Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.     

The role of the interferon gamma release assay in assessing recent tuberculosis transmission in a hospital incident

In 2007, an extensive contact screening investigation into onward transmission of tuberculosis was instigated at a hospital in Northern Ireland following diagnosis of pulmonary multi-drug resistant TB in a healthcare worker. Interferon gamma release assays (IGRAs) were used to test 333 patients and 98 staff. We investigated for evidence of onward transmission and recent infection based on analysis of clinical, demographic and IGRA data. We also described within-patient variability of IGRA results. Among patients and staff, increasing age of patients was the only factor associated with IGRA positivity. Greatest within-subject variability of IU/mL in serially-tested patients/staff was seen in those with a positive IGRA test and this did not correlate with increased exposure to the index case. IGRA positivity being largely explained by increasing age in patients and previous TB contact in staff lends weight to the conclusion that IGRA positivity reflected previous infection rather than recent transmission.     

Inhibition of fibroblast chemotaxis by recombinant human interferon gamma and interferon alpha

Interferons have recently been recognized as potent mediators in inflammatory processes, exerting profound effects on fibroblasts. The influence of interferons gamma and alpha on the chemotactic movement of fibroblasts toward various attractants was, therefore, investigated. Normal human adult and embryonal dermal fibroblasts, fibrosarcoma-derived fibroblasts and SV40-transformed fibroblasts were tested against conditioned medium from fibroblasts, the chemotactic peptide C-140 of fibronectin, platelet-derived growth factor, and leukotriene B4 as attractants in the presence or absence of the interferons. Interferons gamma and alpha inhibited chemotaxis in a dose-dependent manner and at concentrations at least as low as 10(-2) ng/ml. Inhibition was noticeable when the cells were exposed to interferon for as short a period as 60 minutes, and the effect was not readily reversible. Inhibition occurred when the cells came from sparse or dense cultures, but when platelet-derived growth factor was the attractant and the cells had been grown at low density there was no inhibition. It is concluded that this is a specific effect, not to be wholly explained by overall increase in membrane rigidity. Inhibition of fibroblast chemotaxis by interferons may be an important regulatory mechanism during wound healing or fibrosis and metastatic spread of tumor cells.     

Interferon gamma gene +874A/T polymorphism and intracellular interferon gamma expression in pulmonary tuberculosis

We investigated whether IFN-γ gene +874(A/T) polymorphism influences intracellular interferon gamma expression in T-cell subsets of normal healthy subjects (NHS) and pulmonary tuberculosis patients (PTB). Peripheral blood mononuclear cells were stimulated with live Mycobacterium tuberculosis (MTB) and the intracellular IFN-γ expression was studied using flow cytometry. Genotyping of IFN-γ gene +874(A/T) was done using allele specific polymerase chain reaction. Significantly increased IFN-γ expressing CD3+CD4+ and CD3+CD8+ T cells were observed in NHS with AA genotype compared to TT genotype in unstimulated (p = 0.0308 and p = 0.0157) and MTB stimulated (p = 0.0494 and p = 0.0287) cultures and this difference was not observed in PTB patients. The present study suggests that the variant genotypes of IFN-γ (+874) may be associated with altered expression of IFN-γ at the intracellular level and play an immunoregulatory role at the site of M. tuberculosis infection.     

Interferon gamma release assay in diagnosis of pediatric tuberculosis: a meta-analysis

