by enzyme-linked immunosorbent assay at 7, 14, and 21 days after that. Results: The results showed that injection of lysolecithin to the optic chiasm only increased serum concentrations of IL-10 compared to the sham group (P < 0.05 at 7th day, but this increase was prevented by all doses of levothyroxine. IFN-γ was decreased significantly (P < 0.001 21 days after. Comparing to the sham group at all sampling time and with respect to the MS group at the days 7 and 21, levothyroxine decreased serum concentrations of IFN-γ significantly. Conclusion: The results showed that thyroid hormones probably could produce protective effects against induced demyelination through affecting immune responses.

Interleukin-15 differentially enhances the expression of interferon-gamma and interleukin-4 in activated human (CD4(+))T lymphocytes

NARCIS (Netherlands)

Borger, P; Kauffman, HF; Postma, DS; Esselink, MT; Vellenga, E

In this study interleukin (IL)-15 was examined for its ability to modulate the expression of interferon-gamma (IFN-gamma) and IL-4 in activated human T lymphocytes. The effect of IL-15 was compared with IL-2 and IL-7, cytokines all known to use the IL-2 receptor gamma(C) chain. The results

Role of IL-12 and IFN-γ in immune response to toxoplasma gondii infection

International Nuclear Information System (INIS)

Moawad, M.A.F.; ElGawish, M.A.M.

2004-01-01

Interlenkin 12 (IL-12) is a potent immunoregulatory molecule that is critically involved in a wide range of diseases. In several murine models of intracellular infection, endogenous IL-12 has been shown to be crucial for the generation of a protective Th1 response in a primary infection for a intracellular pathogens. Interferon-gamma (IFN-γ) is also an important mediator of cellular immunity against microbial pathogens and tumor cells due to its potent capacity to activate macrophages for enhanced cytotoxicity. The aim of the present study is to evaluate the immune response to toxoplasma gondii after primary inflection (infected groups and secondary infection (re-infected groups for over 19 weeks (the time of the experiment). the evaluation was assessed by measurements of levels of IL-12 and IFN-γ using ELISA technique in the sera of these infected rats. The results demonstrated that the primary immune response induced a fluctuation in the levels of IL-12 in the sera of infected rats, which reached maximum value of 122.6 ±1.4 pg/ml after 15 weeks of primary infection. While, in the challenged groups (secondary immune response, re-infected groups) the levels of IL-12 were generally lower than that of the primary immune response. On the other hand, IFN-γ levels increased significantly in the secondary immune response (re-infected groups) as compared to primary immune response 9 infected groups) In conclusion, the results suggest that IL-12 might have a role in the defense mechanism against intracellular infection with T-gondii especially in primary immune response than in the secondary immune response. This is in contrast to IFN-γ that takes the up-hand in secondary immune response to T-gondii infection

Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

OpenAIRE

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin a...

Efecto de la maca roja (Lepidium meyenii) sobre los niveles de IFN-γ en ratas ovariectomizadas

OpenAIRE

Leiva-Revilla, Johanna; Laboratorio de Endocrinología y Reproducción, Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú.; Guerra-Castañon, Félix; Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú. Facultad de Medicina, Universidad Peruana Cayetano Heredia. Lima-Perú.; Olcese-Mori, Paola; Laboratorio de Endocrinología y Reproducción, Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú.; Lozada, Iván; Laboratorio de Inmunología, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Microbiología, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú.; Rubio, Julio; Laboratorio de Endocrinología y Reproducción, Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú. Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia. Lima-Perú.; Gonzales, Carla; Laboratorio de Endocrinología y Reproducción, Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú. Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia. Lima-Perú.; Gonzales, Gustavo F; Laboratorio de Endocrinología y Reproducción, Facultad de Ciencias y Filosofía. Universidad Peruana Cayetano Heredia. Lima-Perú. Departamento de Ciencias Biológicas y Fisiológicas, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia. Lima-Perú. Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia. Lima-Perú.

2014-01-01

Objectives. Compare the effect of different doses of red maca on gamma interferon (IFN-γ) levels in ovariectomized rats (OVX). Materials and methods. Adult female rats were randomly divided into the following six groups: Group 1: pseudo-ovariectomized rats (PO); Group 2: OVX rats; Group 3: OVX rats treated with 4 ug/kg estradiol; and Group 4, 5 and 6: OVX rats treated with red maca extracts with 2.15, 4.3 and 8.6 mg polyphenols/body weight kilogram, respectively. Results. OVX rats showed low ...

Role of interferon-gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-gamma knock-out mice

DEFF Research Database (Denmark)

Nansen, A; Christensen, Jan Pravsgaard; Röpke, C

1998-01-01

, a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN-gamma knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may...

Contact inhibition and interferon (IFN)-modulated gene expression

Energy Technology Data Exchange (ETDEWEB)

Kulesh, D.A.

1986-01-01

The relationship between cell morphology, proliferation and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes. Cell proliferation was quantitated by labeling indices, which were inferred by autoradiography, and by total cell counts. The normal cells (JHU-1, IMR-90) were dependent on cell shape for proliferation capability while the transformed cells (RT4, HT1080) were shape-dependent for proliferation. Interferon (IFN) induced shape-dependent proliferation and contact inhibition in the transformed cells when used at subantiproliferative concentrations. This ability of B-IFN to confer a level of proliferation control which is characteristic of normal fibroblasts suggests a possible relationship between gene expression mediated by IFN and those genes involved in the maintenance of regulated cell proliferation. To evaluate this possibility, cDNA libraries were constructed from IFN-treated and untreated HT1080 cells. The resulting 10 IFN-induced and 11 IFN-repressed sequences were then differentially rescreened using /sup 32/P-cDNA probes. This screening resulted in the identification of at least four cDNA sequences which appeared to be proliferation regulated as well as IFN-modulated. These cloned, regulated cDNA sequences were then used as /sup 32/P-labeled probes to study both the gene expression at the mRNA level employing Northern blotting and slot blotting techniques.

Cucurbita ficifolia (Cucurbitaceae) modulates inflammatory cytokines and IFN-γ in obese mice.

Science.gov (United States)

Fortis-Barrera, Á; García-Macedo, R; Almanza-Perez, J C; Blancas-Flores, G; Zamilpa-Alvarez, A; Flores-Sáenz, J L; Cruz, M; Román-Ramos, R; Alarcón-Aguilar, F J

2017-02-01

This study investigated the effect of aqueous extract of Cucurbita ficifolia Bouché on systemic chronic inflammation in an obesity model induced by monosodium glutamate (MSG) via modulating the expression of adipokines (TNF-α, IL-6, resistin, and adiponectin) and immune-regulatory cytokines (IFN-γ and IL-10). Cucurbita ficifolia extract was administered daily by gavage to lean and MSG-obese mice for 30 days. At the end of treatment, cytokine mRNA expression in adipose tissue was determined by real-time polymerase chain reaction (PCR), and the protein levels of these cytokines were also quantified by enzyme-linked immunosorbent assay (ELISA). Cucurbita ficifolia extract decreased body mass and inflammation in MSG-obese mice by reducing the expression of TNF-α and IL-6; these decreases were parallel to significant reductions in protein levels. The extract also increased protein levels of IL-10 in lean mice and IFN-γ in both lean and MSG-obese mice. In conclusion, C. ficifolia extract modulates systemic chronic inflammation in MSG-obese mice and could have a beneficial effect on the adaptive immune system in obesity.

Systemic Activation of the IFN System by Exposure of Natural Epithelia to Physiologic (Low Dose) Levels of IFN

Science.gov (United States)

1990-06-20

possibility that epithelial surfaces of the upper alimentary and/or respiratory mucosa exposed to lymphocytes treated with low concentrations of IFN or with IFN... Biotechnology Program Office of Naval Research Office of Naval Research 800 N. Quincy Street Code 1213 Arlington, VA 22217-5000 800 N. Quincy Street Arlington

IFN-Alpha receptor-1 upregulation in PBMC from HCV naïve patients carrying cc genotype. possible role of IFN-lambda.

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Eleonora Lalle

Full Text Available IL-28B gene polymorphisms predict better therapeutic response and spontaneous clearance of HCV. Moreover, higher expression of IFN-lambda has been reported in patients with the rs12979860 CC favourable genotype. The study aim was to establish possible relationships between IL-28B rs12979860 genotypes and expression of IFN-alpha receptor-1 (IFNAR-1 in naïve HCV patients, and to explore the possible role of IFN-lambda.IFNAR-1 mRNA levels were measured in PBMC from naïve patients with chronic hepatitis C with different IL-28 genotypes. The ability of IFN-lambda to up-regulate the expression of IFNAR-1 was established in PBMC from healthy donors carrying different IL-28B genotypes.Lower IFNAR-1 mRNA levels were observed in PBMC from HCV-infected naïve patients as compared to healthy donors. In healthy donors, IFNAR-1 mRNA levels were independent from IL-28B genotype, while in HCV patients, an increasing gradient was observed in TT vs CT vs CC carriers. In the latter group, a direct correlation between IFNAR-1 and endogenous IL-28B expression was observed. Moreover, IFN-lambda up-regulated IFNAR-1 expression in normal PBMC in a time-and dose-dependent manner, with a more effective response in CC vs TT carriers.Endogenous levels of IFN-lambda may be responsible for partial restoration of IFNAR-1 expression in HCV patients with favourable IL-28 genotype. This, in turn, may confer to CC carriers a response advantage to either endogenous or exogenous IFN-alpha, representing the biological basis for the observed association between CC genotype and favourable outcome of either natural infection (clearance vs chronicization or IFN therapy.

Evaluation through comet assay of DNA damage induced in human lymphocytes by alpha particles. Comparison with protons and Co-60 gamma rays

International Nuclear Information System (INIS)

Di Giorgio, M.; Kreiner, A. J.; Schuff, J. A.; Vallerga, M. B.; Taja, M. R.; Lopez, F. O.; Alvarez, D. E.; Saint Martin, G.; Burton, A.; Debray, M. E.; Kesque, J. M.; Somacal, H.; Stoliar, P.; Valda, A.; Davidson, J.; Davidson, M.; Ozafran, M. J.; Vazquez, M. E.

2004-01-01

Several techniques with different sensitivity to single-strand breaks and/or double strand breaks were applied to detect DNA breaks generated by high LET particles. Tests that assess DNA damage in single cells might be the appropriate tool to estimate damage induced by particles, facilitating the assessment of heterogeneity of damage in a cell population. The microgel electrophoresis (comet) assay is a sensitive method for measuring DNA damage in single cells. The objective of this work was to evaluate the proficiency of comet assay to assess the effect of high LET radiation on peripheral blood lymphocytes, compared to protons and Co-60 gamma rays. Materials and methods: Irradiations of blood samples were performed at TANDAR laboratory (Argentina). Thin samples of human peripheral blood were irradiated with different doses (0-2.5 Gy) of 20.2 MeV helium-4 particles in the track segment mode, at nearly constant LET. Data obtained were compared with the effect induced by a MeV protons and Co-60 gamma rays. Alkaline comet assay was applied. Comets were quantified by the Olive tail moment. Distribution of the helium-4 particle and protons were evaluated considering Poisson distribution in lymphocyte nuclei. The mean dose per nucleus per particle result 0.053 Gy for protons and 0.178 Gy for helium-4 particles. When cells are exposed to a dose of 0.1 Gy, the hit probability model predicts that 43% of the nuclei should have experienced and alpha traversal while with protons, 85% of the nuclei should be hit. The experimental results show a biphasic response for helium-4 particles (0.1 Gy), indicating the existence of two subpopulations: unhit and hit. Distributions of tail moment as a function of fluence and experimental dose for comets induced by helium-4 particles, protons and Co-60 gamma rays were analyzed. With helium-4 irradiations, lymphocyte nuclei show an Olive tail moment distribution flattened to higher tail moments a dose increase. However, for irradiations with

Exploratory study on Th1 epitope-induced protective immunity against Coxiella burnetii infection.

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Xiaolu Xiong

Full Text Available Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A(b based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4(+ T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4(+ T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4(+ T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.

Elements of nondestructive assay (NDA) technology

International Nuclear Information System (INIS)

Anon.

1981-01-01

This session provides an introduction to nondestructive assay methods and instruments as they are applied to nuclear safeguards. The purpose of the sessions is to enable participants to: (1) discuss the general principles and major applications of NDA; (2) describe situations in which NDA is particularly useful for nuclear safeguards purposes; (3) distinguish between various passive and active gamma-ray and neutron NDA methods; (4) describe several NDA instruments that measure gamma rays, and identify assay situations particularly suited to gamma-ray techniques; (5) describe several NDA instruments that measure neutrons, and identify assay situations particularly suited to neutron techniques; (6) discuss the role of calorimetry in the NDA of plutonium-bearing materials; and (7) compare the advantages and disadvantages of various NDA methods for different types of nuclear materials

Inhibition of 125I organification and thyroid hormone release by interleukin-1, tumor necrosis factor-alpha, and interferon-gamma in human thyrocytes in suspension culture

International Nuclear Information System (INIS)

Sato, K.; Satoh, T.; Shizume, K.; Ozawa, M.; Han, D.C.; Imamura, H.; Tsushima, T.; Demura, H.; Kanaji, Y.; Ito, Y.

1990-01-01

To elucidate the mechanism of decreased 131I uptake by the thyroid gland in patients with subacute thyroiditis and painless thyroiditis, human thyroid follicles were cultured with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and/or interferon-gamma (IFN gamma), and the effects of these cytokines on thyroid function were studied in vitro. When human thyrocytes were cultured in RPMI-1640 medium containing 0.5% fetal calf serum and TSH for 5-8 days, the cells incorporated 125I, synthesized de novo [125I]iodotyrosines and [125I]iodothyronines, and secreted [125I]T4 and [125I]T3 into the medium. IL-1 alpha and IL-1 beta inhibited 125I incorporation and [125I]iodothyronine release in a concentration-dependent manner. The minimal inhibitory effect was detected at 10 pg/ml. Electron microscopic examination revealed a marked decrease in lysosome formation in IL-1-treated thyrocytes. TNF alpha and IFN gamma also inhibited thyroid function in a concentration-dependent manner. Furthermore, when thyrocytes were cultured with IL-1, TNF alpha and IFN gamma, these cytokines more than additively inhibited thyroid function. Although the main mechanism of 131I uptake suppression in the thyroid gland in subacute thyroiditis is due to cellular damage and suppression of TSH release, our present findings suggest that IL-1, TNF alpha, and IFN gamma produced in the inflammatory process within the thyroid gland further inhibit iodine incorporation and at least partly account for the decreased 131I uptake by the thyroid gland in destruction-induced hyperthyroidism

A novel role for adiponectin in regulating the immune responses in chronic hepatitis C virus infection.

Science.gov (United States)

Palmer, Clovis; Hampartzoumian, Taline; Lloyd, Andrew; Zekry, Amany

2008-08-01

Adipose tissue releases pro-inflammatory and anti-inflammatory mediators, including adiponectin, which elicit a broad range of metabolic and immunological effects. The study aim was to determine in subjects infected with chronic hepatitis C virus (HCV) the effects of total adiponectin and its high-molecular-weight (HMW) and low-molecular-weight isoforms on HCV-specific immune responses. Serum levels of total adiponectin and its isoforms were determined by immunoassay. The ex vivo effect of adiponectin on the HCV-specific T-cell response was examined by interferon gamma (IFN-gamma) enzyme-linked immunosorbent spot and enzyme-linked immunosorbent assay cytokine assays. The role of the mitogen-activated protein kinase (MAPK) signaling pathway in mediating the adiponectin effect on T cells was also evaluated. We found that serum levels of total and HMW adiponectin were significantly decreased in subjects with chronic HCV and increased body mass index (BMI) compared with HCV-infected lean subjects. The presence of an anti-HCV specific immune response was strongly associated with lower BMI (P = 0.004) and higher serum total (P = 0.01) and HMW (P = 0.02) adiponectin. In ex vivo assays, total adiponectin and the HMW adiponectin isoform enhanced HCV-specific IFN-gamma production (P = 0.02 and 0.03, respectively). Adiponectin-R1 receptors were expressed on T cells and monocytes. In depletion experiments, the IFN-gamma response to adiponectin was entirely dependent on the simultaneous presence of both CD4 and CD8 T cells, and to a lesser extent, natural killer cells. Selective inhibition of p38MAPK activity by SB203580 abrogated the IFN-gamma response to adiponectin, whereas extracellular signal-regulated kinase 1/2 inhibition by PD98059 did not affect the response. In chronic HCV, a reciprocal association exists between BMI, adiponectin, and the anti-HCV immune responses, emphasizing the important role played by adiposity in regulating the immune response in HCV infection.

