Inhalation is an important route of infection with Burkholderia pseudomallei, the causative agent of melioidosis. In resistant C57BL/6 mice, activated neutrophils are rapidly recruited to the lungs after intranasal B. pseudomallei infection. Prevention of this response by use of the anti-Gr-1+ cell-depleting monoclonal antibody RBBL/6 interferon (IFN)-gamma(-/-) mice were also acutely susceptible to pulmonary B. pseudomallei infection, dying within 3 days of challenge; this suggests that IFN-gamma is essential for control in the lungs and precedes the protective role of neutrophils in resistance. In neutrophil-depleted mice, lung concentrations of tumor necrosis factor (TNF)-alpha, IFN-gamma, and interleukin-6 were decreased by up to 98%. Natural killer cells were the principle source of IFN-gamma, and monocytes were the principle source of TNF-alpha, suggesting that neutrophils play an important indirect role in the generation of the early cytokine environment in the lungs.

OBJECTIVE

The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis.

METHODS

Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst 33342-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-3beta, activated caspase-3 and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors.

RESULTS

Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-3 (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05).

CONCLUSIONS

The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.

BACKGROUND

Dysfunction of the immune system has been documented in many types of cancers. The precise nature and molecular basis of immune dysfunction in the cancer state are not well defined.

RESULTS

To gain insights into the molecular mechanisms of immune dysfunction in cancer, gene expression profiles of pure sorted peripheral blood lymphocytes from 12 patients with melanoma were compared to 12 healthy controls. Of 25 significantly altered genes in T cells and B cells from melanoma patients, 17 are interferon (IFN)-stimulated genes. These microarray findings were further confirmed by quantitative PCR and functional responses to IFNs. The median percentage of lymphocytes that phosphorylate STAT1 in response to interferon-alpha was significantly reduced (Delta = 16.8%; 95% confidence interval, 0.98% to 33.35%) in melanoma patients (n = 9) compared to healthy controls (n = 9) in Phosflow analysis. The Phosflow results also identified two subgroups of patients with melanoma: IFN-responsive (33%) and low-IFN-response (66%). The defect in IFN signaling in the melanoma patient group as a whole was partially overcome at the level of expression of IFN-stimulated genes by prolonged stimulation with the high concentration of IFN-alpha that is achievable only in IFN therapy used in melanoma. The lowest responders to IFN-alpha in the Phosflow assay also showed the lowest gene expression in response to IFN-alpha. Finally, T cells from low-IFN-response patients exhibited functional abnormalities, including decreased expression of activation markers CD69, CD25, and CD71; TH1 cytokines interleukin-2, IFN-gamma, and tumor necrosis factor alpha, and reduced survival following stimulation with anti-CD3/CD28 antibodies compared to controls.

CONCLUSIONS

Defects in interferon signaling represent novel, dominant mechanisms of immune dysfunction in cancer. These findings may be used to design therapies to counteract immune dysfunction in melanoma and to improve cancer immunotherapy.

As part of a study to identify novel genes associated with macrophage activation, we have cloned a new member of the transforming growth factor beta (TGF-beta) superfamily designated macrophage inhibitory cytokine 1 (MIC-1). MIC-1 is synthesized as a 62-kDa intracellular protein, which, after cleavage by a furin like protease, is secreted as a 25-kDa disulfide-linked dimeric protein. Sequence analysis indicates that it does not cluster within any existing TGF-beta families, suggesting it may be the first member of a new grouping within the TGF-beta superfamily. Tissue Northern blots show that MIC-1 transcripts are only found abundantly in placenta, although smaller amounts are seen in a limited number of other adult and fetal tissues. MIC-1 is not expressed in resting macrophages but is induced by a number of different activation agents, including phorbol myristate acetate, interleukin 1, tumor necrosis factor alpha, and macrophage colony-stimulating factor but not by lipopolysaccharide or interferon-gamma. We have hypothesized that it may be an autocrine inhibitor of macrophage activation but its major biological role is still uncertain.

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.