Although interferon gamma release assays (IGRAs) have been widely used for the diagnosis of latent and active tuberculosis in adults, a relative lack of validation studies in children has led to caution in their clinical interpretation. This meta-analysis systematically evaluated two IGRAs (ELISA and ELISPOT) and the tuberculin skin test (TST). We searched databases (PubMed, MEDLINE, Ovid) between January 2000 and January 2011 using search terms of latent tuberculosis infection or tuberculosis and interferon gamma release assay, or T-SPOT.TB test, or QuantiFERON-TB Gold, or ESAT-6, or CFP-10, and child, or childhood, or pediatrics. We also collected data by performing a manual search of references from relevant articles and communicating with selected authors. The meta-analysis was conducted with random effects models to account for heterogeneity between selected studies. The sensitivities of all three tests in active tuberculosis were similar. The pooled sensitivity was 70% for ELISA studies, 62% for ELISPOT studies and 71% for TST. Calculated sensitivities for IGRAs and the TST differ in culture-confirmed tuberculosis [ELISA (85%) vs. ELISPOT (76%) vs. TST (85%)] and clinical diagnosed cases [ELISA (64%) vs. ELISPOT (58%) vs. TST (66%)]. The pooled specificity was 100% for ELISA and 90% for ELISPOT, but was much lower for TST [56% in all included studies and 49% in children with bacillus Calmette-Guerin (BCG) vaccination]. The agreement between the TST and IGRAs in non-BCG-vaccinated children is higher than that in BCG-vaccinated children. In the diagnosis of active tuberculosis in children, the TST and IGRAs have similar sensitivity. By contrast, the specificity of IGRAs is far greater than the TST, particularly in children with previous BCG vaccination.     

Suppression of bacterial cell wall-induced polyarthritis by recombinant gamma interferon

Group A streptococcal cell wall fragments (SCW) induce erosive polyarthritis, characterized by synovial cell hyperplasia and intense mononuclear cell infiltration, in susceptible rats. Because of the known antiproliferative and immunomodulatory effects of interferon (IFN), we evaluated the effect of systemically administered alpha, beta and gamma IFN on the evolution of these destructive lesions. Treatment with gamma IFN not only reduced the acute response, but had an even greater suppressive effect on the chronic mononuclear cell-mediated destructive phase of the disease (articular index 10.2 +/- 1.2 for SCW only versus 3.8 +/- 0.7 for SCW + gamma IFN; p less than 0.01). Treatment with gamma IFN was more effective in the suppression of the arthritis than alpha, beta IFN. Histopathologic evaluation of the joints demonstrated that gamma IFN-treated animals had significantly fewer inflammatory cells, and less synovial hyperplasia and erosions than the SCW controls. gamma IFN suppression of mononuclear cell prostaglandin synthesis and synovial fibroblast proliferation was consistent with its anti-arthritic effects. These data indicate that the pathophysiology of SCW-induced erosive polyarthritis is subject to regulatory control by gamma IFN and that the mechanisms of suppression may be relevant in the treatment of rheumatoid arthritis.     

Induction of human gamma interferon by Sarcophaga peregrina humoral lectin

Humoral lectin isolated from the hemolymph of injured Sarcophaga peregrina (flesh-fly) larvae was found to activate human peripheral blood cells to produce interferon activity. This interferon was inactivated by dialysis against a solution of pH 2.0 and by heat treatment at 56 degrees C for 30 min, indicating that it was a gamma interferon. The role of this lectin in the defence mechanism is discussed from the viewpoint of comparative immunology.     

Interferon-gamma release assays for the diagnosis of extrapulmonary tuberculosis: a systematic review and meta-analysis

Interferon -gamma release assays (IGRAs) provide a new diagnostic method for Mycobacterium tuberculosis (TB) infection. However, the diagnostic value of IGRAs for extrapulmonary TB (EPTB) has not been clarified. We searched several databases and selected papers with strict inclusion criteria, evaluated the evidence of commercially available IGRAs (QuantiFERON(?) -TB Gold QFT-G or QFT-GIT and T-SPOT(?) .TB) on blood and the tuberculin skin test (TST) using random effects models. Twenty studies with 1711 patients were included. After excluding indeterminate results, pooled sensitivity for the diagnosis of EPTB was 72% [95% confidence interval (CI) 65-79%] for QFT-G or GIT and 90% (95% CI, 86-93%) for T-SPOT; in high-income countries the sensitivity of QFT-G or GIT (79%, 95% CI 72-86%) was much higher than that (29%, 95% CI 14-48%) in low/middle-income countries. Pooled specificity for EPTB was 82% (95% CI 78-87%) for QFT-G or GIT and 68% (95% CI 64-73%) for T-SPOT. Pooled sensitivity of TST from four studies in high-income countries was lower than that of IGRAs. T-SPOT was more sensitive in detecting EPTB than QFT-G or GIT and TST. However, both IGRAs and TST have similar specificity for EPTB. IGRAs have limited value as diagnostic tools to screen and rule out EPTB, especially in low/middle-income countries. The immune status of patients does not affect the diagnostic accuracy of IGRAs for EPTB.     