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

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Gregory G Simon

2010-01-01

Full Text Available This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d and HLA-DR4 (DRA1*0101, DRB1*0401 transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag chimera antigen. Three immunization protocols were compared: 1 primary subcutaneous immunization with 1x10(5 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2 primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3 immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b the value of HLA transgenic mice as a model system for the identification and evaluation

Nitric oxide selectively decreases interferon-gamma expression by activated human T lymphocytes via a cGMP-independent mechanism

NARCIS (Netherlands)

Roozendaal, R; Vellenga, E; Postma, DS; De Monchy, JGR; Kauffman, HF

1999-01-01

The role of exogenous nitric oxide (NO) on the expression of interleukin (IL)-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) by freshly isolated human T lymphocytes was investigated. The presence of NO, generated from any of the NO-donor compounds, S-nitroso-N-acetyl-D,L-penicillamine (NAP),

Immune response to Nocardia brasiliensis extracellular antigens in patients with mycetoma.

Science.gov (United States)

Castro-Matteotti, Bárbara; Vera-Cabrera, Lucio; Ocampo-Candiani, Jorge; Rendón, Adrián; Salinas-Carmona, Mario C; Welsh, Oliverio

2008-03-01

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 x 10(7) CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-gamma, IL-1alpha, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-gamma production in mice, as well as the CFA, were used to stimulate IFN-gamma production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-gamma production in the infected group. In humans, only the CFA-induced IFN-gamma production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.

CD4(+) T cell-mediated protection against a lethal outcome of systemic infection with vesicular stomatitis virus requires CD40 ligand expression, but not IFN-gamma or IL-4

DEFF Research Database (Denmark)

Andersen, C; Jensen, T; Nansen, A

1999-01-01

experiments using B cell- and T cell-deficient recipients revealed that no protection could be obtained in the absence of B cells, whereas treatment with virus-specific immune (IgG) serum controlled viral spreading to the central nervous system (CNS), but did not necessarily accomplish virus elimination......To investigate the mechanism(s) whereby T cells protect against a lethal outcome of systemic infection with vesicular stomatitis virus, mice with targeted defects in genes central to T cell function were tested for resistance to i.v. infection with this virus. Our results show that mice lacking...... the capacity to secrete both IFN-gamma and perforin completely resisted disease. Similar results were obtained using IL-4 knockout mice, indicating that neither cell-mediated nor T(h)2-dependent effector systems were required. In contrast, mice deficient in expression of CD40 ligand were more susceptible than...

Inhibition of sup 125 I organification and thyroid hormone release by interleukin-1, tumor necrosis factor-alpha, and interferon-gamma in human thyrocytes in suspension culture

Energy Technology Data Exchange (ETDEWEB)

Sato, K.; Satoh, T.; Shizume, K.; Ozawa, M.; Han, D.C.; Imamura, H.; Tsushima, T.; Demura, H.; Kanaji, Y.; Ito, Y. (Institute of Clinical Endocrinology, Tokyo (Japan))

1990-06-01

To elucidate the mechanism of decreased 131I uptake by the thyroid gland in patients with subacute thyroiditis and painless thyroiditis, human thyroid follicles were cultured with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and/or interferon-gamma (IFN gamma), and the effects of these cytokines on thyroid function were studied in vitro. When human thyrocytes were cultured in RPMI-1640 medium containing 0.5% fetal calf serum and TSH for 5-8 days, the cells incorporated 125I, synthesized de novo (125I)iodotyrosines and (125I)iodothyronines, and secreted (125I)T4 and (125I)T3 into the medium. IL-1 alpha and IL-1 beta inhibited 125I incorporation and (125I)iodothyronine release in a concentration-dependent manner. The minimal inhibitory effect was detected at 10 pg/ml. Electron microscopic examination revealed a marked decrease in lysosome formation in IL-1-treated thyrocytes. TNF alpha and IFN gamma also inhibited thyroid function in a concentration-dependent manner. Furthermore, when thyrocytes were cultured with IL-1, TNF alpha and IFN gamma, these cytokines more than additively inhibited thyroid function. Although the main mechanism of 131I uptake suppression in the thyroid gland in subacute thyroiditis is due to cellular damage and suppression of TSH release, our present findings suggest that IL-1, TNF alpha, and IFN gamma produced in the inflammatory process within the thyroid gland further inhibit iodine incorporation and at least partly account for the decreased 131I uptake by the thyroid gland in destruction-induced hyperthyroidism.

Obesity Related Alterations in Plasma Cytokines and Metabolic Hormones in Chimpanzees

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Pramod Nehete

2014-01-01

Full Text Available Obesity is characterized by chronic low-grade inflammation and serves as a major risk factor for hypertension, coronary artery disease, dyslipidemias, and type-2 diabetes. The purpose of this study was to examine changes in metabolic hormones, inflammatory cytokines, and immune function, in lean, overweight, and obese chimpanzees in a controlled environment. We observed increased plasma circulating levels of proinflammatory TH-1 cytokines, Interferon gamma, interleukin-6, interleukin-12p40, tumor necrosis factor, soluble CD40 ligand, and Interleukin-1β and anti-inflammatory TH-2 cytokines, Interleukin-4, Interleukin-RA, Interleukin-10, and Interleukin-13 in overweight and obese chimpanzees. We also observed increased levels of metabolic hormones glucagon-like-peptide-1, glucagon, connecting peptide, insulin, pancreatic peptide YY3–36, and leptin in the plasma of overweight and obese chimpanzees. Chemokine, eotaxin, fractalkine, and monocyte chemoattractant protein-1 were higher in lean compared to obese chimpanzees, while chemokine ligand 8 increased in plasma of obese chimpanzees. We also observed an obesity-related effect on immune function as demonstrated by lower mitogen induced proliferation, and natural killer activity and higher production of IFN-γ by PBMC in Elispot assay, These findings suggest that lean, overweight, and obese chimpanzees share circulating inflammatory cytokines and metabolic hormone levels with humans and that chimpanzees can serve as a useful animal model for human studies.

Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages.

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Taha Salim

Full Text Available Macrophage produced inducible nitric oxide synthase (iNOS is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS, iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ and tumor necrosis factor alpha (TNF-α. To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced.

Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages.

Science.gov (United States)

Salim, Taha; Sershen, Cheryl L; May, Elebeoba E

2016-01-01

Macrophage produced inducible nitric oxide synthase (iNOS) is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO) by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS), iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced.

The production of lymphokines by primary alloreactive T-cell clones: a co-ordinate analysis of 233 clones in seven lymphokine assays.

Science.gov (United States)

Sanderson, C J; Strath, M; Warren, D J; O'Garra, A; Kirkwood, T B

1985-01-01

A total of 233 primary alloreactive T-cell clones have been tested for the production of interleukin-2 (IL-2), interleukin-3 (IL-3), immune(gamma) interferon (IFN) and granulocyte-macrophage colony-stimulating factor (CSF-2), B-cell growth factor I and II (BCGFI, BCGFII), and eosinophil differentiation factor (EDF). EDF was assayed by means of the eosinophil differentiation assay (EDA). Two principal correlations were observed: IL-3 was shown to be the major lymphokine detected in the bone marrow proliferation assay (BMPA) used to detect CSF-2, and there was a high correlation between the EDA and BCGFII. Subsequent work has suggested that this latter correlation is because a single factor is responsible for both activities. Apart from these two exceptions, and low level correlations probably due to the fact that different assays detect more than one lymphokine, there was no evidence for co-ordinate expression of lymphokines. There was a large variation in amounts of individual lymphokines produced. More clones produced multiple lymphokines than would be expected from independent control. Taken together, this pattern of regulation is consistent with the hypothesis that antigen stimulation of T cells results in the activation of all the lymphokine genes, but the amount of each produced is determined by secondary controlling mechanisms. PMID:3935571

Comparison of histopathology, cultivation of tissues and rectal contents, and interferon-gamma and serum antibody responses for the diagnosis of bovine paratuberculosis

DEFF Research Database (Denmark)

Huda, A.; Jensen, H.E.

2003-01-01

contents, and (3) examination of repeated blood samples for interferon-gamma (IFN-gamma) and antibody responses. Tissue samples were taken from the small and large intestine and corresponding mesenteric lymph nodes, and from the pharyngeal tonsil and other lymphoid nodes (retropharyngeal, mediastinal...

Nondestructive assay methods for irradiated nuclear fuels

International Nuclear Information System (INIS)

Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

1978-01-01

This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed

SH2 modified STAT1 induces HLA-I expression and improves IFN-γ signaling in IFN-α resistant HCV replicon cells.

Directory of Open Access Journals (Sweden)

Bret Poat

2010-09-01

Full Text Available We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2 domains of STAT1 (at Ala-656 and Asn-658 efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

DEFF Research Database (Denmark)

Andersen, Rikke Sick; Sørensen, Rikke Bæk; Ritter, Cathrin

2011-01-01

. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition......, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion......, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors....

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

DEFF Research Database (Denmark)

Andersen, Rikke Sick; Sørensen, Rikke Bæk; Ritter, Cathrin

2011-01-01

. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-¿ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition......, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion......, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors....

Interferon gamma release assays for the diagnosis of latent TB infection in HIV-infected individuals in a low TB burden country.

LENUS (Irish Health Repository)

Cheallaigh, Clíona Ní

2013-01-01

Interferon gamma release assays (IGRAs) are used to diagnose latent tuberculosis infection. Two IGRAs are commercially available: the Quantiferon TB Gold In Tube (QFT-IT) and the T-SPOT.TB. There is debate as to which test to use in HIV+ individuals. Previous publications from high TB burden countries have raised concerns that the sensitivity of the QFT-IT assay, but not the T-SPOT.TB, may be impaired in HIV+ individuals with low CD4+ T-cell counts. We sought to compare the tests in a low TB burden setting.

IFN-γ-producing Th1-like regulatory T cells may limit acute cellular renal allograft rejection: Paradoxical post-transplantation effects of IFN-γ.

Science.gov (United States)

Xu, Xiaoguang; Huang, Haiyan; Wang, Qiang; Cai, Ming; Qian, Yeyong; Han, Yong; Wang, Xinying; Gao, Yu; Yuan, Ming; Xu, Liang; Yao, Chen; Xiao, Li; Shi, Bingyi

2017-02-01

IFN-γ is a protypical proinflammatory cytokine that plays a central role in inflammation and acute graft rejection. Accumulating evidence indicates that IFN-γ can exert previously unexpected immunoregulatory activities. However, little is known about the role of IFN-γ secreted by Th1-like regulatory T cells in human kidney transplantation. To determine the function of IFN-γ in acute T cell-mediated renal allograft rejection (ACR), we examined serum cytokine expression profiles in ACR patients by human cytokine multiplex immunoassay and analyzed the cellular origins of IFN-γ in peripheral blood and renal allograft biopsies from ACR cases and controls by flow cytometry and immunohistochemistry, respectively. The results showed significant reduction in serum concentrations of Th1-inducing cytokines IL-12p70 and IFN-γ as well as Th2-related cytokine IL-4 in ACR patients compared with stable controls. However, levels of several Th1-, Th2- and Th17-related cytokines, such as IL-2, TNF-α, TNF-β, IL-12 (p40), IL-10, IL-15, IL-17, IL-21, and IL-23, as well as the frequencies of Th1 and Th17 cell, did not differ between ACR cases and stable controls. Moreover, we found the levels of IFN-γ were correlated with those of the anti-inflammatory factor, IL-1 receptor antagonist (IL-1Ra) in ACR. Notably, the Th1-like Treg cell-to-Foxp3 - Th1 cell ratio was significantly lower in ACR patients compared with that in stable controls. In graft biopsies from ACR patients, Treg cells and Th1-like Treg cells were less abundant than those without ACR. Our study indicates that IFN-γ secreted from Th1-like Treg cells negatively modulates ACR. Copyright © 2016 Elsevier GmbH. All rights reserved.

PRMT5-Mediated Methylation of NF-κB p65 at Arg174 Is Required for Endothelial CXCL11 Gene Induction in Response to TNF-α and IFN-γ Costimulation.

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Daniel P Harris

Full Text Available Inflammatory agonists differentially activate gene expression of the chemokine family of proteins in endothelial cells (EC. TNF is a weak inducer of the chemokine CXCL11, while TNF and IFN-γ costimulation results in potent CXCL11 induction. The molecular mechanisms underlying TNF plus IFN-γ-mediated CXCL11 induction are not fully understood. We have previously reported that the protein arginine methyltransferase PRMT5 catalyzes symmetrical dimethylation of the NF-κB subunit p65 in EC at multiple arginine residues. Methylation of Arg30 and Arg35 on p65 is critical for TNF induction of CXCL10 in EC. Here we show that PRMT5-mediated methylation of p65 at Arg174 is required for induction of CXCL11 when EC are costimulated with TNF and IFN-γ. Knockdown of PRMT5 by RNAi reduced CXCL11 mRNA and protein levels in costimulated cells. Reconstitution of p65 Arg174Ala or Arg174Lys mutants into EC that were depleted of endogenous p65 blunted TNF plus IFN-γ-mediated CXCL11 induction. Mass spectrometric analyses showed that p65 Arg174 arginine methylation is enhanced by TNF plus IFN-γ costimulation, and is catalyzed by PRMT5. Chromatin immunoprecipitation assays (ChIP demonstrated that PRMT5 is necessary for p65 association with the CXCL11 promoter in response to TNF plus IFN-γ. Further, reconstitution of p65 Arg174Lys mutant in EC abrogated this p65 association with the CXCL11 promoter. Finally, ChIP and Re-ChIP assays revealed that symmetrical dimethylarginine-containing proteins complexed with the CXCL11 promoter were diminished in p65 Arg174Lys-reconstituted EC stimulated with TNF and IFN-γ. In total, these results indicate that PRMT5-mediated p65 methylation at Arg174 is essential for TNF plus IFN-γ-mediated CXCL11 gene induction. We therefore suggest that the use of recently developed small molecule inhibitors of PRMT5 may present a therapeutic approach to moderating chronic inflammatory pathologies.

Interferon Gamma and PSA-Restricted Expression of FAS Ligand: A Novel Gene Therapy Strategy for Prostate Cancer

National Research Council Canada - National Science Library

Hall, Simon

2003-01-01

Introduction: Preliminary studies pointed to the ability for IFN-gamma to enhance sensitivity and/or reverse resistance to Fas transactivation on prostate cancer cells and work during the past 2 years illustrated...

Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania☆,☆☆

Science.gov (United States)

Bakari, Muhammad; Aboud, Said; Nilsson, Charlotta; Francis, Joel; Buma, Deus; Moshiro, Candida; Aris, Eric A.; Lyamuya, Eligius F.; Janabi, Mohamed; Godoy-Ramirez, Karina; Joachim, Agricola; Polonis, Victoria R.; Bråve, Andreas; Earl, Patricia; Robb, Merlin; Marovich, Mary; Wahren, Britta; Pallangyo, Kisali; Biberfeld, Gunnel; Mhalu, Fred; Sandström, Eric

2016-01-01

Background We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. Methods Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1 mg intradermally (id), n = 20, or 3.8 mg intramuscularly (im), n = 20, or placebo, n = 20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. Results The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01 AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher

ISG15 Inhibits IFN-α-Resistant Liver Cancer Cell Growth

Directory of Open Access Journals (Sweden)

Xin-xing Wan

2013-01-01

Full Text Available Hepatocellular carcinoma (HCC is one of the most prevalent tumors worldwide. Interferon-α (IFN-α has been widely used in the treatment of HCC, but patients eventually develop resistance. ISG15 ubiquitin-like modifier (ISG15 is a ubiquitin-like protein transcriptionally regulated by IFN-α which shows antivirus and antitumor activities. However, the exact role of ISG15 is unknown. In the present study, we showed that IFN-α significantly induced ISG15 expression but failed to induce HepG2 cell apoptosis, whereas transient overexpression of ISG15 dramatically increased HepG2 cell apoptosis. ISG15 overexpression increased overall protein ubiquitination, which was not observed in cells with IFN-α-induced ISG15 expression, suggesting that IFN-α treatment not only induced the expression of ISG15 but also inhibited ISG15-mediated ubiquitination. The tumor suppressor p53 and p21 proteins are the key regulators of cell survival and death in response to stress signals such as DNA damage. We showed that p53 or p21 is only up regulated in HepG2 cells ectopically expressing ISG15, but not in the presence of IFN-α-induced ISG15. Our results suggest that ISG15 overexpression could be developed into a powerful gene-therapeutic tool for treating IFN-α-resistant HCC.

Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

International Nuclear Information System (INIS)

Smith, J.R.

1990-08-01

The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

Interferon gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central nervous system and lymphoid cells

DEFF Research Database (Denmark)

Issazadeh-Navikas, Shohreh; Mustafa, M; Ljungdahl, A

1995-01-01

, the target organ in EAE, cells expressing mRNA for IFN-gamma, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.) and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal...... to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63-88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63-88-induced production of both IFN-gamma and IL-4...... cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-beta did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-gamma, while TGF-beta may act...