G to A hypermutation of the human immunodeficiency virus type 1 (HIV-1) is induced by a deaminase APOBEC3G and is related to host antiviral defense. APOBEC3G has also been found to reduce the replication of HIV-1 by an unknown mechanism. This enzyme also reduces the production of hepatitis B virus, although the mechanism for this action has not been clearly elucidated. The hypermutated hepatitis B virus (HBV) is rarely found in usual sequencing analyses. Using peptide nucleic acid mediated by polymerase chain reaction clamping, we detected the hypermutated HBV DNA in 1 of 8 patients with acute HBV infection and 4 of 10 with chronic HBV infection. In the latter group, hypermutated genomes were found only in eAb-positive patients. As much as 72.5% of G residues were mutated in the hypermutated clones. G to A substitutions were predominant in almost all clones sequenced compared with other substitutions. G to A mutated viral genomes also were found in HepG2-derived cell lines that continuously produced HBV into the supernatant. Both alpha and gamma interferon reduced virus production in these cell lines, but they did not alter the frequency of the hypermutation. Transcripts of APOBEC3G, as well as some other deaminases, were found in these cell lines. In conclusion, our results show that part of the minus strand DNA of HBV is hypermutated both in vitro (HepG2 cell lines) and in vivo. The role and mechanism of hypermutation in reducing HBV replication should be further investigated to understand the anti-HBV defense system.

Transcriptional induction of genes is an essential part of the cellular response to interferons. To isolate yet unidentified IFN-regulated genes we have performed a differential screening on a cDNA library prepared from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta interferon (Hu-alpha/beta IFN). In the course of these studies we have isolated a human cDNA which codes for a protein sharing homology with the mouse Rpt-1 gene; it will be referred as Staf-50 for Stimulated Trans-Acting Factor of 50 kDa. Amino acid sequence analysis revealed that Staf-50 is a member of the Ring finger family and contains all the features of a transcriptional regulator able to initiate a second cascade of gene induction (secondary response). Staf-50 is induced by both type I and type II IFN in various cell lines and down-regulates the transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type 1 in transfected cells. These data are consistent with a role of Staf-50 in the mechanism of transduction of the IFN antiviral action.

Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural killer cell proliferation and displays unusually tight controls of secretion. Even though IL-15 mRNA is constitutively expressed in monocytes/macrophages and is upregulated by a variety of stimuli, evidence for IL-15 cytokine secretion is only found exceptionally, eg, conditions of pathological, chronic inflammation. This raises the possibility that monocytes express membrane-bound IL-15 rather than secrete it. The current study explores this hypothesis. We demonstrate here that biologically active IL-15 is indeed detectable in a constitutively expressed, membrane-bound form on normal human monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI, RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore, cell surface-bound IL-15 is upregulated upon interferon-gamma stimulation. Interestingly, monocyte/macrophage inhibitory cytokines such as IL-4 and IL-13 fail to downregulate both constitutive and induced cell-surface expression of IL-15. Membrane-bound IL-15 does not elute with acetate buffer or trypsin treatment, suggesting that it is an integral membrane protein and that it is not associated with the IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T lymphocytes to proliferate in vitro, indicating that it is biologically active. These findings enlist IL-15 in the fairly small family of cytokines for which the presence of a biologically active membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis factor-alpha, and IL-10) and invites the speculation that most of the biological effects of IL-15 under physiological conditions are exerted by the cell surface-bound form.

Plasmacytoid dendritic cells (pDCs), the professional producers of type I interferons (IFN-alpha/beta), play a pivotal role in innate and adaptive immune responses against viral infections. Although functional impairment of circulating pDCs in chronic hepatitis B (CHB) patients has been reported previously, the mechanism responsible for these defects remains unclear. We hypothesize that HBsAg circulating in high amounts during HBV infection may interact with pDC and contribute to pDC dysfunction. In support of this hypothesis we show that pDCs treated with HBsAg secreted much less IFN-alpha than control pDCs. Furthermore, suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-alpha secretion. HBsAg inhibited TLR9-mediated IRF-7 expression and nuclear translocation, which are important for induction of IFN-alpha gene transcription. HBsAg upregulated the SOCS-1 expression and bound to BDCA-2 receptors on the plasma membrane of pDCs, resulting in the inhibition of the IFN-alpha production. In conclusion, the above data suggested that HBsAg may directly interfere with the function of pDC through HBsAg-mediated upregulation of SOCS-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.

Theiler's virus is a picornavirus responsible for a persistent infection of the central nervous system of the mouse, leading to a chronic demyelinating disease considered to be a model for multiple sclerosis. The leader (L) protein encoded by Theiler's virus is a 76-amino-acid-long peptide containing a zinc-binding motif. This motif is conserved in the L proteins of all cardioviruses, including encephalomyocarditis virus. The L protein of Theiler's virus was suggested to interfere with the alpha/beta interferon (IFN-alpha/beta) response (W.-P. Kong, G. D. Ghadge, and R. P. Roos, Proc. Natl. Acad. Sci. USA 91:1796-1800, 1994). We show that expression of the L protein indeed inhibits the production of alpha/beta interferon by infected L929 cells. The L protein specifically inhibits the transcription of the IFN-alphabeta genes, which are known to be activated early in response to viral infection. Mutation of the zinc finger was sufficient to block the anti-interferon activity, outlining the importance of this motif in the L protein function. In agreement with the anti-interferon role of the L protein, a virus bearing a mutation in the zinc-binding motif was dramatically impaired in its ability to persist in the central nervous system of SJL/J mice.