The ongoing challenge of latent tuberculosis

The global health community has set itself the task of eliminating tuberculosis (TB) as a public health problem by 2050. Although progress has been made in global TB control, the current decline in incidence of 2% yr⁻¹ is far from the rate needed to achieve this. If we are to succeed in this endeavour, new strategies to reduce the reservoir of latently infected persons (from which new cases arise) would be advantageous. However, ascertainment of the extent and risk posed by this group is poor. The current diagnostics tests (tuberculin skin test and interferon-gamma release assays) poorly predict who will develop active disease and the therapeutic options available are not optimal for the scale of the intervention that may be required. In this article, we outline a basis for our current understanding of latent TB and highlight areas where innovation leading to development of novel diagnostic tests, drug regimens and vaccines may assist progress. We argue that the pool of individuals at high risk of progression may be significantly smaller than the 2.33 billion thought to be immune sensitized by Mycobacterium tuberculosis and that identifying and targeting this group will be an important strategy in the road to elimination.     

Risk stratification in hemangiomas of infancy

Hemangiomas of infancy are very common tumors, but they are heterogeneous in their behavior. A small, but significant, subset causes medical complications or permanent disfigurement, but due to their heterogeneity, there is no appropriate "one size fits all" approach to management. In addition, the rapid evolution of this tumor over the first weeks-to-months of infancy renders even more difficult the task of predicting which infants will have medical complications or permanent disfigurement. This article outlines the clinical characteristics that help to stratify hemangiomas into those which are high risk, and likely to require either active treatment or closer scrutiny, and those which are low risk, and likely to behave in an innocuous manner. Five major factors are emphasized: the age of the child, the location of the hemangioma(s), the total number of hemangiomas present, the hemangioma subtype, and the presence and nature of dermal involvement.     

Suppression of rat cytomegalovirus replication by antibodies against gamma interferon.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.     

Identification of genes modulated by interferon gamma in breast cancer cells

Interferon gamma (IFNγ) plays a context-dependent dual tumor-suppressor and pro-tumorigenic roles in cancer. IFNγ induces morphological changes in breast cancer (BC) cells with or without estrogen receptor alpha (ERα) expression. However, IFNγ-regulated genes in BC cells remain unexplored. Here, we performed a cDNA microarray analysis of MCF-7 (ERα+) and MDA-MB-231 (HER2-/PR-/ERα-) cells with and without IFNγ treatment. We identified specific IFNγ?modulated genes in each cell type, and a small group of genes regulated by IFNγ common in both cell types. IFNγ treatment for an extended time mainly repressed gene expression shared by both cell types. Nonetheless, some of these IFNγ-repressed genes were seemingly deregulated in human mammary tumor samples, along with decreased IFNGR1 (an IFNγ receptor) expression. Thus, IFNγ signaling-elicited anti-tumor activities may be mediated by the downregulation of main IFNγ target genes in BC; however, it may be deregulated by the tumor microenvironment in a tumor stage-dependent manner.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.     