Diminished interferon-gamma production and responsiveness after endotoxin administration to healthy humans

NARCIS (Netherlands)

Weijer, Sebastiaan; Lauw, Fanny N.; Branger, Judith; van den Blink, Bernt; van der Poll, Tom

2002-01-01

To obtain insight in the capacity of the lipopolysaccharide (LPS)-tolerant host to produce interferon (IFN)-gamma and to respond to this cytokine, whole blood was obtained from healthy humans before and 4 h after intravenous injection of LPS (4 ng/kg) and stimulated ex vivo. LPS exposure in vivo

Fundamentals of passive nondestructive assay of fissionable material: laboratory workbook

International Nuclear Information System (INIS)

Reilly, T.D.; Augustson, R.H.; Parker, J.L.; Walton, R.B.; Atwell, T.L.; Umbarger, C.J.; Burns, C.E.

1975-02-01

This workbook is a supplement to LA-5651-M, ''Fundamentals of Passive Nondestructive Assay of Fissionable Material'' which is the text used during the Nondestructive Assay Training Session given by Group A-1 of the Los Alamos Scientific Laboratory. It contains the writeups used during the six laboratory sessions covering basic gamma-ray principles, quantitative gamma-ray measurements, uranium enrichment measurements, equipment holdup measurements, basic neutron principles, and quantitative neutron assay

The cost of successful antiviral therapy in hepatitis C patients: a comparison of IFN-free versus IFN-based regimens at an individual patient level in Australia

Directory of Open Access Journals (Sweden)

Lee AS

2017-10-01

Full Text Available Allister Sebastian Lee,1 Mieke L van Driel,2 Darrell HG Crawford3,4 1Faculty of Medicine, 2Primary Care Clinical Unit, Faculty of Medicine, 3School of Clinical Medicine, Faculty of Medicine, University of Queensland, 4Gallipoli Medical Research Foundation, Greenslopes Private Hospital, Brisbane, Australia Background: Chronic hepatitis C remains a major global health burden with serious long-term consequences if left untreated. Recently the treatment standard of care has shifted to new interferon (IFN-free drug regimens, which have been shown to be safe and effective. The aim of our study was to assess and compare medical resource utilization and costs of successfully treating patients with IFN-based and IFN-free therapies in Australia. Methods: We performed a retrospective chart review of 30 HCV-infected patients successfully treated with IFN-based therapy between 2013 and 2015. We also generated a model for a virtual group of 100 genotype 1 (GT1 and 100 genotype 3 (GT3 patients treated with IFN-free therapy derived from national guidelines and clinical trial data. Results: In comparison to virtual patients receiving IFN-free therapy, our IFN-treated patients on average had distinctively more liver clinic visits and blood tests. However, mean total cost per patient was $19,164 and $85,300 (AUD more for GT1 and GT3 patients receiving IFN-free therapy, respectively. This difference was largely accounted for by higher antiviral drug costs. Of our 30 patients treated with IFN, total mean cost per patient during the study period was $33,595. Conclusion: Resource utilization is lower with IFN-free treatment, which reflects the reduced need for patient monitoring and improved side-effect profile of these new drugs. However, total costs are still largely dominated by antiviral drug costs, representing a huge burden on national budgets. Our insight into resource utilization and costs associated with both types of treatment can serve as a reference for

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2.

Directory of Open Access Journals (Sweden)

Zhongshuai Xin

Full Text Available Interferon-α (IFN-α is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b. However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

Science.gov (United States)

Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression using experimental data

Science.gov (United States)

Available diagnostic assays for Mycobacterium avium subsp paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of the infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN- gamma, ELI...

Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence

Directory of Open Access Journals (Sweden)

Stefan Seeger

2013-02-01

Full Text Available We present an immunoassay for Interferon-γ (IFN-γ with a limit of detection of 1.9 pM (30 pg/mL and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA. The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O : 9 in pigs

DEFF Research Database (Denmark)

Riber, Ulla; Jungersen, Gregers

2007-01-01

Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype 0:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers...... of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon......-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week postinoculation...

Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

Science.gov (United States)

Dhar, Jayeeta; Barik, Sailen

2016-12-01

Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

Polymorphisms in an interferon-gamma receptor-1 gene marker and susceptibility to periodontitis

NARCIS (Netherlands)

Fraser, DA; Loos, BG; Boman, U; van Winkelhoff, AJ; van der Velden, U; Schenck, K; Dembic, Z

2003-01-01

Chronic marginal periodontitis is an inflammatory condition in which the supporting tissues of the teeth are destroyed. Interferon (IFN)-gamma is a cytokine that plays a pivotal role in the defense against infection, and mutations in the gene coding for the ligand binding chain (alpha, RI) of the

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

Science.gov (United States)

Schoggins, John W.; MacDuff, Donna A.; Imanaka, Naoko; Gainey, Maria D.; Shrestha, Bimmi; Eitson, Jennifer L.; Mar, Katrina B.; Richardson, R. Blake; Ratushny, Alexander V.; Litvak, Vladimir; Dabelic, Rea; Manicassamy, Balaji; Aitchison, John D.; Aderem, Alan; Elliott, Richard M.; García-Sastre, Adolfo; Racaniello, Vincent; Snijder, Eric J.; Yokoyama, Wayne M.; Diamond, Michael S.; Virgin, Herbert W.; Rice, Charles M.

2014-01-01

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Selection of non-destructive assay methods: Neutron counting or calorimetric assay?

International Nuclear Information System (INIS)

Cremers, T.L.; Wachter, J.R.

1994-01-01

The transition of DOE facilities from production to D ampersand D has lead to more measurements of product, waste, scrap, and other less attractive materials. Some of these materials are difficult to analyze by either neutron counting or calorimetric assay. To determine the most efficacious analysis method, variety of materials, impure salts and hydrofluorination residues have been assayed by both calorimetric assay and neutron counting. New data will be presented together with a review of published data. The precision and accuracy of these measurements are compared to chemistry values and are reported. The contribution of the gamma ray isotopic determination measurement to the overall error of the calorimetric assay or neutron assay is examined and discussed. Other factors affecting selection of the most appropriate non-destructive assay method are listed and considered

Fundamentals of passive nondestructive assay of fissionable material: laboratory workbook

Energy Technology Data Exchange (ETDEWEB)

Reilly, T.D.; Augustson, R.H.; Parker, J.L. Walton, R.B.; Atwell, T.L.; Umbarger, C.J.; Burns, C.E.

1975-02-01

This workbook is a supplement to LA-5651-M, ''Fundamentals of Passive Nondestructive Assay of Fissionable Material'' which is the text used during the Nondestructive Assay Training Session given by Group A-1 of the Los Alamos Scientific Laboratory. It contains the writeups used during the six laboratory sessions covering basic gamma-ray principles, quantitative gamma-ray measurements, uranium enrichment measurements, equipment holdup measurements, basic neutron principles, and quantitative neutron assay.

Comparative study of gamma-ray logging and radiometric core assay of boreholes in block-I of Tummalapalle area, Cuddapah District, Andhra Pradesh using statistical methods

International Nuclear Information System (INIS)

Sreenivasa Reddy, M.; Mandal, G.; Bhaumik, B.K.

2012-01-01

A comparative study has been carried out between gamma-ray logging and radiometric core assay data of boreholes in Block-I of Tummalapalle area, South Cuddapah Basin, Andhra Pradesh using statistical methods. The product of average grade (in %eU 3 O 8 ) and thickness (in m) of uranium mineralization has been used for this purpose. The outcome of statistical analysis revealed a strong relation between gamma-ray logging and radiometric core assay data. Slope of the line is close to 1.0 and intercept is not different from 0.0. It ensures homogeneity of uranium ore distribution in the host rock. The calculated value from t-test is well within the limit and ensures the negligibility in the difference of means. This indicates that either of the data can be used for ore reserve calculation. (author)

Interferon lambda (IFN-λ) efficiently blocks norovirus transmission in a mouse model.

Science.gov (United States)

Rocha-Pereira, Joana; Jacobs, Sophie; Noppen, Sam; Verbeken, Eric; Michiels, Thomas; Neyts, Johan

2018-01-01

Human noroviruses are highly efficient in person to person transmission thus associated with explosive outbreaks of acute gastroenteritis. Outbreak control is limited to disinfection and isolation measures. Strategies to control the spread of noroviruses should be developed and models to study norovirus transmission will greatly facilitate this. Here, a mouse-to-mouse transmission model, in which mice develop acute murine norovirus (MNV)-induced diarrhea, was used to explore the role of interferon lambda (IFN-λ) in the control of a norovirus infection. Sentinel AG129 mice [deficient in IFN-α/β and IFN-γ receptors] that were co-housed with MNV-infected mice shedding high amounts of virus in their stool, developed a MNV-infection with associated diarrhea. Inoculation of such sentinel mice with an IFN-λ expression plasmid resulted in the production of circulating IFN-λ and upregulation of the expression of IFN-stimulated genes (ISGs) of the gut. Injection of the IFN-λ-expressing plasmid to sentinels prevents MNV-induced disease upon exposure to MNV-infected mice, as well as MNV replication in the small intestine, the associated signs of inflammation and the mounting of a specific IgG-based immune response. This demonstrates that IFN-λ can alone mediate protection against transmission of norovirus. The development of a simple delivery method for IFN-λ could be explored as a strategy to control norovirus outbreaks and protect vulnerable populations such as the elderly and immunocompromised. Copyright © 2017. Published by Elsevier B.V.

An interferon-gamma release assay test performs well in routine screening for tuberculosis

DEFF Research Database (Denmark)

Vestergaard Danielsen, Allan; Fløe, Andreas; Lillebæk, Troels

2014-01-01

Introduction: A positive interferon-gamma release assay (IGRA) is regarded as proof of latent Mycobacterium tuberculosis infection. We conducted an evaluation of the IGRA test “T-SPOT.TB” to test its performance during clinical routine use by analysing the positivity rate and odds, effect of season...... and sensitivity. Material and methods: Data from T-SPOT.TB testing together with age and test indications (anti-tumour necrosis factor alpha (TNFα) candidate, contact investigation or suspicion of tuberculosis (TB)) were combined with mycobac­teria culture results. Results: A total of 1,809 patients were tested....... Conclusive results were achieved for 1,780 patients (98.4%). Among these, 4.6% of anti-TNFα candidates, 19.3% of contacts and 24.4% of TB suspects tested positive. Compared with anti-TNFα candidates, the odds for a positive result were significantly higher for contact investigations (odds ratio (OR), mean...

IFN-beta inhibits T cell activation capacity of central nervous system APCs

DEFF Research Database (Denmark)

Teige, Ingrid; Liu, Yawei; Issazadeh-Navikas, Shohreh

2006-01-01

We have previously investigated the physiological effects of IFN-beta on chronic CNS inflammation and shown that IFN-beta(-/-) mice develop a more severe experimental autoimmune encephalomyelitis than their IFN-beta(+/-) littermates. This result was shown to be associated with a higher activation...... state of the glial cells and a higher T cell cytokine production in the CNS. Because this state suggested a down-regulatory effect of IFN-beta on CNS-specific APCs, these results were investigated further. We report that IFN-beta pretreatment of astrocytes and microglia (glial cells) indeed down......-modulate their capacity to activate autoreactive Th1 cells. First, we investigated the intrinsic ability of glial cells as APCs and report that glial cells prevent autoreactive Th1 cells expansion while maintaining Ag-specific T cell effector functions. However, when the glial cells are treated with IFN-beta before...

Capability for identification of gamma-irradiated bovine liver by new high sensitivity comet assay

International Nuclear Information System (INIS)

Miyahara, Makoto; Toyoda, Masatake; Saito, Akiko; Ito, Hitoshi

2000-01-01

DNA in food will sustain damage by gamma radiation. The detection capability of the high sensitivity comet assay was studied using fluorescence-microscopy. Beef liver was irradiated at a range of 1 Gy to 8 kGy. Single cells were obtained from the irradiated liver, then analyzed by agaros-gel electrophoresis. The pH of the buffer for electrophoresis was pH 13, which is generally utilized for sensitive detection of DNA damage. The pattern formed by DNA was visualized by staining with ethidium bromide. The resulting comets were evaluated with a scale we developed, and Influence Scores were calculated based on the Tice method. It is possible to detect irradiation damage to beef liver at 10 Gy. Together with Influence Score, histogram of comet type is used for detection of irradiation. We elucidated those histograms were useful for distinguishing damage caused by irradiation from that of others. DNA damage can be caused not only by irradiation, but also by the other treatments. Therefore, the respective influences of freezing, preservation, irradiating temperature, atmosphere of irradiation, cooking, and homogenizing devices were also examined. This new comet assay will be a useful method of detecting DNA damage to identify irradiated foods. (author)

Interferon-gamma and interleukin-10 profile of children with tuberculosis in North Sumatera, Indonesia

Science.gov (United States)

Daulay, R. S.; Daulay, R. M.

2018-03-01

Cellular immunity was mediated the host immune response against Mycobacterium tuberculosis, in which cytokine and T-helper (Th) 1 cells play an important role. Interferon-gamma (IFN-γ) is a leading cytokine involved in the immune response of tuberculosis (TB).The primary function of IFN-γ is to activate macrophages in opposition Mycobacterium tuberculosis. Contrast from IFN-γ, interleukin-10 (IL-10) is considered inhibitory cytokine, important to an adequate balance between inflammatory responses. To analyze cytokine profile, particularly IFN-γ and IL-10 of the children with TB in Indonesia, a cross-sectional study was conducted at two general hospitals and seven primary health care located in Medan and Batubara, North Sumatera, Indonesia. Among 51 children with TB disease and 51 healthy children, found that IFN-γ and IL-10 levels were lower in TB patients compared to healthy children. Statistically significant decreased production of the IFN-γ levels (p=0.042) were found in TB patients 9.41 (1.10-28.06) pg/ml contrast to healthy children 6.30 (1.30-89.76) pg/ml. Homologue finding of the IL-10 levels were also found in TB patients 4.93 (0.22-48.01) pg/ml and 4.93 (0.07-81.60) pg/ml in healthy children, but not statistically significant (p=0.784). High levels of IL-10 were not proven to suppress the levels production of IFN-γ in TB patients.

Direct Assay of Filter Media following DEOX Testing

International Nuclear Information System (INIS)

R.P. Lind; J.J. Giglio; D.G. Cummings; M.W. Huntley; C.D. Morgan; K.J. Bateman; D.L. Wahlquist; D.A. Sell

2007-01-01

The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs-137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media

Estimated Uncertainty in Segmented Gamma Scanner Assay Results due to the Variation in Drum Tare Weights

International Nuclear Information System (INIS)

Bosko, A.; Croft, St.; Gulbransen, E.

2009-01-01

General purpose gamma scanners are often used to assay unknown drums that differ from those used to create the default calibration. This introduces a potential source of bias into the matrix correction when the correction is based on the estimation of the mean density of the drum contents from a weigh scale measurement. In this paper we evaluate the magnitude of this bias that may be introduced by performing assay measurements with a system whose matrix correction algorithm was calibrated with a set of standard drums but applied to a population of drums whose tare weight may be different. The matrix correction factors are perturbed in such cases because the unknown difference in tare weight gets reflected as a bias in the derived matrix density. This would be the only impact if the difference in tare weight was due solely to the weight of the lid or base, say. But in reality the reason for the difference may be because the steel wall of the drum is of a different thickness. Thus, there is an opposing interplay at work which tends to compensate. The purpose of this work is to evaluate and bound the magnitude of the resulting assay uncertainty introduced by tare weight variation. We compare the results obtained using simple analytical models and the 3-D ray tracing with ISOCS software to illustrate and quantify the problem. The numerical results allow a contribution to the Total Measurement Uncertainty (TMU) to be propagated into the final assay result. (authors)

A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide

DEFF Research Database (Denmark)

Barington, T; Sparholt, S; Juul, L

1992-01-01

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...... antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality...... and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection...

Association of TNF-alpha (-308 A/G) and IFN-gamma (+874 A/T) gene polymorphisms in response to spontaneous and treatment induced viral clearance in HCV infected multitransfused thalassemic patients.