Cytokine release at the cartilage/pannus junction (CPJ) may be involved in cartilage destruction and tissue repair in rheumatoid arthritis (RA). Tissue samples of CPJ from 12 RA patients were examined for the presence of cytokines using immunohistochemical techniques with immunoaffinity purified F(ab')2 antibodies raised against recombinant human cytokines. Twenty-four areas of distinct CPJ at which a discrete junction between cartilage and overlying pannus exists were observed. In all specimens, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha. IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta 1 were detected in cells in pannus particularly along the surface of cartilage and at the site of cartilage erosion. Double immunofluorescence staining showed that most cytokine containing cells also labelled with a macrophage marker (CD68). About 50% of blood vessel endothelial cells stained for GM-CSF. Twelve areas of diffuse fibroblastic CPJ, at which an indistinct margin is seen between cartilage and pannus were examined. At this site, TGF-beta 1 was the only cytokine detected in fibroblast-like cells. None of these cytokines were detected in synovial tissue at the normal synovium/cartilage junction. Chondrocytes from all 11 normal specimens as well as those from RA patients stained for IL-1 alpha, TNF-alpha, IL-6, GM-CSF and TGF-beta 1, especially those close to subchondral bone. However, IL-1 beta, interferon-gamma and lymphotoxin were not detected in either the normal synovium/cartilage junction or rheumatoid CPJ.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferons (IFNs) inhibit the replication of a wide range of animal viruses by acting at various steps of the life cycle. Interferons display a particularly potent antiviral effect on HIV-1 replication in primary human macrophages. A high virus-to-cell multiplicity of infection was used to investigate which steps in a single replicative cycle in these primary human cells were affected by IFNs. Monocyte-derived macrophages from healthy seronegative donors were infected with HIV-1BaL. Virus production was assessed by immunoassay for p24 antigen. Viral DNA was detected by PCR while mRNA was detected specifically by RT-PCR with primers bracketing the 5' introns of HIV-1 to detect only spliced transcripts such as tat, rev, nef, and env mRNAs. Macrophages pretreated with IFN-alpha, -beta or -gamma had a reduced viral DNA signal while the spliced mRNA signal was essentially abolished. No virus was produced. To test whether IFNs could reduce HIV transcripts in cells with established productive infection, macrophages were infected and reinfection was then prevented by azidothymidine before starting interferon treatment. Under such conditions, the addition of interferons did not affect significantly the levels of HIV spliced transcripts. No intracellular accumulation of p24 antigen was observed. Therefore, the major effect of IFNs was at an early step of the virus life cycle and resulted in a reduced viral DNA synthesis.

We investigated the cis-acting sequences that function in the B-cell-specific and interferon-gamma-inducible expression of the HLA-DR alpha gene, a human class II major histocompatibility complex gene. The effects of 5' deletions on the activity of the DR alpha promoter and the influence of upstream DR alpha promoter elements on the activity of the herpes simplex virus thymidine kinase promoter were examined by a transient transfection assay in human B-, T-, and fibroblast cell lines. We show that the DR alpha gene is regulated by positive and negative cis-acting sequences between positions -1300 and +31 from the site of initiation of transcription. We also demonstrate that the DR alpha promoter sequences from positions -116 to -92 and from -136 to -80 are the minimal sequences required for conferring B-cell specificity and interferon-gamma inducibility upon the Herpes simplex virus thymidine kinase promoter, respectively.