Morphological changes of human neuroblastoma cells induced by human gamma interferon

The treatment of GOTO cells, originated from human neuroblastoma, with recombinant human interferon-gamma (rHuIFN-gamma) induced the morphological changes: the extension and bifurcation of neurites and the multinucleated giant cell formation. The treatment of KP-N-RT cells, originated from human neuroblastoma, with rHuIFN-gamma also induced the similar morphological changes. The treatment of these cells with natural HuIFN-gamma also induced the same morphological changes, but those with recombinant human leukocyte interferon (rHuIFN-alpha A), recombinant human fibroblast interferon (rHuIFN-beta) and recombinant murine interferon-gamma (rMuIFN-gamma) did not induce it. The rHuIFN-gamma and the rHuIFN-beta inhibited more strongly the growth of GOTO and KP-N-RT cells than the rHuIFN-alpha A. This suggests that the morphological changes of these neuroblastoma cells are not simply due to the cell growth inhibition, but due to the property which only the rHuIFN-gamma possesses.     

Human interferon gamma is encoded by a single class of mRNA

Polyadenylated RNA from human peripheral blood lymphocytes and spleen cells, treated with different inducers for IFN-gamma production, was fractionated on denaturing sucrose gradients. Two IFN-gamma mRNA peaks at 12S and 16S were consistently observed. Nucleotide sequence analysis of cDNA clones showed that the 12S IFN-gamma mRNA from the different sources is identical to the gel fractionated 18S IFN-gamma mRNA which gave rise to the IFN-gamma cDNA clone p69 (1). Nucleotide sequence analysis of several IFN-gamma cDNA clones showed the presence of a CGA (Arg) codon at position 140 of mature IFN-gamma in contrast with the CAA (Gln) codon, which is found in p69 (1). Specifically primed cDNA extension on total induced polyadenylated RNA revealed the presence of a single mRNA species having a 5' untranslated region of 125-130 nucleotides. The nucleotide sequence of this region has been obtained. These data suggest that a single human IFN-gamma gene, which has very little polymorphism, gives rise to a single size class of mRNA.     

Mechanisms in the in vivo release of lymphokines. II. Regulation of in vivo release of type II interferon IFN gamma

The regulation of IFNγ release was studied in vivo in mice intravenously sensitized with cell walls of BCG (CW/Dr) and challenged with OT. Subcutaneous inoculation with CFA, but not with CW/Dr, following intravenous sensitization, reduced the titers of IFNγ. Similar inoculation prior to intravenous sensitization enhanced the titers of IFNγ in high- and low-responder mice. A substance that suppressed the activity of interferon was detected in the sera of sensitized and challenged low-responder strains.     

Comprehensive network map of interferon gamma signaling

Interferon gamma (IFN-γ), is a cytokine, which is an important regulator of host defense system by mediating both innate and adaptive immune responses. IFN-γ signaling is primarily associated with inflammation and cell-mediated immune responses. IFN-γ is also represented as antitumor cytokine which facilitates immunosurveillance in tumor cells. In addition, IFN-γ mediated signaling also elicits pro-tumorigenic transformations and promotes tumor progression. Impact of IFN-γ signaling in mammalian cells has been widely studied which indicate that IFN-γ orchestrates distinct cellular functions including immunomodulation, leukocyte trafficking, apoptosis, anti-microbial, and both anti- and pro-tumorigenic role. However, a detailed network of IFN-γ signaling pathway is currently lacking. Therefore, we systematically curated the literature information pertaining to IFN-γ signaling and develop a comprehensive signaling network to facilitate better understanding of IFN-γ mediated signaling. A total of 124 proteins were catalogued that were experimentally proven to be involved in IFN-γ signaling cascade. These 124 proteins were found to participate in 81 protein-protein interactions, 94 post-translational modifications, 20 translocation events, 54 activation/inhibiton reactions. Further, 236 differential expressed genes were also documented in IFN-γ mediated signaling. IFN-γ signaling pathway is made freely available to scientific audience through NetPath at (http://www.netpath.org/pathways?path_id=NetPath_32). We believe that documentation of reactions pertaining to IFN-γ signaling and development of pathway map will facilitate further research in IFN-γ associated human diseases including cancer.