Science.gov (United States)

Biswas, Aritra; Gupta, Nabyendu; Gupta, Debanjali; Datta, Abira; Firdaus, Rushna; Chowdhury, Prosanto; Bhattacharyya, Maitreyee; Sadhukhan, Provash C

2018-06-01

Multitransfused thalassemic individuals are at high risk of developing transfusion transmitted Hepatitis C virus (HCV) infection. The aim of the study was to correlate the effects of host cytokine single nucleotide polymorphisms of TNF-α (-308 A/G) and IFN-γ (+874 A/T) in spontaneous or IFN induced treatment response in the HCV infected thalassemic individuals. A total of 427 HCV sero-reactive thalassemic individuals were processed for HCV viral genomic diversity and host gene polymorphisms analysis of TNF-α (-308 A/G) and IFN-γ (+874 A/T). Out of 427 HCV sero-reactive individuals, 69.09% were found to be HCV RNA positive with genotype 3 as the predominant infecting strain (94.29%). Study highlighted that, A allele was significantly associated with (p < .05) spontaneous clearance of HCV infection and G allele was correlated with viral persistence at TNF-α (-308) gene polymorphism. Whereas in case of IFN-γ (+874) SNPs, A allele was significantly responsible (p < .05) for spontaneous clearance than T allele. Our study also indicated that in relapsed cases, IFN-γ (+874) T allele is more responsible than A allele. Though no significant correlation was found at both TNF-α (-308) and IFN-γ (+874) gene polymorphism among SVR and relapsed thalassemic patients. A allele at both TNF-α (-308) and IFN-γ (+874) were strongly associated with spontaneous clearance among this population. But in case of SVR and relapsed cases no significant association was found. This cytokine gene polymorphisms pattern will help clinicians to take an informed decision about therapeutic management of HCV infected thalassemic individuals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radioactive waste package assay facility. Volume 2. Investigation of active neutron and active gamma interrogation

International Nuclear Information System (INIS)

Bailey, M.; Bunce, L.J.; Findlay, D.J.S.; Jolly, J.E.; Parsons, T.V.; Sene, M.R.; Swinhoe, M.T.

1992-01-01

Volume 2 of this report describes the theoretical and experimental work carried out at Harwell on active neutron and active gamma interrogation of 500 litre cemented intermediate level waste drums. The design of a suitable neutron generating target in conjunction with a LINAC was established. Following theoretical predictions of likely neutron responses, an experimental assay assembly was built. Responses were measured for simulated drums of ILW, based on CAGR, Magnox and PCM wastes. Good correlations were established between quantities of 235 -U, nat -U and D 2 O contained in the drums, and the neutron signals. Expected sensitivities are -1g of fissile actinide and -100g of total actinide. A measure of spatial distribution is obtainable. The neutron time spectra obtained during neutron interrogation were more complex than expected, and more analysis is needed. Another area of discrepancy is the difference between predicted and measured thermal neutron flux in the drum. Clusters of small 3 He proportional counters were found to be much superior for fast neutron detection than larger diameter counters. It is necessary to ensure constancy of electron beam position relative to target(s) and drum, and prudent to measure the target neutron or gamma output as appropriate. 59 refs., 77 figs., 11 tabs

A randomized, double-blind, phase I/II trial of tumor necrosis factor and interferon-gamma for treatment of AIDS-related complex (Protocol 025 from the AIDS Clinical Trials Group).

Science.gov (United States)

Agosti, J M; Coombs, R W; Collier, A C; Paradise, M A; Benedetti, J K; Jaffe, H S; Corey, L

1992-05-01

To determine safety and efficacy of tumor necrosis factor (TNF) and interferon-gamma (IFN gamma) in the treatment of patients with acquired immunodeficiency syndrome (AIDS)-related complex, a randomized, double-blind study was conducted. Twenty-five patients with AIDS-related complex and CD4 lymphocytes less than or equal to 500 x 10(6)/L attended an AIDS Clinical Trials Unit of a tertiary referral center. Patients were administered tumor necrosis factor (TNF) (10 micrograms/m2) or IFN gamma (10 micrograms/m2), or both intramuscularly three times weekly for 16 weeks. Side effects from all three preparations included fever, constitutional symptoms, and local reactions. No significant hematologic, hepatic, renal, or coagulation abnormalities were observed. CD4 lymphocyte counts, beta 2-microglobulin, p24 antigen levels, and anti-p24 antibody did not change significantly during therapy. Similarly, no significant change was noted in rates of HIV isolation from peripheral blood mononuclear cells or plasma. TNF and IFN gamma were tolerable after premedication with acetaminophen; however, no significant change in markers of human immunodeficiency virus infection was demonstrated. These cytokines alone do not appear to be of benefit, nor do they appear to hasten the progression of HIV infection.

Deficiency of the AIM2-ASC Signal Uncovers the STING-Driven Overreactive Response of Type I IFN and Reciprocal Depression of Protective IFN-γ Immunity in Mycobacterial Infection.

Science.gov (United States)

Yan, Shanshan; Shen, Hongbo; Lian, Qiaoshi; Jin, Wenlong; Zhang, Ronghua; Lin, Xuan; Gu, Wangpeng; Sun, Xiaoyu; Meng, Guangxun; Tian, Zhigang; Chen, Zheng W; Sun, Bing

2018-02-01

The nucleic acids of Mycobacterium tuberculosis can be detected by intracellular DNA sensors, such as cyclic GMP-AMP synthase and absent in melanoma 2 (AIM2), which results in the release of type I IFN and the proinflammatory cytokine IL-1β. However, whether cross-talk occurs between AIM2-IL-1β and cyclic GMP-AMP synthase-type I IFN signaling upon M. tuberculosis infection in vivo is unclear. In this article, we demonstrate that mycobacterial infection of AIM2 -/- mice reciprocally induces overreactive IFN-β and depressive IFN-γ responses, leading to higher infection burdens and more severe pathology. We also describe the underlying mechanism whereby activated apoptosis-associated speck-like protein interacts with a key adaptor, known as stimulator of IFN genes (STING), and inhibits the interaction between STING and downstream TANK-binding kinase 1 in bone marrow-derived macrophages and bone marrow-derived dendritic cells, consequently reducing the induction of type I IFN. Of note, apoptosis-associated speck-like protein expression is inversely correlated with IFN-β levels in PBMCs from tuberculosis patients. These data demonstrate that the AIM2-IL-1β signaling pathway negatively regulates the STING-type I IFN signaling pathway by impeding the association between STING and TANK-binding kinase 1, which protects the host from M. tuberculosis infection. This finding has potential clinical significance. Copyright © 2018 by The American Association of Immunologists, Inc.

Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

Directory of Open Access Journals (Sweden)

Tobias Manigold

2010-02-01

Full Text Available In man, infection with South American Andes virus (ANDV causes hantavirus cardiopulmonary syndrome (HCPS. HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+CD8(+ T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473 years after the acute infection. Remarkably, Gn(465-473-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+CD27(-CD28(-CCR7(-CD127(- effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T

Stat1-independent regulation of gene expression in response to IFN-γ

Science.gov (United States)

Ramana, Chilakamarti V.; Gil, M. Pilar; Han, Yulong; Ransohoff, Richard M.; Schreiber, Robert D.; Stark, George R.

2001-01-01

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling. PMID:11390994

Gamma-irradiated influenza A virus provides adjuvant activity to a co-administered poorly immunogenic SFV vaccine in mice.

Directory of Open Access Journals (Sweden)

Rachelle eBabb

2014-06-01

Full Text Available Many currently available inactivated vaccines require 'adjuvants' to maximise the protective immune responses generated against the antigens of interest. Recent studies in mice with gamma-irradiated influenza A virus (γ-FLU have shown its superior efficacy compared to other forms of inactivated FLU vaccines and its ability to induce both potent type-I interferon (IFN-I responses and the IFN-I associated partial lymphocyte activation. Commonly, IFN-I responses induced by adjuvants, combined in vaccine preparations, have been shown to effectively enhance the immunogenicity of the antigens of interest. Therefore, we investigated the potential adjuvant activity of γ-FLU and the possible effect on antibody responses against co-administrated antigens, using gamma-irradiated Semliki Forest Virus (γ-SFV as the experimental vaccine in mice. Our data show that co-vaccination with γ-FLU and γ-SFV resulted in enhanced SFV-specific antibody responses in terms of increased titres by 6 fold and greater neutralisation efficacy, when compared to vaccination with γ-SFV alone. This study provides promising evidence related to the possible use of γ-FLU as an adjuvant to poorly immunogenic vaccines without compromising the vaccine efficacy of γ-FLU.

Evaluation of Granulysin and Perforin as Candidate Biomarkers for Protection Following Vaccination with Mycobacterium bovis BCG or M. bovisDeltaRD1

Science.gov (United States)

Tuberculosis remains a major health problem worldwide. Cell mediated immunity based on a Th1 response plays an important role in the outcome of the disease. Measurements of immune responsiveness after TB vaccination include the standard skin test, IFN gamma-based diagnostic assays and T cell prolif...

Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

Direct assay of filter media following DEOX testing

International Nuclear Information System (INIS)

Westphal, B.R.; Lind, R.P.; Giglio, J.J.; Cummings, D.G.; Huntley, M.W.; Morgan, C.D.; Bateman, K.J.; Wahlquist, D.L.; Sell, D.A.

2007-01-01

The direct assay of filter media by gamma spectrometry following DEOX testing has distinct advantages over analytical chemistry. Prior to using gamma spectrometry for the quantification of cesium (Cs- 137), a calibration must be established with known sources since gamma spectrometry yields relative results. Quantitative analytical chemistry, in particular ICP-MS, has been performed on the filter media for comparison to the gamma spectrometry data. The correlation of gamma spectrometry to ICP-MS data is presented to justify the continued use of gamma spectrometry for filter media. (authors)

Interferon-gamma and tumor necrosis factor-alpha sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

DEFF Research Database (Denmark)

Fluhr, Herbert; Krenzer, Stefanie; Stein, Gerburg M

2007-01-01

The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas......-mediated signaling during early implantation. Here we show that ESCs are primarily resistant to Fas-mediated apoptosis independently of their state of hormonal differentiation. Pre-treatment of ESCs with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha sensitizes them to become apoptotic upon stimulation...... of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-gamma and TNF-alpha was accompanied by a significant upregulation of Fas and FLICE...

We Can Still Be Friends: IFN-γ Breaks Up Macrophage Enhancers.

Science.gov (United States)

Novakovic, Boris; Wang, Cheng; Logie, Colin

2017-08-15

Interferon (IFN)-γ can prime macrophages for inflammatory responses by several mechanisms, including enhancer establishment and gene activation. In this issue of Immunity, Kang et al. (2017) provide insight into the mechanisms of IFN-γ-mediated gene repression as they show that IFN-γ promotes the disassembly of select active enhancers by interfering with enhancer-binding transcription factor MAF. Copyright © 2017 Elsevier Inc. All rights reserved.

Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells

Directory of Open Access Journals (Sweden)

Xin Liu

2017-11-01

Full Text Available For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α, the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

Biodistribution analysis of 125I-albumin-IFN-alpha2b fusion protein in rats

International Nuclear Information System (INIS)

Zhou Yaoyuan; Zhang Rongjun; Cai Gangming; Gu Xiaobo; Jiang Mengjun; Zhang Bo; Yang Min; Cao Guoxian; Yang Jianliang

2009-01-01

125 I-albumin-IFN-alpha2b was prepared with the methods of Ch-T and purified with PD-10 column. The radiochemical purity was measured with TCA (trichloroacetic acid) precipitation. The antiviral activities of 125 I-albumin-IFN-alpha2b and albumin-IFN-alpha2b were compared with WISH/VSV system in vitro. SD rats were injected with 125 I-albumin-IFN-alpha2b subcutaneously and sacrificed at 0.5, 2, 6, 24, 48, 90, 180 and 300 h post-injection. Selected organs were dissected, weighed and their radioactivity was measured using γ-counter. The accumulated radioactivity in the tissues was calculated in terms of percentage of injected dose per gram organ (%ID·g -1 ). The labeling yield was 82.72%. The radiochemical purity of 125 I-albumin-IFN-alpha2b was 95.53%, and its radioactivity was 0.26 MBq/μg. The antiviral bioactivities of albumin-IFN-alpha2b and 125 I-albumin- IFN-alpha2b did not change. Biodistribution analysis of 125 I-albumin-IFN-alpha2b in rats showed that concentrated 125 I-albumin-IFN-alpha2b in blood reached maximum at 6 h post injection, and eliminated slowly. No specific accumulation was seen in other tissues. 125 I-albumin-IFN-alpha2b could maintain in peripheral blood for a long time and it meant albumin-IFN-alpha2b would be an effective long-term interferon. (authors)

Benefit of hepatitis C virus core antigen assay in prediction of therapeutic response to interferon and ribavirin combination therapy.

Science.gov (United States)

Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

2005-01-01

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay.

Development of an integrated assay facility

International Nuclear Information System (INIS)

Molesworth, T.V.; Bailey, M.; Findlay, D.J.S.; Parsons, T.V.; Sene, M.R.; Swinhoe, M.T.

1990-01-01

The I.R.I.S. concept proposed the use of passive examination and active interrogation techniques in an integrated assay facility. A linac would generate the interrogating gamma and neutron beams. Insufficiently detailed knowledge about active neutron and gamma interrogation of 500 litre drums of cement immobilised intermediate level waste led to a research programme which is now in its main experimental stage. Measurements of interrogation responses are being made using simulated waste drums containing actinide samples and calibration sources, in an experimental assay assembly. Results show that responses are generally consistent with theory, but that improvements are needed in some areas. A preliminary appraisal of the engineering and economic aspects of integrated assay shows that correct operational sequencing is required to achieve the short cycle time needed for high throughput. The main engineering features of a facility have been identified

The self-absorption effect of gamma rays in 239Pu

International Nuclear Information System (INIS)

Hsiaohua Hsu

1989-01-01

Nuclear materials assay with gamma-ray spectrum measurement is a well-established method for safeguards. However, for a thick source, the self-absorption of characteristic low-energy gamma rays has been a handicap to accurate assay. The author has carried out Monte Carlo simulations to study this effect using the 239 Pu α-decay gamma-ray spectrum as an example. The thickness of a plutonium metal source can be considered a function of gamma-ray intensity ratios. In a practical application, gamma-ray intensity ratios can be obtained from a measured spectrum. With the help of calculated curves, scientists can find the source thickness and make corrections to gamma-ray intensities, which then lead to an accurate quantitative determination of radioactive isotopes in the material

Is TB Testing Associated With Increased Blood Interferon-Gamma Levels?

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Aideen E. Kennedy

2017-10-01

Full Text Available The Republic of Ireland reports a relatively low prevalence of Johne’s disease (JD compared to international counterparts. Postulated reasons for this include a lower average herd size and a grass-based production system. Ireland also engages in high levels of bovine tuberculosis (bTB testing. As interferon-gamma (IFN-γ is believed to play a key role in protecting against JD, it is our hypothesis that administration of purified protein derivative (PPD, as part of the bTB test, is associated with a systemic increase in IFN-γ production, which may potentially limit clinical progression of the disease. We studied 265 cows (202 Friesian and 63 “Non-Friesian,” e.g., JerseyX, Norwegian Red to assess IFN-γ levels and Mycobacterium avium subspecies paratuberculosis (MAP antibody response before and after the bTB test. As part of the compulsory annual bTB test, avian and bovine PPD were administered at two separate cervical sites. To assess IFN-γ production, blood samples were taken before and 72 h after PPD administration. MAP antibody response was assessed before and 10 days post-PPD administration. A significant increase in MAP antibody response was identified post-bTB compared to pre-bTB response (p < 0.001. Additionally, IFN-γ production significantly increased at the post-bTB time point (p < 0.001 compared to the pre-bTB test readings. This may indicate a beneficial effect of bTB testing in controlling JD.

Type I and type II interferons upregulate functional type I interleukin-1 receptor in a human fibroblast cell line TIG-1.

Science.gov (United States)

Takii, T; Niki, N; Yang, D; Kimura, H; Ito, A; Hayashi, H; Onozaki, K

1995-12-01

The regulation of type I interleukin-1 receptor (IL-1R) expression by type I, interferon (IFN)-alpha A/D, and type II IFN, IFN-gamma, in a human fibroblast cell line TIG-1 was investigated. After 2 h stimulation with human IFN-alpha A/D or IFN-gamma, the levels of type I IL-1R mRNA increased. We previously reported that IL-1 upregulates transcription and cell surface molecules of type I IL-1R in TIG-1 cells through induction of prostaglandin (PG) E2 and cAMP accumulation. However, indomethacin was unable to inhibit the effect of IFNs, indicating that IFNs augment IL-1R expression through a pathway distinct from that of IL-1. The augmentation was also observed in other fibroblast cell lines. Nuclear run-on assays and studies of the stability of mRNA suggested that the increase in IL-1R mRNA was a result of the enhanced transcription of IL-1R gene. Binding studies using 125I-IL-1 alpha revealed that the number of cell surface IL-1R increased with no change in binding affinity by treatment with these IFNs. Pretreatment of the cells with IFNs enhanced IL-1-induced IL-6 production, indicating that IFNs upregulate functional IL-1R. IL-1 and IFNs are produced by the same cell types, as well as by the adjacent different cell types, and are concomitantly present in lesions of immune and inflammatory reactions. These results therefore suggest that IFNs exhibit synergistic effects with IL-1 through upregulation of IL-1R. Augmented production of IL-6 may also contribute to the reactions.