Diabetes susceptibility in non-obese diabetic (NOD) mice may entail faulty activation of immunoregulatory cells resulting from cytokine deficiencies. Depletion of T cells prevents disease onset in these mice. Since we had previously shown that IL-2 treatment in vivo restored the ability of NOD/Lt mice to produce self-restricted suppressor T cells (Ts) in a syngeneic mixed lymphocyte reaction (SMLR), we investigated the possibility that diabetes could be circumvented by treatment with immunostimulatory agents that increase cytokine production. By 20 weeks of age, 75% of vehicle-treated NOD/Lt female controls had become glycosuric, while glycosuria developed in only 17% of NOD/Lt females injected with human recombinant interleukin-2 (rIL-2, 250 U twice weekly) beginning at 6 weeks of age. Treatment of mice with Poly [I:C] alone [50 micrograms twice weekly, an inducer of Interferon (IFN) alpha/beta] or in conjunction with rIL-2 was even more effective, completely preventing glycosuria for 20 weeks. However, therapeutic effects required continuous administration of the immunostimulants since pancreatic insulin content declined and severity of insulitis increased following cessation of treatment. IL-2 treatment increased transcription of interleukin-1 (IL-1) mRNA in peritoneal macrophages and increased lipopolysaccharide (LPS)-stimulated IL-1 secretion in comparison to controls. In the presence of stimulators from IL-2-treated mice, T lymphocytes isolated from both controls and IL-2-treated NOD/Lt mice proliferated in a SMLR and acquired Ts function. Peritoneal macrophages from Poly [I:C]-treated mice exhibited increased IFN alpha gene transcription and LPS-stimulated IL-1 secretion. T cells isolated from Poly [I:C]-treated mice were capable of suppressing NOD-Lt T cell responses to alloantigens in a mixed lymphocyte culture without prior activation in a SMLR. Thus, Poly [I:C] treatment may recruit a different population of regulatory cells than those elicited by treatment with IL-2. However, the mechanisms by which autoreactive T-cell clones may be regulated by these two treatments in NOD/Lt mice may be synergistic. These results indicate that in addition to T-cell depletion protocols, diabetes in NOD mice can be prevented by treatment with immunostimulatory agents.

Interferon (IFN)-alpha, IFN-beta, and IFN-gamma are currently available for the treatment of malignancies, viral infections (e.g., hepatitis C virus), multiple sclerosis (MS), and skin conditions. In addition to their therapeutic effects, IFNs commonly cause various side effects. Most common among the side effects of IFN are "flu-like" symptoms such as chills, fever, and muscle soreness. However, IFN can also cause significant neuropsychiatric side effects, particularly symptoms of depression. A literature search was conducted in order to summarize current information on (1) the frequency, characteristics, and risk factors of IFN-induced depression, (2) possible biochemical mechanisms associated with IFN-induced depression, and (3) the treatment strategies for IFN-induced depression. Review of the literature suggests that symptoms of depression induced by IFN therapy, in particular IFN-alpha therapy, are common and can limit the treatment utility, often necessitating discontinuation of IFN therapy or the use of psychopharmacologic agents. Depression is also a suspected side effect of therapy with IFN-beta and IFN-gamma; however, the association has not been as convincingly confirmed. Importantly, IFNs affect neurochemical pathways putatively involved in the etiology of depression. While these depressive side effects usually resolve after the completion of IFN therapy, they can persist or reappear with dose escalations. It is recommended that health care providers, patients and their families be informed about the potential risk of the psychiatric disturbances that can occur with IFN-alpha therapy. Screening and monitoring, ideally using symptom rating scales for depression, and early antidepressant treatment intervention appear necessary to optimize IFN therapy for the majority of patients.

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

The type I interferon (IFN) system responds to viral infection and induces an "antiviral state" in cells, providing an important first line of defense against virus infection. Interaction of type I IFNs (IFN alpha and IFN beta) with their receptor induces hundreds of cellular genes. Of the proteins induced by IFN, the antiviral function of only a few is known, and their mechanisms of action are only partly understood. Additionally, although viral-encoded mechanisms that counteract specific components of the type I IFN response have been known for some time, it has recently become clear that many (if not most) viruses encode some form of IFN-antagonist. Understanding the interplay between viral-encoded IFN antagonists and the interferon response will be essential if the therapeutic potential of IFNs is to be fully exploited.

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3-11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirBB. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-alpha and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4+ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88-/- mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-gamma-dependent CD4+ T cell polarization and antibody switching.

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-alpha genes. A network-based analysis suggests that the synergy between IFN-beta and TNF-alpha results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.

Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-alpha/beta production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.

