We have previously shown that the nonstructural (NS) proteins NS1 and NS2 of bovine respiratory syncytial virus (BRSV) mediate resistance to the <em>alpha</em>/beta <em>interferon</em> (IFN)-mediated antiviral response. Here, we show that they, in addition, are able to prevent the induction of beta IFN (IFN-beta) after virus infection or double-stranded RNA stimulation. In BRSV-infected MDBK cells upregulation of IFN-stimulated genes (ISGs) such as MxA did not occur, although IFN signaling via JAK/STAT was found intact. In contrast, infection with recombinant BRSVs lacking either or both NS genes resulted in efficient upregulation of ISGs. Biological IFN activity and IFN-beta were detected only in supernatants of cells infected with the NS deletion mutants but not with wild-type (wt) BRSV. Subsequent analyses of IFN-beta promoter activity showed that infection of cells with the double deletion mutant BRSV DeltaNS1/2, but not with BRSV wt, resulted in a significant increase in IFN-beta gene promoter activity. Induction of the IFN-beta promoter depends on the activation of three distinct transcription factors, NF-kappaB, ATF-2/c-Jun, and IFN regulatory factor <em>3</em> (IRF-<em>3</em>). Whereas NF-kappaB and ATF-2/c-Jun activities were readily detectable and comparable in both wt BRSV- and BRSV DeltaNS1/2-infected cells, phosphorylation and transcriptional activity of IRF-<em>3</em>, however, were observed only after BRSV DeltaNS1/2 infection. NS protein-mediated inhibition of IRF-<em>3</em> activation and IFN induction should have considerable impact on the pathogenesis and immunogenicity of BRSV.

Viral double-stranded RNA (dsRNA) generated during the course of infection leads to the activation of a latent transcription factor, dsRNA-activated factor 1 (DRAF1). DRAF1 binds to a DNA target containing the type I <em>interferon</em>-stimulated response element and induces transcription of responsive genes. DRAF1 is a multimeric transcription factor containing the <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) protein and one of the histone acetyl transferases, CREB binding protein (CBP) or p<em>3</em>00 (CBP/p<em>3</em>00). In uninfected cells, the IRF-<em>3</em> component of DRAF1 resides in the cytoplasm. The cytoplasmic localization of IRF-<em>3</em> is dependent on a nuclear export signal, and we demonstrate IRF-<em>3</em> recognition by the chromosome region maintenance 1 (CRM1) (also known as exportin 1) shuttling receptor. Following infection and specific phosphorylation, IRF-<em>3</em> accumulates in the nucleus where it associates with CBP and p<em>3</em>00. We identify a nuclear localization signal (NLS) in IRF-<em>3</em> that is critical for nuclear accumulation. Mutation of the NLS abrogates nuclear localization even following infection. The NLS appears to be active constitutively, but it is recognized by only a subset of importin-<em>alpha</em> shuttling receptors. Evidence is presented to support a model in which IRF-<em>3</em> normally shuttles between the nucleus and the cytoplasm but cytoplasmic localization is dominant prior to infection. Following infection, phosphorylated IRF-<em>3</em> can bind to the CBP/p<em>3</em>00 proteins resident in the nucleus. We provide the evidence of a role for CBP/p<em>3</em>00 binding in the nuclear sequestration of a transcription factor that normally resides in the cytoplasm.

OBJECTIVE

Commensal bacteria are crucial for the development of the mucosal immune system. Probiotics are commensals with special characteristics and may protect mucosal surfaces against pathogens. Pathogens cause significant phenotypic alterations in infected epithelial cells, and probiotics reverse these deleterious responses. We hypothesized that probiotics and/or commensals may also reverse epithelial damage produced by cytokines.

METHODS

Human intestinal epithelial cells were exposed basolaterally to <em>interferon</em> (IFN)-gamma (10(<em>3</em>) U/mL) or tumor necrosis factor (TNF)-<em>alpha</em> (10 ng/mL) for up to 48 hours and assessed for ion transport, transepithelial resistance (TER), and epithelial permeability in the presence or absence of probiotics (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]), or the commensal, Bacteroides thetaiotaomicron (BT).

RESULTS

Agonist-stimulated chloride secretion was inhibited by IFN-gamma, an effect prevented by ST/LA or BT. The ability of ST/LA or BT to restore Cl(-) secretion was blocked by inhibitors of p<em>3</em>8 MAPK, ERK1, 2, and PI<em>3</em>K. The cystic fibrosis transmembrane conductance regulator (CFTR) and the NKCC1 cotransporter were down-regulated by IFN-gamma, and ST/LA pretreatment reversed this effect. Both TNF-<em>alpha</em> and IFN-gamma significantly reduced TER and increased epithelial permeability, effects prevented by ST/LA or BT. A Janus kinase (JAK) inhibitor synergistically potentiated effects of ST/LA or BT on TER and permeability, but p<em>3</em>8, ERK1, 2, or PI<em>3</em>K inhibition did not. Finally, only probiotic-treated epithelial cells exposed to cytokines showed reduced activation of SOCS<em>3</em> and STAT1,<em>3</em>.

CONCLUSIONS

Deleterious effects of TNF-alpha and IFN-gamma on epithelial function are prevented by probiotic, and to a lesser extent, commensal pretreatment. These data extend the spectrum of effects of such bacteria on intestinal epithelial function and may justify their use in inflammatory disorders.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (<em>3</em> mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor <em>alpha</em> and <em>interferon</em> gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.

<em>Interferon</em> gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or <em>3</em> weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (<em>alpha</em>-smooth-muscle actin [<em>alpha</em>-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by <em>alpha</em>-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.