Potential predictive assay using RAR-β for radiosensitization by 13-cis-retinoic acid and interferonα2a in human carcinoma cells

International Nuclear Information System (INIS)

Ryu, Samuel; Nevaldine, Barbara H.; Unguraneau, Carmen; Chung, Chung T.; King, Gerald A.; Stein, Joseph P.

1997-01-01

Purpose: Cell culture studies have shown increased cytotoxicity and inhibition of cell proliferation by combined use of 13-cis-retinoic acid (CRA) and interferon-α2a (IFN). Clinically, a direct antitumor activity has been observed by combined use of CRA and IFN in patients with cancer of the cervix and skin. Since IFN is known to enhance radiation response in selected human carcinoma cell lines, we carried out a series of experiments in an effort to improve the efficacy of radiation therapy by CRA and IFN in combination. Materials and Methods: Human cervical carcinoma ME-180 and HeLa cell lines were exposed to 10 μM CRA and 1000 unit/ml IFN in combination for 48 hours prior to radiation. Endpoint of radiosensitization study was colony-forming ability of single cells. Retinoic acid receptors (RAR and RXR) were detected by RNAse protection assay. Apoptosis was quantitated by using immunohistochemical staining method (Apop Tag). The cells were also transfected with bcl-2 or RAR-β gene to explore the mechanism of radiation-induced cell killing. Results: A substantial radiosensitization was observed by the combined use of CRA and IFN in human cervical carcinoma ME-180 cells in culture. The radiation enhancement ratio was 2.0 at 1% cell survival level. The principal mode of radiation-induced cell killing was apoptosis since more than 90% of the ME-180 cells showed evidence of apoptosis by combined treatment of CRA, IFN, and radiation. Both apoptosis and radiosensitization were blocked by transfecting bcl-2 gene into ME-180 cells. In contrast to these results with ME-180 cells, no radiosensitization was observed in HeLa cells by CRA and IFN under the same experimental conditions. Both cell lines express various RXR and RAR mRNAs. However, the RAR-β was undetectable in HeLa cells but present at high levels in ME-180 cells, as determined by RNAse protection assay. Because of this differential expression of RAR-β mRNA, we hypothesized that RAR-β may mediate the

Wild type measles virus attenuation independent of type I IFN

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Horvat Branka

2008-02-01

Full Text Available Abstract Background Measles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt. Results The adaptation of a measles virus isolate (G954-PBL by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13 differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene. While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain. Conclusion Thus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system.

Assay of uranium in fused salt cake generated at the natural uranium metal fuel fabrication plants by gamma-ray spectrometry

International Nuclear Information System (INIS)

Kalsi, P.C.; Bhanu, A.U.; Sahoo, S.; Iyer, R.H.

1986-01-01

A passive gamma-ray spectroscopic method is employed for the assay of uranium in fused salt cake, a scrap produced at the natural uranium metal fuel fabrication plants. The method makes use of NaI(TI) detector coupled with a multichannel analyser. The 1 MeV gamma-ray of 238 U was used for the calibration. The calibration curve was made by counting synthetic mixtures made of U 3 O 8 powder, the heat treatment salt and iron in the form of fine powder. The uranium content in these synthetic mixtures was kept in the range of 1-11 per cent. 23 lots of the fused salt cake taken from three different batches of the salt cake were then analysed by this method. The uranium content of fused salt cake was found to be in the range of 1.70-11.43 per cent. To compare the gamma spectrometric results with a completely independent method, chemical analysis of all the fused salt cakes were also carried out. The NDA results were found to agree within ± 17 per cent with the chemical analysis results. (author)

DNA vaccine delivered by a needle-free injection device improves potency of priming for antibody and CD8+ T-cell responses after rAd5 boost in a randomized clinical trial.

Directory of Open Access Journals (Sweden)

Barney S Graham

Full Text Available DNA vaccine immunogenicity has been limited by inefficient delivery. Needle-free delivery of DNA using a CO2-powered Biojector® device was compared to delivery by needle and syringe and evaluated for safety and immunogenicity.Forty adults, 18-50 years, were randomly assigned to intramuscular (IM vaccinations with DNA vaccine, VRC-HIVDNA016-00-VP, (weeks 0, 4, 8 by Biojector® 2000™ or needle and syringe (N/S and boosted IM at week 24 with VRC-HIVADV014-00-VP (rAd5 with N/S at 10(10 or 10(11 particle units (PU. Equal numbers per assigned schedule had low (≤500 or high (>500 reciprocal titers of preexisting Ad5 neutralizing antibody.120 DNA and 39 rAd5 injections were given; 36 subjects completed follow-up research sample collections. IFN-γ ELISpot response rates were 17/19 (89% for Biojector® and 13/17 (76% for N/S delivery at Week 28 (4 weeks post rAd5 boost. The magnitude of ELISpot response was about 3-fold higher in Biojector® compared to N/S groups. Similar effects on response rates and magnitude were observed for CD8+, but not CD4+ T-cell responses by ICS. Env-specific antibody responses were about 10-fold higher in Biojector-primed subjects.DNA vaccination by Biojector® was well-tolerated and compared to needle injection, primed for greater IFN-γ ELISpot, CD8+ T-cell, and antibody responses after rAd5 boosting.ClinicalTrials.gov NCT00109629.

A genetically engineered live-attenuated simian-human immunodeficiency virus that co-expresses the RANTES gene improves the magnitude of cellular immunity in rhesus macaques

International Nuclear Information System (INIS)

Shimizu, Yuya; Inaba, Katsuhisa; Kaneyasu, Kentaro; Ibuki, Kentaro; Himeno, Ai; Okoba, Masashi; Goto, Yoshitaka; Hayami, Masanori; Miura, Tomoyuki; Haga, Takeshi

2007-01-01

Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4 + Th cell-proliferative response and by inducing an antigen-specific IFN-γ ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4 + Th responses and IFN-γ ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4 + T cell responses

Effect of alpha-interferon alone and combined with other antineoplastic agents on renal cell carcinoma determined by the tetrazolium microculture assay.

Science.gov (United States)

Homma, Y; Aso, Y

1994-01-01

The antiproliferative effect of various alpha-interferons (alpha-IFNs), alone or combined with other agents, on a renal cell carcinoma cell line was evaluated by the tetrazolium microculture assay to examine the rationale for combination therapies. Cells incubated in 96-week microculture plates at 5 x 10(3)/well were exposed to various agents for 3 days. There were no obvious differences in the growth inhibition caused by the 5 kinds of alpha-IFN examined as single agents. The combination of alpha-IFN with the following agents was also assessed: 5-fluorouracil (5FU), methotrexate (MTX), mitomycin C, bleomycin, cis-diaminedichloroplatinum (CDDP), vinblastine, etoposide (ETOP), alpha-IFN, tumor necrosis factor-alpha (TNF), and alpha-difluoromethylornithine. Synergism was observed for the combination of alpha-IFN+TNF, while the other combinations had additive or subadditive effects. No interference or antagonism was found. Trimodal combinations of alpha-IFN+MTX with either 5FU, ETOP, or CDDP all showed subadditive effects. These results indicated that an increased antiproliferative effect, although not necessarily synergistic, was obtained by the combination of alpha-IFN with a variety of antineoplastic agents, providing a rationale to seek for combination therapies including alpha-IFN for treating renal cell carcinoma.

Effects of interferon gamma and specific polyclonal antibody on the infection of murine peritoneal macrophages and murine macrophage cell line PMJ2-R with Encephalitozoon cuniculi

Czech Academy of Sciences Publication Activity Database

Jelínek, Jiří; Salát, Jiří; Sak, Bohumil; Kopecký, Jan

2007-01-01

Roč. 54, č. 3 (2007), s. 172-176 ISSN 0015-5683 R&D Projects: GA ČR GP524/03/D167 Institutional research plan: CEZ:AV0Z60220518 Keywords : microsporidia * Encephalitozoon cuniculi * antibody * macrophage s * interferon gamma (IFN-gamma) Subject RIV: EC - Immunology Impact factor: 1.000, year: 2007

Development of a simplified and convenient assay for cell-mediated immunity to the mumps virus.

Science.gov (United States)

Otani, Naruhito; Shima, Masayuki; Nakajima, Kazuhiko; Takesue, Yoshio; Okuno, Toshiomi

2014-09-01

Because methods for measuring cell-mediated immunity (CMI) to the mumps virus are expensive, time-consuming, and technically demanding, the role of CMI in mumps virus infection remains unclear. To address this issue, we report here the development of a simplified method for measuring mumps virus-specific CMI that is suitable for use in diverse laboratory and clinical settings. A mumps vaccine was cultured with whole blood, and interferon (IFN)-γ released into the culture supernatant was measured using an enzyme-linked immunosorbent assay. IFN-γ production in blood from vaccinated subjects markedly increased in response to the vaccine and decreased before the antibody titer decreased in some cases, suggesting that this assay may be used as a simple surrogate method for measuring CMI specific for the mumps virus. Copyright © 2014 Elsevier B.V. All rights reserved.

Circadian variations of interferon-induced enhancement of human natural killer (NK) cell activity.

Science.gov (United States)

Gatti, G; Cavallo, R; Sartori, M L; Carignola, R; Masera, R; Delponte, D; Salvadori, A; Angeli, A

1988-01-01

We searched for circadian changes in the enhancement of the NK activity after exposure to IFN-gamma of peripheral blood mononuclear (PBM) cells obtained serially throughout the 24-h cycle. In August-October 1986, blood was drawn from 7 healthy, diurnally active and nocturnally resting male volunteers (22-34 yr) at 4-h intervals for 24 h starting at 08:00. PBM cells were immediately separated and assayed for NK cell activity, using K 562 cultured cells as a target in a 4-h 51Cr release assay after prior incubation for 20 h with buffer or 300 IU rIFN-gamma. Circadian variations of the spontaneous NK cell cytotoxicity were apparent; the activity was at its maximum at the end of the night or in the early morning and then declined in the afternoon. The 24-h rhythmic pattern was validated with statistical significance by the Cosinor method (p less than 0.02; acrophase 04:22). Maximum enhancement by IFN-gamma was attained in the second part of the night or in the early morning, i.e. in phase with the peak of the spontaneous NK cell activity. A significant circadian rhythm of the percent increase above control levels was validated by the Cosinor method (p less than 0.01; acrophase 04:03). Our findings may be of relevance to a better understanding of the mechanisms of control of human NK activity and warrant consideration as an approach to improve the effectiveness of time-qualified immunotherapy.

Potentiating day-old blood samples for detection of interferon-gamma responses following infection with Mycobacterium avium subsp. paratuberculosis

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Nielsen, Søren Saxmose; Jungersen, Gregers

time interval from blood sampling to culture. The objective of the study was to assess options for use of day-old blood samples for early-stage diagnosis of MAP infections. Bovine interleukin 12 (IL-12) can induce, and IL-10 reduce, IFN-γ production. Therefore, addition of IL-12 and anti-IL-10 could...... result in production of IFN-γ in samples previously exposed to MAP antigens. Whole blood samples were collected from heifers in a Danish dairy herd known to be infected with MAP. The samples were collected on three sample dates, and on each date the blood samples were stimulated with PPDj and recombinant......The interferon gamma (IFN-γ) test measuring specific cell-mediated immune responses in whole blood can be used for diagnosis at an early stage of Mycobacterium avium subsp. paratuberculosis (MAP) infection. A major obstacle for the practical use of IFN-γ testing is the recommended maximum 8 hour...

Identification of Two Subgroups of Type I IFNs in Perciforme Fish Large Yellow Croaker Larimichthys crocea Provides Novel Insights into Function and Regulation of Fish Type I IFNs

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Yang Ding

2016-09-01

Full Text Available Like mammals, fish possess an interferon regulatory factor 3 (IRF3/IRF7-dependent type I IFN responses, but the exact mechanism by which IRF3/IRF7 regulate the type I IFNs remains largely unknown. In this study, we identified two type I IFNs in the Perciforme fish large yellow croaker Larimichthys crocea, one of which belongs to the fish IFNd subgroup, and the other is assigned to a novel subgroup of group I IFNs in fish, tentatively termed IFNh. The two IFN genes are constitutively expressed in all examined tissues, but with varied expression levels. Both IFN genes can be rapidly induced in head kidney and spleen tissues by polyinosinic-polycytidylic acid. The recombinant IFNh was shown to be more potent to trigger a rapid induction of the antiviral genes MxA and PKR than the IFNd, suggesting that they may play distinct roles in regulating early antiviral immunity. Strikingly, IFNd, but not IFNh, could induce the gene expression of itself and IFNh through a positive feedback loop mediated by the IFNd-dependent activation of IRF3 and IRF7. Furthermore, our data demonstrate that the induction of IFNd can be enhanced by the dimeric formation of IRF3 and IRF7, while the IFNh expression mainly involves IRF3. Taken together, our findings demonstrate that the IFN responses are diverse in fish and are likely to be regulated by distinct mechanisms.

Control of HIV infection by IFN-α: implications for latency and a cure.

Science.gov (United States)

Bourke, Nollaig M; Napoletano, Silvia; Bannan, Ciaran; Ahmed, Suaad; Bergin, Colm; McKnight, Áine; Stevenson, Nigel J

2018-03-01

Viral infections, including HIV, trigger the production of type I interferons (IFNs), which in turn, activate a signalling cascade that ultimately culminates with the expression of anti-viral proteins. Mounting evidence suggests that type I IFNs, in particular IFN-α, play a pivotal role in limiting acute HIV infection. Highly active anti-retroviral treatment reduces viral load and increases life expectancy in HIV positive patients; however, it fails to fully eliminate latent HIV reservoirs. To revisit HIV as a curable disease, this article reviews a body of literature that highlights type I IFNs as mediators in the control of HIV infection, with particular focus on the anti-HIV restriction factors induced and/or activated by IFN-α. In addition, we discuss the relevance of type I IFN treatment in the context of HIV latency reversal, novel therapeutic intervention strategies and the potential for full HIV clearance.

Nondestructive assay methodologies in nuclear forensics analysis

International Nuclear Information System (INIS)

Tomar, B.S.

2016-01-01

In the present chapter, the nondestructive assay (NDA) methodologies used for analysis of nuclear materials as a part of nuclear forensic investigation have been described. These NDA methodologies are based on (i) measurement of passive gamma and neutrons emitted by the radioisotopes present in the nuclear materials, (ii) measurement of gamma rays and neutrons emitted after the active interrogation of the nuclear materials with a source of X-rays, gamma rays or neutrons

Effector/memory T cells of the weanling mouse exhibit Type 2 cytokine polarization in vitro and in vivo in the advanced stages of acute energy deficit.

Science.gov (United States)

Steevels, Tessa A M; Hillyer, Lyn M; Monk, Jennifer M; Fisher, Megan E; Woodward, Bill D

2010-06-01

Our objective was to determine whether the polarizing cytokine profile of the effector/memory T-cell compartment reflects the profound decline of cell-mediated inflammatory competence that characterizes acute prepubescent malnutrition. Weanling C57BL/6J mice were permitted free access to a complete purified diet, free access to an isocaloric low-protein diet or restricted intake of the complete diet for 14 days. First, interleukin (IL)-4 and interferon (IFN)-gamma concentrations generated in vitro by splenic and nodal effector/memory T cells were assessed following exposure to plate-bound anti-CD3. Second, net systemic production of IFN-gamma and IL-4 by the effector/memory T-cell compartment was assessed by the in vivo cytokine capture assay following anti-CD3 stimulation. In vitro stimulation generated less IFN-gamma (P=.002) but more IL-4 (P=.05) by T cells from the restricted-intake group relative to the age-matched control group. Similarly, in vivo stimulation generated low serum levels of antibody-captured IFN-gamma in the restricted-intake group vis-à-vis the age-matched control group (P=.01), while the IL-4 response was sustained (P=.39). By contrast, the 14-day low-protein model exhibited no change in T-cell cytokine signature either in vitro or in vivo. However, following extended consumption of the low-protein diet (26 days), carcass energy losses exceeded those of the 14-day protocol and serum levels of in vivo antibody-captured IFN-gamma were low after anti-CD3 challenge relative to the age-matched control group (P=.02), while levels of captured IL-4 remained unaffected (P=.07). Acute weanling malnutrition elicits a Type 2 polarizing cytokine character on the part of the effector/memory T-cell compartment, but only in the most advanced stages of energy decrement. Copyright (c) 2010 Elsevier Inc. All rights reserved.