Pulmonary fibrosis can complicate diverse pulmonary and systemic pathologies. In many cases the underlying cause remains unidentified. Mortality from the disease is increasing steadily in the UK and USA. The clinical features are well-described, but patients frequently present at an advanced stage, and current treatments have not improved the poor prognosis. There is a compelling need to identify the fibrotic process earlier and to develop new therapeutic agents. Increased collagen deposition is central to the pathology and interest over the last decade has focused on the role of cytokines in this process. These polypeptide mediators are believed to be released from both circulating inflammatory and resident lung cells in response to endothelial and epithelial injury. Key cytokines currently implicated in the fibrotic process are transforming growth factor-beta, tumour necrosis factor-alpha and endothelin-1. This article outlines the evidence implicating these mediators in the pathogenesis of pulmonary fibrosis and also considers the possible role of cytokines with antifibrotic effects, such as interferon-gamma. The "balance" of positively and negatively regulating cytokines is discussed, and the potential for interaction with other factors including viruses, hormones and altered antioxidant status is also considered. Finally, potential novel therapeutic approaches are discussed, together with suggestions for future studies and clinical trials. As the outcomes of different avenues of research over the last ten years are brought together, it is clear that there is now a hitherto unrivalled opportunity to begin to tackle the treatment of this devastating disease.

The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L-/- mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L-/- mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-alpha/beta signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.

Spontaneous inflammatory bowel disease (IBD) resembling human ulcerative colitis develops in mice mutant for the T cell receptor alpha gene (TCR-alpha-/-). TCR-alpha-/- mice lack TCR-alpha/beta+ cells but contain TCR-gamma/delta+ cells and a small population of a unique CD4+, TCR-alpha-/beta+(low) cells. Since all the immunoglobulin (Ig) classes are present in these mice, help to B cells must be provided by cells other than TCR-alpha/beta+ cells. In the present study, we found serum levels of IgG1 and IgG2 to be markedly increased in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD or TCR-alpha+/- controls. An increase in IgG1-, IgG2a- and IgA- but not IgM-secreting mesenteric lymph node (MLN) B cells was detected in TCR-alpha-/- mutant mice. There was also a marked increase in MLN B cells secreting autoantibody (IgG) to tropomyosin, a cytoskeletal protein. Examination of the hyperplastic MLN showed a marked increase in the number of B, TCR-delta+, and CD4+ TCR-alpha-/beta+ cells, similar to the cell population observed at the site of colonic inflammation. Analysis of spontaneous cytokine production by MLN cells using an enzyme-linked immunospot assay, immunohistochemistry, and reverse transcription/polymerase chain reaction showed a decrease of interleukin 2 (IL-2) but a marked increase of IL-4 and interferon gamma (IFN-gamma) production in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD and TCR alpha+/- control mice. Both TCR-alpha-/beta+ and TCR-delta+ cells were found to be capable of producing IL-4; IFN-gamma was produced mostly by non-T cells, many of which were shown to be CD3- NK 1.1+ cells. We propose that the cytokine imbalance present in these mice results in expansion of B cells, production and switching of autoantibodies to IgG2 subclass, and development of IBD. It is possible that the unusual CD4+ TCR-alpha-/beta+ population and expanded TCR-gamma/delta+ population present in TCR-alpha-/- mice plays a central role in this abnormal immune response.

To investigate the mechanisms of exercise-induced immune perturbations, we measured promising immunomodulatory hormones and cytokines in plasma of 16 male marathon runners before and after a competitive 42.195-km race. Interleukin 1-beta (IL-1beta) and interferon gamma (IFN-gamma) concentrations remained unchanged after the marathon. The cytokines IL-12, IFN-alpha and tumour necrosis factor alpha (TNF-alpha) could not be detected even using highly sensitive specific immunoassays, indicating at least that overshooting responses of these cytokines had not occurred after exercise. As mechanisms for the small changes in these cytokines, we demonstrated for the first time a significant rise in concentrations of inhibitory cytokine IL-10 in addition to the immunosuppressive hormone cortisol, although concentrations of IL-4 and transforming growth factor-beta (TGF-beta) were unaffected by the race. Furthermore, concentrations of IL-1 receptor antagonist (IL-1ra) and IL-6, which are negative-feedback inhibitors of cytokine production, increased by more than 100 times. As for humoral mediators of neutrophil mobilization, concentrations of growth hormone (GH), cortisol and granulocyte colony-stimulating factor (G-CSF) increased significantly. In addition, concentrations of neutrophil-priming substances (IL-6, IL-8, G-CSF, GH and prolactin) also increased significantly and the induction of IL-8 and G-CSF with exercise was demonstrated for the first time in the present study. In contrast, IL-2 concentration decreased, by 32%, and this was correlated with the induction of nitric oxide (NO) production. Muscle damage, monitored using changes in concentrations of creatine kinase and myoglobin, was also observed. These results suggested that exercise-induced pathogenesis including previously reported immunosuppression and neutrophil hyper-reactivity might be attributed, at least partly, to the systemic dynamics of the above bioactive substances.