IFN signaling: how a non-canonical model led to the development of IFN mimetics

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Howard M Johnson

2013-07-01

Full Text Available The classical model of cytokine signaling dominates our view of specific gene activation by cytokines such as the interferons (IFNs. The importance of the model extends beyond cytokines and applies to hormones such as growth hormone (GH and insulin, and growth factors such as epidermal growth factor (EGF and fibroblast growth factor (FGF. According to this model, ligand activates the cell via interaction with the extracellular domain of the receptor. This results in activation of receptor or receptor-associated tyrosine kinases, primarily of the Janus kinase (JAK family, phosphorylation and dimerization of the STAT transcription factors, which dissociate from the receptor cytoplasmic domain and translocate to the nucleus. This view ascribes no further role to the ligand, JAK kinase, or receptor in either specific gene activation or the associated epigenetic events. The presence of dimeric STATs in the nucleus essentially explains it all. Our studies have resulted in the development of a non-canonical, more complex model of IFNγ signaling that is akin to that of steroid hormone/steroid receptor signaling. We have shown that ligand, receptor, activated JAKs and STATs are associated with specific gene activation, where the receptor subunit IFNGR1 functions as a co-transcription factor and the JAKs are involved in associated epigenetic events. We found that the type I IFN system functions similarly. The fact that GH receptor, insulin receptor, EGF receptor, and FGF receptor undergo nuclear translocation upon ligand binding suggests that they may also function similarly. The steroid hormone/steroid receptor nature of type I and II IFN signaling provides insight into the specificity of signaling by members of cytokine families. The non-canonical model could also provide better understanding to more complex cytokine families such as those of IL-2 and IL-12, whose members often use the same JAKs and STATs, but also have different functions and

The influence of royal jelly and human interferon-alpha (HuIFN-αN3) on proliferation, glutathione level and lipid peroxidation in human colorectal adenocarcinoma cells in vitro.

Science.gov (United States)

Filipič, Bratko; Gradišnik, Lidija; Rihar, Klemen; Šooš, Eugen; Pereyra, Adriana; Potokar, Jana

2015-12-01

Among royal jelly's (RJ) various biological activities, its possible antitumour activity deserves particular attention. The purpose of this study was to investigate the influence of RJ, its bioactive component 10-hydroxy-2-decenoic acid (10- HDA), and human interferon-alpha (HuIFN-αN3) on the proliferation of human colorectal adenocarcinoma cells (CaCo- 2), and ascertain their effect on intracellular glutathione (GSH) level and lipid peroxidation. We studied the antiproliferative (AP) activity of RJ [(0.1 g/10 mL phosphate buffer saline (PBS)], HuIFN-αN3 (1000 I.U. mL⁻¹), 10-HDA at 100.0 μmol L⁻¹, and their different combinations, in the ratio 1:1, 1:2, and 2:1 on CaCo-2 cells. The GSH level was measured by glutathione assay. The lipid peroxidation was measured by malondialdehyde (MDA) assay. Single RJ had a low AP activity: 2.0 (0.5 mg mL⁻¹). HuIFN-αN3 had an AP activity of 2.5 (208.33 I.U. mL⁻¹), while 10-HDA had an AP activity of 1.5 (37.5 μmol mL⁻¹). The highest AP activity of 3.8 was obtained when RJ and HuIFN-αN3 were applied at the ratio 2:1. In that combination the level of GSH was 24.9±2.4 nmol g⁻³ of proteins (vs. 70.2±3.2 nmol g⁻³ in the control) and the level of MDA was 72.3±3.1 nmol g⁻³ (vs. 23.6±9.1 nmol g⁻³ in the control). It is generally assumed that 10-HDA, an important constituent of RJ, together with HuIFN-αN3, is responsible for the inhibition of CaCo-2 cells proliferation in vitro. In our study, however, RJ and HuIFN-αN3 applied at 2:1 decreased the level of GSH the most and significantly increased lipid peroxidation via MDA in CaCo-2 cells. Future studies should show whether these GSH- and MDA-related activities of RJ, HuIFN-αN3, 10-HDA, and their combinations may decrease the tumorigenicity index and tumorigenic potential of various tumour cells in vitro.

Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

Science.gov (United States)

Murray, Paul D.; McGavern, Dorian B.; Pease, Larry R.; Rodriguez, Moses

2017-01-01

IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4+ and CD8+ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4+ and CD8+ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4+ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination. PMID:11857334

Biologic consequences of Stat1-independent IFN signaling

Science.gov (United States)

Gil, M. Pilar; Bohn, Erwin; O'Guin, Andrew K.; Ramana, Chilakamarti V.; Levine, Beth; Stark, George R.; Virgin, Herbert W.; Schreiber, Robert D.

2001-01-01

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences. PMID:11390995

Interferon gamma, interferon-gamma-induced-protein 10, and tuberculin responses of children at high risk of tuberculosis infection

DEFF Research Database (Denmark)

Petrucci, Roberta; Abu Amer, Nabil; Gurgel, Ricardo Queiroz

2008-01-01

BACKGROUND: Children in contact with adults with pulmonary tuberculosis (TB) are at risk for infection and disease progression, and chemoprophylaxis may reduce this risk. The identification of infection is based on the tuberculin skin test (TST) and interferon-gamma (INF-gamma) release assays...

Diagnosis of Coxiella burnetii infection: comparison of a whole blood interferon-gamma production assay and a Coxiella ELISPOT

NARCIS (Netherlands)

Schoffelen, T.; Limonard, G.J.; Bleeker-Rovers, C.P.; Bouwman, J.J.; Meer, J.W. van der; Nabuurs-Franssen, M.H.; Sprong, T.; Deuren, M. van

2014-01-01

Diagnosis of ongoing or past infection with Coxiella burnetii, the causative agent of Q fever, relies heavily on serology: the measurement of C. burnetii-specific antibodies, reflecting the host's humoral immune response. However, cell-mediated immune responses play an important, probably even more

High-Yield Production in Escherichia coli of Fungal Immunomodulatory Protein Isolated from Flammulina velutipes and Its Bioactivity Assay in Vivo

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Shenkui Liu

2013-01-01

Full Text Available A fungal immunomodulatory protein isolated from Flammulina velutipes (FIP-fve has structural similarity to the variable region of the immunoglobulin heavy chain. In the present study, the recombinant bioactive FIP-fve protein with a His-tag in N-terminal of recombinant protein was expressed in transetta (DE3 at a high level under the optimized culturing conditions of 0.2 mM IPTG and 28 °C. The efficiency of the purification was improved with additional ultrasonication to the process of lysozyme lysis. The yield of the bioactive FIP-fve protein with 97.1% purity reached 29.1 mg/L with a large quantity for industrial applications. Enzyme-linked immunosorbent assay showed a maximum increase in interleukin-2 (IL-2 and gamma interferon (IFN-γ for the mice serum group of 5 mg/kg body mass (p < 0.01 with three doses of His-FIP-fve. However, the production of IL-4 had no apparent difference compared to the control.

Interpretation of the gamma interferon test for diagnosis of subclinical paratuberculosis in cattle

DEFF Research Database (Denmark)

Jungersen, Gregers; Huda, A.; Hansen, J.J.

2002-01-01

A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma...... interferon (IFN-gamma) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64...

In silico and in vivo analysis of Toxoplasma gondii epitopes by correlating survival data with peptide-MHC-I binding affinities.

Science.gov (United States)

Huang, Si-Yang; Jensen, Maria Risager; Rosenberg, Carina Agerbo; Zhu, Xing-Quan; Petersen, Eskild; Vorup-Jensen, Thomas

2016-07-01

Protein antigens comprising peptide motifs with high binding affinity to major histocompatibility complex class I (MHC-I) molecules are expected to induce a stronger cytotoxic T-lymphocyte response and thus provide better protection against infection with microorganisms where cytotoxic T-cells are the main effector arm of the immune system. Data on cyst formation and survival were extracted from past studies on the DNA immunization of mice with plasmids coding for Toxoplasma gondii antigens. From in silico analyses of the vaccine antigens, the correlation was tested between the predicted affinity for MHC-I molecules of the vaccine peptides and the survival of immunized mice after challenge with T. gondii. ELISPOT analysis was used for the experimental testing of peptide immunogenicity. Predictions for the Db MHC-I molecule produced a strong, negative correlation between survival and the dissociation constant of vaccine-derived peptides. The in silico analyses of nine T. gondii antigens identified peptides with a predicted dissociation constant in the interval from 10nM to 40μM. ELISPOT assays with splenocytes from T. gondii-infected mice further supported the importance of the peptide affinity for MHC-I. In silico analysis clearly helped the search for protective vaccine antigens. The ELISPOT analysis confirmed that the predicted T-cell epitopes were immunogenic by their ability to release interferon gamma in spleen cells. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

Energy Technology Data Exchange (ETDEWEB)

Chen, Chia-Ling [Translational Research Center, Taipei Medical University, Taipei 110, Taiwan (China); Chiang, Tzu-Hui; Tseng, Po-Chun [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Wang, Yu-Chih [Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin2014@tmu.edu.tw [Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China)

2015-10-23

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cells also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ. - Highlights: • This study demonstrates that PTEN decrease causes cellular unresponsive to IFN-γ. • Lung cancer cells with PTEN deficiency show unresponsive to IFN-γ signaling. • PTEN decrease inhibits IFN-γ-induced CD54, cell proliferation inhibition, and cytotoxicity. • ROS-mediated SHP2 activation makes PTEN-deficient cells unresponsive to IFN-γ.

IFN-induced modulation of histocompatibility antigens on human cells. Background, mechanisms and perspectives

DEFF Research Database (Denmark)

Hokland, M; Basse, P; Justesen, J

1989-01-01

IFN proteins are a family of lymphokines with anti-viral effects. Several other effects of IFNs have also been described, including enhancement of natural killer (NK) cell activity, enhancement of cytotoxic T-lymphocyte activity, and enhancement of the expression of major histocompatibility compl...... to the classical anti-viral mechanism. This concept proposes that the MHC-enhancing effect of IFNs is a vital part of the immunological defense against virus infections and an integral part of the anti-viral effects of IFN proteins. Udgivelsesdato: 1988-Nov...

Field experience with a mobile tomographic nondestructive assay system

International Nuclear Information System (INIS)

Prettyman, T.H.; Betts, S.E.; Taggart, D.P.; Estep, R.J.; Nicholas, N.J.; Lucas, M.C.; Harlan, R.A.

1995-01-01

A mobile tomographic gamma-ray scanner (TGS) developed by Los Alamos National Laboratory was recently demonstrated at the Rocky Flats Environmental Technology Site and is currently in use at Los Alamos waste storage areas. The scanner was developed to assay radionuclides in low-level, transuranic, and mixed waste in containers ranging in size from 2 ft 3 boxes to 83-gallon overpacks. The tomographic imaging capability provides a complete correction for source distribution and matrix attenuation effects, enabling accurate assays of Pu-239 and other gamma-ray emitting isotopes. In addition, the system can reliably detect self-absorbing material such as plutonium metal shot, and can correct for bias caused by self-absorption. The system can be quickly configured to execute far-field scans, segmented gamma-ray scans, and a host of intermediate scanning protocols, enabling higher throughput (up to 20 drums per 8-hour shift). In this paper, we will report on the results of field trials of the mobile system at Rocky Flats and Los Alamos. Assay accuracy is confirmed for cases in which TGS assays can be compared with assays (e.g. with calorimetry) of individual packages within the drums. The mobile tomographic technology is expected to considerably reduce characterization costs at DOE production and environmental technology sites

Test plan for Digface Chemical and Radiation Assay System

International Nuclear Information System (INIS)

Akers, D.W.

1993-07-01

The Digface Chemical and Radiation Assay System (CRAS) Project will develop a sensor using Prompt Gamma Neutron Activation Analysis (PGNAA) that can detect the present of hazardous chemicals and radioactive materials. The CRAS is being designed for in situ assay of closed drums and contaminated soils for gamma-ray emitting radionuclides and hazardous elements. The CRAS is based upon the use of 252 Cf PGNAA with a germanium gamma-ray spectrometer as the analyzer. Tasks being performed include determining detection limits for a number of hazardous chemicals and assessing matrix and transmission effects through soil. Initial analyses suggest that the technique is applicable to a number of hazardous materials such as trichloroethane and carbon tetrachloride

DMPD: What is disrupting IFN-alpha's antiviral activity? [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 15283983 What is disrupting IFN-alpha's antiviral activity? Mbow ML, Sarisky RT. Tr...ends Biotechnol. 2004 Aug;22(8):395-9. (.png) (.svg) (.html) (.csml) Show What is disrupting IFN-alpha's ant...iviral activity? PubmedID 15283983 Title What is disrupting IFN-alpha's antiviral activity? Authors Mbow ML,

IFN-β: A Contentious Player in Host–Pathogen Interaction in Tuberculosis

Science.gov (United States)

Sabir, Naveed; Hussain, Tariq; Shah, Syed Zahid Ali; Zhao, Deming; Zhou, Xiangmei

2017-01-01

Tuberculosis (TB) is a major health threat to the human population worldwide. The etiology of the disease is Mycobacterium tuberculosis (Mtb), a highly successful intracellular pathogen. It has the ability to manipulate the host immune response and to make the intracellular environment suitable for its survival. Many studies have addressed the interactions between the bacteria and the host immune cells as involving many immune mediators and other cellular players. Interferon-β (IFN-β) signaling is crucial for inducing the host innate immune response and it is an important determinant in the fate of mycobacterial infection. The role of IFN-β in protection against viral infections is well established and has been studied for decades, but its role in mycobacterial infections remains much more complicated and debatable. The involvement of IFN-β in immune evasion mechanisms adopted by Mtb has been an important area of investigation in recent years. These advances have widened our understanding of the pro-bacterial role of IFN-β in host–pathogen interactions. This pro-bacterial activity of IFN-β appears to be correlated with its anti-inflammatory characteristics, primarily by antagonizing the production and function of interleukin 1β (IL-1β) and interleukin 18 (IL-18) through increased interleukin 10 (IL-10) production and by inhibiting the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome. Furthermore, it also fails to provoke a proper T helper 1 (Th1) response and reduces the expression of major histocompatibility complex II (MHC-II) and interferon-γ receptors (IFNGRs). Here we will review some studies to provide a paradigm for the induction, regulation, and role of IFN-β in mycobacterial infection. Indeed, recent studies suggest that IFN-β plays a role in Mtb survival in host cells and its downregulation may be a useful therapeutic strategy to control Mtb infection. PMID:29258190

Interferon gamma, interferon-gamma-induced-protein 10, and tuberculin responses of children at high risk of tuberculosis infection

DEFF Research Database (Denmark)

Petrucci, Roberta; Abu Amer, Nabil; Gurgel, Ricardo Queiroz

2008-01-01

BACKGROUND: Children in contact with adults with pulmonary tuberculosis (TB) are at risk for infection and disease progression, and chemoprophylaxis may reduce this risk. The identification of infection is based on the tuberculin skin test (TST) and interferon-gamma (INF-gamma) release assays. Ot...

Enhancement of nonspecific resistance by liposome-encapsulated immunomodulators does not affect skin graft rejection in mice

NARCIS (Netherlands)

Hagen, ten T.L.M.; Vossen, A.C.T.M.; Vianen, van W.; Tibbe, G.J.M.; Savelkoul, H.F.J.; Heremans, H.; Bakker-Woudenberg, I.A.J.M.

1995-01-01

Administration of liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidyl ethanolamine (LE-MTPPE) or interferon-gamma (LE-IFN-gamma), or co-encapsulated MTPPE and IFN-gamma (LE-(MTPPE/IFN-gamma)) resulted in a dramatic increase of the nonspecific antimicrobial resistance in

Effects of IFN-β1a and IFN-β1b treatment on the expression of cytokines, inducible NOS (NOS type II), and myelin proteins in animal model of multiple sclerosis.

Science.gov (United States)

Lubina-Dąbrowska, Natalia; Stepień, Adam; Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Langfort, Józef; Chalimoniuk, Małgorzata

2017-08-01

The aim of this study was to investigate the effects of interferon (IFN)-β1a and IFN-β1b treatment on inflammatory factors and myelin protein levels in the brain cortex of the Lewis rat experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis. To induce EAE, rat were immunized with inoculums containing spinal cord guinea pig homogenized in phosphate-buffered saline and emulsified in Freund's complete adjuvant containing 110 µg of the appropriate antigen in 100 µl of an emulsion and additionally 4-mg/ml Mycobacterium tuberculosis (H37Ra). The rats were treated three times per week with subcutaneous applications of 300,000 units IFN-β1a or IFN-β1b. The treatments were started 8 days prior to immunization and continued until day 14 after immunization. The rats were killed on the 14th day of the experiment. EAE induced dramatic increase in interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-concentrations and inducible nitric oxide synthase (iNOS) expression in the brain, which closely corresponded to the course of neurological symptoms and the loss of weight. Both IFN-β1b and IFN-β1a treatments inhibited the pro-inflammatory cytokines (IL-6, IL-1β, TNF-α and IFN-γ), decreased the activation of astrocytes, increased the myelin protein level in the brain cortex, and improved the neurological status of EAE rats by different mechanisms; IFN-β1a reduced iNOS expression, at least in part, by the enhancement of IL-10, while IFN-β1b diminished IL-10 concentration and did not decrease EAE-induced iNOS expression.

A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

Science.gov (United States)

Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

2015-10-01

During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

On the influence of matrix's heterogeneity on uncertainty of gamma-spectrometry at activity assay of radioactive waste

Directory of Open Access Journals (Sweden)

V. S. Prokopenko

2009-09-01

Full Text Available The influence of the waste matrix heterogeneity on the flux density value of initial gamma quanta at the transport of quanta in the matrix was considered. It is shown that the waste heterogeneity leads to the positive shift of the average flux density value comparing with corresponding value for homogeneous waste if average value of the attenuation factor in heterogeneous matrix is equal to the attenuation factor of homogeneous matrix. Due to this the activity assay of heterogeneous waste by a technique which was calibrated by using a homogeneous standard (surrogate container the measurement results will be positively shifted, or, in other words, conservative estimation of the waste activity will be obtained.

Characterization of the bovine type I IFN locus: rearrangements, expansions, and novel subfamilies

Directory of Open Access Journals (Sweden)

Walker Angela M

2009-04-01

Full Text Available Abstract Background The Type I interferons (IFN have major roles in the innate immune response to viruses, a function that is believed to have led to expansion in the number and complexity of their genes, although these genes have remained confined to single chromosomal region in all mammals so far examined. IFNB and IFNE define the limits of the locus, with all other Type I IFN genes except IFNK distributed between these boundaries, strongly suggesting that the locus has broadened as IFN genes duplicated and then evolved into a series of distinct families. Results The Type I IFN locus in Bos taurus has undergone significant rearrangement and expansion compared to mouse and human, however, with the constituent genes separated into two sub-loci separated by >700 kb. The IFNW family is greatly expanded, comprising 24 potentially functional genes and at least 8 pseudogenes. The IFNB (n = 6, represented in human and mouse by one copy, are also present as multiple copies in Bos taurus. The IFNT, which encode a non-virally inducible, ruminant-specific IFN secreted by the pre-implantation conceptus, are represented by three genes and two pseudogenes. The latter have sequences intermediate between IFNT and IFNW. A new Type I IFN family (IFNX of four members, one of which is a pseudogene, appears to have diverged from the IFNA lineage at least 83 million years ago, but is absent in all other sequenced genomes with the possible exception of the horse, a non-ruminant herbivore. Conclusion In summary, we have provided the first comprehensive annotation of the Type I IFN locus in Bos taurus, thereby providing an insight into the functional evolution of the Type I IFN in ruminants. The diversity and global spread of the ruminant species may have required an expansion of the Type I IFN locus and its constituent genes to provide broad anti-viral protection required for foraging and foregut fermentation.

Safeguards and Non-destructive Assay

International Nuclear Information System (INIS)

Carchon, R.; Bruggeman, M.

2001-01-01

SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

Structure alteration and immunological properties of {sup 60}Co gamma rays irradiated bothropstoxin-I

Energy Technology Data Exchange (ETDEWEB)

Baptista, Janaina A.; Yonamine, Camila Myiagui; Caproni, Priscila; Casare, Murilo; Spencer, Patrick Jack; Nascimento, Nanci do [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: janabap@gmail.com; Andrade Junior, Heitor Franco de; Vieira, Daniel Perez; Galisteo Junior, Andres Jimenez [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Instituto de Medicina Tropical de Sao Paulo, SP (Brazil). Lab. de Protozoologia

2007-07-01

About 20000 ophidic accidents are registered every year in Brazil. Serum therapy with equine antisera is the only efficient treatment. The venoms employed for immunization are fairly toxic and some venoms present low immunogenicity. Thus, the obtention of modified antigens with lower toxicity and preserved or improved immunogenicity would be useful. These toxins, when submitted to gamma radiation, in aqueous solution, present structural modifications. This occurs due to reactions with the radiolysis products of water. Some scavenger substances, such as NaNO{sub 3} and t-butanol, remove selectively the water radiolysis products. Ionizing radiation has proven to be a powerful tool to attenuate snake venoms toxicity without affecting and even increasing their immunogenic properties. However, the immune mechanisms involved in recognition, processing and presentation of irradiated antigens are yet unclear. In the present work, we investigated the immunological behavior of bothropstoxin-I (Bthx-1), before and after irradiation, in the presence of selective scavengers. Isogenic mice were immunized with either the native or the irradiated toxin, either with or without scavengers. After three immunizations, serum samples were collected and the antibody titers and isotypes were determined by Enzyme Linked Immuno Sorbent Assay. The antigenic characterization of native and irradiated bothropstoxin-I was performed by Western blot. The detection of expression of murine cytokines (IFN-{gamma} and IL-10) was analyzed by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). According to our data, irradiation process has promoted structural modifications in the toxin, characterized by higher molecular weight forms of the protein (aggregates and oligomers). Our data also indicate that irradiated toxins, alone or in the presence of NaNO{sub 3}, an aqueous electron scavenger, were immunogenic and the antibodies elicited by them were able to recognize the native toxin. On the other

Post-transcriptional regulation of macrophage ABCA1, an early response gene to IFN-γ

International Nuclear Information System (INIS)

Alfaro Leon, Martha Leticia; Evans, Glenn F.; Farmen, Mark W.; Zuckerman, Steven H.

2005-01-01

Interferon-γ (IFN-γ) down-regulates receptors associated with reverse cholesterol transport including ABCA1. In the present study, the kinetics and mechanism of ABCA1 down-regulation were determined in mouse peritoneal macrophages. IFN-γ decreased ABCA1 mRNA 1 h following IFN-γ addition and was maximally reduced by 3 h. Down-regulation was protein synthesis dependent and involved post-transcriptional processes. ABCA1 message had a T 1/2 of 115 min in actinomycin treated cells that was reduced to a T 1/2 of 37 min by IFN-γ. The decrease in message stability was also associated with a rapid loss of ABCA1 protein, significant 3 h following IFN-γ addition. The kinetics of ABCA1 message and protein decrease was consistent with the early IFN-γ-induced changes in Stat1 phosphorylation and nuclear translocation observed in these cells. Therefore, ABCA1 can be considered as an early response gene to macrophage activation by IFN-γ with down-regulation occurring by message destabilization

Endogenous IFN-β signaling exerts anti-inflammatory actions in experimentally induced focal cerebral ischemia

DEFF Research Database (Denmark)

Inácio, Ana R; Liu, Yawei; Clausen, Bettina H

2015-01-01

of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-βKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation......BACKGROUND: Interferon (IFN)-β exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-β, by limiting inflammation, decreases neuronal death...... strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. RESULTS: Here, we report alterations in local and systemic inflammation in IFN-β knockout (IFN-βKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-βKO mice showed a higher number...

Q{sub {gamma}-H2AX}, an analysis method for partial-body radiation exposure using {gamma}-H2AX in non-human primate lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Redon, Christophe E., E-mail: redonc@mail.nih.gov [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Nakamura, Asako J.; Gouliaeva, Ksenia [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Rahman, Arifur; Blakely, William F. [Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603 (United States); Bonner, William M. [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States)

2011-09-15

We previously used the {gamma}-H2AX assay as a biodosimeter for total-body irradiation (TBI) exposure ({gamma}-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant {gamma}-H2AX responses days after total-body exposure to 1-8.5 Gy ({sup 60}Co {gamma}-rays at 55 cGy min{sup -1}). Here, we introduce a partial-body exposure analysis method, Q{sub {gamma}-H2AX}, which is based on the number of {gamma}-H2AX foci per damaged cells as evident by having one or more {gamma}-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q{sub {gamma}-H2AX} dose-response calibration curves to assess acute partial-body exposures. {gamma}-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of {gamma}-H2AX may provide a robust biodosimeter for analyzing partial-body exposures to ionizing radiation in humans.

Use of delayed gamma rays for active non-destructive assay of {sup 235}U irradiated by pulsed neutron source (plasma focus)

Energy Technology Data Exchange (ETDEWEB)

Andola, Sanjay; Niranjan, Ram [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kaushik, T.C., E-mail: tckk@barc.gov.in [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Rout, R.K. [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kumar, Ashwani; Paranjape, D.B.; Kumar, Pradeep; Tomar, B.S.; Ramakumar, K.L. [Radioanalytical Chemistry Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Gupta, S.C. [Applied Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

2014-07-01

A pulsed neutron source based on plasma focus device has been used for active interrogation and assay of {sup 235}U by monitoring its delayed high energy γ-rays. The method involves irradiation of fissile material by thermal neutrons obtained after moderation of a burst of neutrons emitted upon fusion of deuterium in plasma focus (PF) device. The delayed gamma rays emitted from the fissile material as a consequence of induced fission were detected by a large volume sodium iodide (NaI(Tl)) detector. The detector is coupled to a data acquisition system of 2k input size with 2k ADC conversion gain. Counting was carried out in pulse height analysis mode for time integrated counts up to 100 s while the temporal profile of delayed gamma has been obtained by counting in multichannel scaling mode with dwell time of 50 ms. To avoid the effect of passive (natural) and active (from surrounding materials) backgrounds, counts have been acquired for gamma energy between 3 and 10 MeV. The lower limit of detection of {sup 235}U in the oxide samples with this set-up is estimated to be 14 mg.

Study of gamma irradiation effects on the etching and optical properties of CR-39 solid state nuclear track detector and its application to uranium assay in soil samples

International Nuclear Information System (INIS)

Amol Mhatre; Kalsi, P.C.

2011-01-01

The gamma irradiation effects in the dose range of 2.5-43.0 Mrad on the etching and optical characteristics of CR-39 solid state nuclear track detector (SSNTD) have been studied by using etching and UV-Visible spectroscopic techniques. From the measured bulk etch rates at different temperatures, the activation energies for bulk etching at different doses have also been determined. It is seen that the bulk etch rates increase and the activation energies for bulk etching decrease with the increase in gamma dose. The optical band gaps of the unirradiated and the gamma -irradiated detectors determined from the UV-Visible spectra were found to decrease with the increase in gamma dose. These results have been explained on the basis of scission of the detector due to gamma irradiation. The present studies can be used for the estimation of gamma dose in the range of 2.5-43.0 Mrad and can also be used for estimating track registration efficiency in the presence of gamma dose. The CR-39 detector has also been applied for the assay of uranium in some soil samples of Jammu city. (author)

Immunostimulatory effects of natural human interferon-alpha (huIFN-alpha) on carps Cyprinus carpio L.

Science.gov (United States)

Watanuki, Hironobu; Chakraborty, Gunimala; Korenaga, Hiroki; Kono, Tomoya; Shivappa, R B; Sakai, Masahiro

2009-10-15

Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.

Kinetics of IFN-gamma and TNF-alpha gene expression and their relationship with disease progression after infection with Mycobacterium tuberculosis in guinea pigs.

Science.gov (United States)

Roh, In Soon; Cho, Sungae; Eum, Seok-Yong; Cho, Sang-Nae

2013-05-01

Guinea pig is one of the most suitable animal models for Mycobacterium tuberculosis (M. tb) infection since it shows similarities to pulmonary infection in humans. Although guinea pig shows hematogenous spread of M. tb infection into the whole body, immunological studies have mainly focused on granulomatous tissues in lungs and spleens. In order to investigate the time-course of disease pathogenesis and immunological profiles in each infected organ, we performed the following approaches with guinea pigs experimentally infected with M. tb over a 22-week post-infection period. We examined body weight changes, M. tb growth curve, cytokine gene expression (IFN-γ and TNF-α), and histopathology in liver, spleen, lungs and lymph nodes of infected guinea pigs. The body weights of infected guinea pigs did not increase as much as uninfected ones and the number of M. tb bacilli in their organs increased except bronchotracheal lymph node during the experimental period. The gene expression of IFN-γ and TNF-α was induced between 3 and 6 weeks of infection; however, kinetic profiles of cytokine gene expression showed heterogeneity among organs over the study period. Histophathologically granulomatous lesions were developed in all four organs of infected guinea pigs. Although IFN-γ and TNF-α gene expression profiles showed heterogeneity, the granuloma formation was clearly observed in every organ regardless of whether the number of bacilli increased or decreased. However, this protective immunity was accompanied with severe tissue damage in all four organs, which may lead to the death of guinea pigs.

Culture supernatants of oral cancer cells induce impaired IFN-α production of pDCs partly through the down-regulation of TLR-9 expression.

Science.gov (United States)

Han, Nannan; Zhang, Zun; Jv, Houyu; Hu, Jingzhou; Ruan, Min; Zhang, Chenping

2018-06-05

The aim of the present study was to investigate whether tumor-derived supernatants down-regulate the immune function of plasmacytoid dendritic cells (pDCs) in oral cancer and the potential molecular mechanisms of this effect. Immunohistochemistry (IHC) and flow cytometry were used to detect tumor-infiltrating and peripheral blood pDCs. MTS and flow cytometry were employed to evaluate the immune response of CD4 + T cells. Real-time PCR and ELISA assays were used to identify TLR-7 and TLR-9 expression, IFN-α production and tumor-secreted soluble cytokines. The proportion of pDCs (0.121%±0.043%) was significantly higher in Oral squamous cell carcinoma (OSCC) samples than in normal tissue (0.023%±0.016%) (P = 0.021). TLR9 mRNA was significantly lower in tumor-infiltrating pDCs and positively correlated to low IFN-α production (r = 0.956; Poral cancer cells negatively regulated TLR9 mRNA expression and the subsequent IFN-α production of pDCs, which inhibited the immune response of CD4 + T cells. The neutralizing antibodies blocking assay showed that the specific inhibitory effect of pDC functionality was associated with the soluble fraction of the oral cancer environment, which is mainly mediated by IL-10 and TGF-β cooperation. Tumor-derived supernatants may impair the function of tumor-infiltrating pDCs, which subsequently decreases the immune response of CD4 + T cells in human oral cancer through TGF-β- and IL-10- dependent mechanisms. Careful manipulation of these impaired pDCs may help develop an important alternative immunotherapy for the treatment of oral cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

Involvement of T cells in enhanced resistance to Klebsiella pneumoniae septicemia in mice treated with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine or gamma interferon

NARCIS (Netherlands)

Hagen, ten T.L.; Vianen, van W.; Savelkoul, H.F.J.; Heremans, H.; Buurman, W.A.; Bakker-Woudenberg, I.A.

1998-01-01

We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-) results in strongly increased survival of mice from a normally lethal septicemia with Klebsiella pneumoniae.

Attenuation correction for the collimated gamma ray assay of cylindrical samples

International Nuclear Information System (INIS)

Patra, Sabyasachi; Agarwal, Chhavi; Goswami, A.; Gathibandhe, M.

2015-01-01

The Hybrid Monte Carlo (HMC) method developed earlier for attenuation correction of non-collimated samples [Agarwal et al., 2008, Nucl. Instrum. Methods A 597, 198], has been extended to the segmented gamma ray assay of cylindrical samples. The method has been validated both experimentally and theoretically. For experimental validation, the results of HMC calculation have been compared with the experimentally obtained attenuation correction factors. The HMC attenuation correction factors have also been compared with the results obtained from literature available near-field and far-field formulae at two sample-to-detector distances (10.3 cm and 20.4 cm). The method has been found to be valid at all sample-to-detector distances over a wide range of transmittance. On the other hand, the literature available near-field and far-field formulae have been found to work over a limited range of sample-to detector distances and transmittances. The HMC method has been further extended to circular collimated geometries where analytical formula for attenuation correction does not exist. - Highlights: • Hybrid Monte Carlo method for attenuation correction developed for SGA system. • Method found to work for all sample-detector geometries for all transmittances. • The near-field formula applicable only after certain sample-detector distance. • The far-field formula applicable only for higher transmittances (>18%). • Hybrid Monte Carlo method further extended to circular collimated geometry

Accounting for segment correlations in segmented gamma-ray scans

International Nuclear Information System (INIS)

Sheppard, G.A.; Prettyman, T.H.; Piquette, E.C.

1994-01-01

In a typical segmented gamma-ray scanner (SGS), the detector's field of view is collimated so that a complete horizontal slice or segment of the desired thickness is visible. Ordinarily, the collimator is not deep enough to exclude gamma rays emitted from sample volumes above and below the segment aligned with the collimator. This can lead to assay biases, particularly for certain radioactive-material distributions. Another consequence of the collimator's low aspect ratio is that segment assays at the top and bottom of the sample are biased low because the detector's field of view is not filled. This effect is ordinarily countered by placing the sample on a low-Z pedestal and scanning one or more segment thicknesses below and above the sample. This takes extra time, however, We have investigated a number of techniques that both account for correlated segments and correct for end effects in SGS assays. Also, we have developed an algorithm that facilitates estimates of assay precision. Six calculation methods have been compared by evaluating the results of thousands of simulated, assays for three types of gamma-ray source distribution and ten masses. We will report on these computational studies and their experimental verification

In-plant measurements of gamma-ray transmissions for precise K-edge and passive assay of plutonium concentration and isotopic abundance in product solutions at the Tokai Reprocessing Plant

International Nuclear Information System (INIS)

Asakura, Y.; Kondo, I.; Masui, J.; Shoji, K.; Russo, P.A.; Hsue, S.T.; Sprinkle, J.K. Jr.; Johnson, S.S.

1982-01-01

A field test has been carried out for more than 2 years for determination of plutonium concentration by K-edge absorption densitometry and for determination of plutonium isotopic abundance by transmission-corrected passive gamma-ray spectrometry. This system was designed and built at Los Alamos National Laboratory and installed at the Tokai reprocessing plant of the Power Reactor and Nuclear Fuel Development Corporation as a part of the Tokai Advanced Safeguards Technology Exercise (TASTEX). For K-edge measurement of plutonium concentration, the transmissions at two discrete gamma-ray energies are measured using the 121.1- and 122.1-keV gamma rays from 75 Se and 57 Co. Intensities of the plutonium passive gamma rays in the energy regions between 38 and 51 keV and between 129 and 153 keV are used for determination of the isotopic abundances. More than 200 product solution samples have been measured in a timely fashion during these 2 years. The relative precisions and accuracies of the plutonium concentration measurement are shown to be within 0.6% (1 sigma) in these applications, and those for plutonium isotopic abundances are within 3% for 238 Pu, 0.4% for 239 Pu, 1.2% for 240 Pu, 1.3% for 241 Pu, and 7% for 242 Pu. The time required is 10 min for the concentration assay, 10 min for the isotopics assay, and about 15 min for handling procedures in the laboratory

Delayed Gamma-Ray Spectroscopy for Non-Destructive Assay of Nuclear Materials

Energy Technology Data Exchange (ETDEWEB)

Ludewigt, Bernhard [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Mozin, Vladimir [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Campbell, Luke [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Favalli, Andrea [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Hunt, Alan W. [Iowa State Univ., Ames, IA (United States); Reedy, Edward T.E. [Iowa State Univ., Ames, IA (United States); Seipel, Heather A. [Iowa State Univ., Ames, IA (United States)

2015-09-28

This project has been a collaborative effort of researchers from four National Laboratories, Lawrence Berkley National Laboratory (LBNL), Lawrence Livermore National Laboratory (LLNL), Los Alamos National Laboratory (LANL), Pacific Northwest National Laboratory (PNNL), and Idaho State University’s (ISU) Idaho Accelerator Center (IAC). Experimental measurements at the Oregon State University (OSU) were also supported. The research included two key components, a strong experimental campaign to characterize the delayed gamma-ray signatures of the isotopes of interests and of combined targets, and a closely linked modeling effort to assess system designs and applications. Experimental measurements were performed to evaluate fission fragment yields, to test methods for determining isotopic fractions, and to benchmark the modeling code package. Detailed signature knowledge is essential for analyzing the capabilities of the delayed gamma technique, optimizing measurement parameters, and specifying neutron source and gamma-ray detection system requirements. The research was divided into three tasks: experimental measurements, characterization of fission yields, and development of analysis methods (task 1), modeling in support of experiment design and analysis and for the assessment of applications (task 2), and high-rate gamma-ray detector studies (task 3).

Safety and immunogenicity of adenovirus-vectored near-consensus HIV type 1 clade B gag vaccines in healthy adults.

Science.gov (United States)

Harro, Clayton D; Robertson, Michael N; Lally, Michelle A; O'Neill, Lori D; Edupuganti, Srilatha; Goepfert, Paul A; Mulligan, Mark J; Priddy, Frances H; Dubey, Sheri A; Kierstead, Lisa S; Sun, Xiao; Casimiro, Danilo R; DiNubile, Mark J; Shiver, John W; Leavitt, Randi Y; Mehrotra, Devan V

2009-01-01

Vaccines inducing pathogen-specific cell-mediated immunity are being developed using attenuated adenoviral (Ad) vectors. We report the results of two independent Phase I trials of similar replication-deficient Ad5 vaccines containing a near-consensus HIV-1 clade B gag transgene. Healthy HIV-uninfected adults were enrolled in two separate, multicenter, dose-escalating, blinded, placebo-controlled studies to assess the safety and immunogenicity of a three-dose homologous regimen of Ad5 and MRKAd5 HIV-1 gag vaccines given on day 1, week 4, and week 26. Adverse events were collected for 29 days following each intradeltoid injection. The primary immunogenicity endpoint was the proportion of subjects with a positive unfractionated Gag-specific IFN-gamma ELISPOT response measured 4 weeks after the last dose (week 30). Analyses were performed after combining data for each dose group from both protocols, stratifying by baseline Ad5 titers. Overall, 252 subjects were randomized to receive either vaccine or placebo, including 229 subjects (91%) who completed the study through week 30. Tolerability and immunogenicity did not appear to differ between the Ad5 and MRKAd5 vaccines. The frequency of injection-site reactions was dose dependent. Systemic adverse events were also dose dependent and more frequent in subjects with baseline Ad5 titers or =200, especially after the first dose. The percent of ELISPOT responders and the ELISPOT geometric means overall were significantly higher for all four vaccine doses studied compared to placebo, and were generally higher in vaccine recipients with baseline Ad5 titers or = 200. Ad5 titers increased after vaccination in a dose-dependent fashion. Both Ad5-vectored HIV-1 vaccines were generally well tolerated and induced cell-mediated immune responses against HIV Gag-peptides in the majority of healthy adults with baseline Ad5 titers vaccine-induced immunity to the Ad5 vector may dampen the CMI response to HIV Gag.

Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

International Nuclear Information System (INIS)

Cheon, Soyoung; Song, Seok Bean; Jung, Minkyung; Park, Yoorim; Bang, Jung-Wook; Kim, Tae Sung; Park, Hyunjeong; Kim, Cherl-hyun; Yang, Yool-hee; Bang, Sa Ik; Cho, Daeho

2008-01-01

Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-γ inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-γ production, we measured IL-18-induced IFN-γ production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-γ expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-γ production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-γ production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-γ production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-γ production via p38 MAPK

Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

OpenAIRE

Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

2005-01-01

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) ...

Achieving Higher Accuracy in the Gamma-Ray Spectrocopic Assay of Holdup

International Nuclear Information System (INIS)

Russo, P.A.; Wenz, T.R.; Smith, S.E.; Harris, J.F.

2000-01-01

Gamma-ray spectroscopy is an important technique for the measurement of quantities of nuclear material holdup in processing equipment. Because the equipment in large facilities dedicated to uranium isotopic enrichment, uranium/plutonium scrap recovery or various stages of fuel fabrication is extensive, the total holdup may be large by its distribution alone, even if deposit thicknesses are small. Good accountability practices require unbiased measurements with uncertainties that are as small as possible. This paper describes new procedures for use with traditional holdup analysis methods based on gamma-ray spectroscopy. The procedures address the two sources of bias inherent in traditional gamma-ray measurements of holdup. Holdup measurements are performed with collimated, shielded gamma-ray detectors. The measurement distance is chosen to simplify the deposit geometry to that of a point, line or area. The quantitative holdup result is based on the net count rate of a representative gamma ray. This rate is corrected for contributions from room background and for attenuation by the process equipment. Traditional holdup measurements assume that the width of the point or line deposit is very small compared to the measurement distance, and that the self-attenuation effects can be neglected. Because each point or line deposit has a finite width and because self-attenuation affects all measurements, bias is incurred in both assumptions. In both cases the bias is negative, explaining the systematically low results of gamma-ray holdup measurements. The new procedures correct for bias that arises from both the finite-source effects and the gamma-ray self-attenuation. The procedures used to correct for both of these effects apply to the generalized geometries. One common empirical parameter is used for both corrections. It self-consistently limits the total error incurred (from uncertain knowledge of this parameter) in the combined correction process, so that it is

The influence of drinking-water pollution with heavy metal on the expression of IL-4 and IFN-γ in mice by real-time polymerase chain reaction.

Science.gov (United States)

Radbin, Rayhaneh; Vahedi, Fatemeh; Chamani, JamshidKhan

2014-10-01

In recent years, water pollution has been converted to a challenging discussion in health area of human being. Heavy elements are one of the most important water pollutants and their negative adverse effects on body systems have been confirmed. In this study, investigation of effects of two heavy elements including lead (Pb) and copper (Cu) on expression of interlukin-4 (IL-4) and interferon-gamma (IFN-γ) as humoral and cellular immunity biomarkers, respectively, was aimed and PCR, real-time PCR and electrophoresis techniques were used. In this study, BALB/c mice were studied that had free access to drinking water which contained Cu or Pb salts. After 2 weeks, spleens of mice were removed, RNA extracted, and cDNA was prepared for RT-PCR. Then the expression of IL-4 and IFN-γ genes were assessed by real-time PCR. The expression of IFN-γ was up-regulated in both treated groups and the expression of IL-4 was only up-regulated in the group treated with Cu and down-regulated in the group treated with Pb. This study shows that the presence of heavy elements as drinking-water pollutants results in a disproportion of natural cytokines balances, and thus may result in a negative effect on immune system.

Cytokine gene expression profiles in chicken spleen and intestinal tissues during Ascaridia galli infection

DEFF Research Database (Denmark)

Pleidrup, Janne A.; Norup, Liselotte R.; Dalgaard, Tina S.

2014-01-01

In the poultry production industry, chickens with access to outdoor areas are exposed to a wide range of parasites e.g. the helminth Ascaridia galli. By real-time quantitative RTPCR, the relative gene expression of the T helper 1 (Th1) cytokine IFN-gamma, the T helper 2 (Th2) cytokine IL-13...... expression of jejunal IFN-gamma and IL-13 was observed. Finally, at the expected period of an adaptive immune response (days 14-21) a general decreased expression of IFN-gamma and TGF-beta 4 in spleen and IFN-gamma in jejunum was followed by a decreased expression of IFN-gamma and IL-10 at day 21 in caecal...

Immune-enhancing activities of low molecular weight β-glucan depolymerized by gamma irradiation

Science.gov (United States)

Sung, Nak-Yun; Byun, Eui-Hong; Kwon, Sun-Kyu; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Yoo, Young-Choon; Kim, Mee-Ree; Lee, Ju-Woon

2009-07-01

β-glucans are structural cell wall polymers of many microorganisms and cereals which possess immunomodulatory properties and have been used in the food, cosmetic and medical industry. In our previous study, β-glucan was depolymerized by gamma irradiation and leads to improve the solubility and viscosity. This study was carried out to evaluate the functional properties, mainly immune-enhancing activities of low molecular weight β-glucan fragmented by gamma irradiation. The results showed that RAW 264.7 macrophage cell stimulation activities of irradiated β-glucan were higher than that of non-irradiated β-glucan. In addition, the oral administration of gamma-irradiated β-glucan significantly increased the proliferation and cytokine (IFN-γ and IL-2) release of spleen and Peyer's patch cells compared with non-irradiated β-glucan. In conclusion, gamma irradiation could be used as an effective method for the production of depolymerized β-glucan improved functional property such as immunomodulatory activity.

Immune-enhancing activities of low molecular weight β-glucan depolymerized by gamma irradiation

International Nuclear Information System (INIS)

Sung, Nak-Yun; Byun, Eui-Hong; Kwon, Sun-Kyu; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Yoo, Young-Choon; Kim, Mee-Ree; Lee, Ju-Woon

2009-01-01

β-glucans are structural cell wall polymers of many microorganisms and cereals which possess immunomodulatory properties and have been used in the food, cosmetic and medical industry. In our previous study, β-glucan was depolymerized by gamma irradiation and leads to improve the solubility and viscosity. This study was carried out to evaluate the functional properties, mainly immune-enhancing activities of low molecular weight β-glucan fragmented by gamma irradiation. The results showed that RAW 264.7 macrophage cell stimulation activities of irradiated β-glucan were higher than that of non-irradiated β-glucan. In addition, the oral administration of gamma-irradiated β-glucan significantly increased the proliferation and cytokine (IFN-γ and IL-2) release of spleen and Peyer's patch cells compared with non-irradiated β-glucan. In conclusion, gamma irradiation could be used as an effective method for the production of depolymerized β-glucan improved functional property such as immunomodulatory activity.

Characterization of the binding of radioiodinated hybrid recombinant IFN-alpha A/D to murine and human lymphoid cell lines

International Nuclear Information System (INIS)

Faltynek, C.R.; Princler, G.L.; Schwabe, M.; Shata, M.T.; Lewis, G.K.; Kamin-Lewis, R.M.

1990-01-01

The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors

A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.

Science.gov (United States)

Graham, Simon P; Honda, Yoshikazu; Pellé, Roger; Mwangi, Duncan M; Glew, E Jane; de Villiers, Etienne P; Shah, Trushar; Bishop, Richard; van der Bruggen, Pierre; Nene, Vishvanath; Taracha, Evans L N

2007-02-09

Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.

Dual isotope notch observer for isotope identification, assay and imaging with mono-energetic gamma-ray sources

Science.gov (United States)

Barty, Christopher P.J.

2013-02-05

A dual isotope notch observer for isotope identification, assay and imaging with mono-energetic gamma-ray sources includes a detector arrangement consists of three detectors downstream from the object under observation. The latter detector, which operates as a beam monitor, is an integrating detector that monitors the total beam power arriving at its surface. The first detector and the middle detector each include an integrating detector surrounding a foil. The foils of these two detectors are made of the same atomic material, but each foil is a different isotope, e.g., the first foil may comprise U235 and second foil may comprise U238. The integrating detectors surrounding these pieces of foil measure the total power scattered from the foil and can be similar in composition to the final beam monitor. Non-resonant photons will, after calibration, scatter equally from both foils.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

Science.gov (United States)

Niessen, Anna; Heyder, Petra; Krienke, Stefan; Blank, Norbert; Tykocinski, Lars-Oliver; Lorenz, Hanns-Martin; Schiller, Martin

2015-07-15

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. © 2015. Published by The Company of Biologists Ltd.

Portable gamma-ray holdup and attributes measurements of high- and variable-burnup plutonium

International Nuclear Information System (INIS)

Wenz, T.R.; Russo, P.A.; Miller, M.C.; Menlove, H.O.; Takahashi, S.; Yamamoto, Y.; Aoki, I.

1991-01-01

High burnup-plutonium holdup has been assayed quantitatively by low resolution gamma-ray spectrometry. The assay was calibrated with four plutonium standards representing a range of fuel burnup and 241 Am content. Selection of a calibration standard based on its qualitative spectral similarity to gamma-ray spectra of the process material is partially responsible for the success of these holdup measurements. The spectral analysis method is based on the determination of net counts in a single spectral region of interest (ROI). However, the low-resolution gamma-ray assay signal for the high-burnup plutonium includes unknown amounts of contamination from 241 Am. For most needs, the range of calibration standards required for this selection procedure is not available. A new low-resolution gamma-ray spectral analysis procedure for assay of 239 Pu has been developed. The procedure uses the calculated isotope activity ratios and the measured net counts in three spectral ROIs to evaluate and remove the 241 Am contamination from the 239 Pu assay signal on a spectrum-by-spectrum basis. The calibration for the new procedure requires only a single plutonium standard. The procedure also provides a measure of the burnup and age attributes of holdup deposits. The new procedure has been demonstrated using portable gamma-ray spectroscopy equipment for a wide range of plutonium standards and has also been applied to the assay of 239 Pu holdup in a mixed oxide fuel fabrication facility. 10 refs., 5 figs., 3 tabs

Gamma-ray measurements with the segmented gamma scan

International Nuclear Information System (INIS)

Martin, E.R.; Jones, D.F.; Parker, J.L.

1977-12-01

A revised and updated operation and maintenance manual for the segmented gamma-scan instrument is presented, which describes routine assay techniques as well as the theory of operation in sufficient depth that an experienced assayist can make nonroutine assays on a wide variety of materials and samples. In addition, complete electronic and electrical schematics of the Los Alamos Scientific Laboratory (LASL)-designed portions of the system are presented, along with sufficient system and circuit description to facilitate maintenance and troubleshooting. Complete software system descriptions are included, although detailed listings would have to be obained from LASL in order to make machine-language code changes

Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

Science.gov (United States)

Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

Association of interferon-gamma and interleukin 10 genotypes and serum levels with partial clinical remission in type 1 diabetes

DEFF Research Database (Denmark)

Alizadeh, B Z; Hanifi-Moghaddam, P; Eerligh, P

2006-01-01

We studied whether serum interferon (IFN)-gamma or interleukin (IL)-10 levels and their corresponding functional polymorphic genotypes are associated with partial remission of type 1 diabetes (T1D). A multi-centre study was undertaken in patients with newly diagnosed T1D and matched controls. T1D...

Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor.

Directory of Open Access Journals (Sweden)

Tatsunari Kondoh

Full Text Available It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35 is found in the little brown bat (Myotis lucifugus genome. Putative mlEFL35-derived protein (mlEFL35p contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor.

Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

DEFF Research Database (Denmark)

Li, Yiping; Kang, H.N.; Babiuk, L.A.

2006-01-01