BACKGROUND

The pathogenesis of myocarditis that occurs in Trypanosoma cruzi-infected mice is still poorly understood. Therefore, it is important to know the mediators that trigger leukocyte migration to the heart as well as the cellular source of these possible mediators. In this study, we investigated (1) NO synthase (NOS) induction, (2) NO synthesis, (<em>3</em>) trypanocidal activity, and (4) chemokine and cytokine mRNA expression by isolated cardiomyocytes infected with T cruzi.

RESULTS

Mouse cardiomyocytes were isolated, infected with T cruzi, and evaluated for induction of inducible NOS (iNOS), nitrite production, trypanocidal activity, and cytokine and chemokine mRNA expression. We found that T cruzi-infected murine embryonic cardiomyocytes produced nitrite and expressed mRNAs for the chemokines chemokine growth-related oncogene, monokine induced by interferon-gamma, macrophage inflammatory protein-2, interferon-gamma-inducible protein, RANTES, and monocyte chemotactic protein, for iNOS, and for the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Separate addition of IL-1beta, interferon-gamma, TNF-alpha or monocyte chemotactic protein, macrophage inflammatory protein-2, and interferon-gamma-inducible protein, to cultured cardiomyocytes resulted in NO production but low trypanocidal activity. However, simultaneous addition of IL-1beta, interferon-gamma, and TNF-alpha or the chemokines to cultures resulted in the induction of iNOS, high levels of nitrite, and a marked trypanocidal activity. The iNOS/L-arginine pathway mediated the latter activity, inasmuch as it was inhibited by treatment with N:(G)-monomethyl-L-arginine.

CONCLUSIONS

These results indicate that iNOS activation and the proinflammatory cytokines and chemokines produced by cardiomyocytes are likely to control parasite growth and cell influx, thus contributing to the pathogenesis of chagasic cardiomyopathy seen in T cruzi-infected mice.

BACKGROUND

Prospective data on incidence, characteristics, and risk factors for cryptococcal meningitis immune reconstitution inflammatory syndrome (CM-IRIS) are lacking.

METHODS

Prospective study of 65 antiretroviral therapy (ART)-naive HIV-infected cryptococcal meningitis (CM) patients, who started ART after initiation of antifungal treatment. CM-IRIS definition: (1) cerebrospinal fluid (CSF) culture-confirmed CM, (2) symptom resolution before starting ART, (<em>3</em>) adherence to fluconazole and ART, (4) recurrence of CM symptoms after starting ART, (5) immunologic and/or virologic response to ART, (6) no alternative diagnosis.

RESULTS

ART was started at a median of 47 days from CM diagnosis. CM-IRIS developed in 11 of 65 (17%), at a median 29 days from starting ART. No factors at first CM episode (fungal burden, rate of clearance, CSF, or HIV parameters) predicted those at risk of CM-IRIS. At 6 months on ART, IRIS patients had greater CD4 rise from baseline (220 vs. 124 x 10 cells /L in non-IRIS, P = 0.01), and 4 of 11 CM-IRIS patients died compared with 14 of 54 non-IRIS patients (P = 0.5). For those developing CM-IRIS, CSF proinflammatory cytokines interferon gamma, tumour necrosis factor alpha, and interleukin 6, did not differ between first CM and CM-IRIS episode.

CONCLUSIONS

Patients with CM-IRIS had greater immune restoration in response to ART. Although common and potentially fatal, larger prospective studies are needed to determine whether CM-IRIS, in patients treated initially with amphotericin B, is associated with any increase in overall mortality.

PML nuclear bodies (NBs) are nuclear matrix-associated structures altered by viruses and oncogenes. We show here that PML overexpression induces rapid cell death, independent of de novo transcription and cell cycling. PML death involves cytoplasmic features of apoptosis in the absence of caspase-<em>3</em> activation, and caspase inhibitors such as zVAD accelerate PML death. zVAD also accelerates <em>interferon</em> (IFN)-induced death, suggesting that PML contributes to IFN-induced apoptosis. The death effector BAX and the cdk inhibitor p27KIP1 are novel NB-associated proteins recruited by PML to these nuclear domains, whereas the acute promyelocytic leukaemia (APL) PML/RAR <em>alpha</em> oncoprotein delocalizes them. Arsenic enhances targeting of PML, BAX and p27KIP1 to NBs and synergizes with PML and IFN to induce cell death. Thus, cell death susceptibility correlates with NB recruitment of NB proteins. These findings reveal a novel cell death pathway that neither requires nor induces caspase-<em>3</em> activation, and suggest that NBs participate in the control of cell survival.

Th1 and Th2 lymphocytes express a different repertoire of chemokine receptors (CCRs). CXCR<em>3</em>, the receptor for I-TAC (<em>interferon</em>-inducible T cell <em>alpha</em>-chemoattractant), Mig (monokine induced by gamma-<em>interferon</em>), and IP10 (<em>interferon</em>-inducible protein 10), is expressed preferentially on Th1 cells, whereas CCR<em>3</em>, the receptor for eotaxin and several other CC chemokines, is characteristic of Th2 cells. While studying responses that are mediated by these two receptors, we found that the agonists for CXCR<em>3</em> act as antagonists for CCR<em>3</em>. I-TAC, Mig, and IP10 compete for the binding of eotaxin to CCR<em>3</em>-bearing cells and inhibit migration and Ca(2+) changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-<em>3</em>, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A hybrid chemokine generated by substituting the first eight NH(2)-terminal residues of eotaxin with those of I-TAC bound CCR<em>3</em> with higher affinity than eotaxin or I-TAC (<em>3</em>- and 10-fold, respectively). The hybrid was 5-fold more potent than I-TAC as an inhibitor of eotaxin activity and was effective at concentrations as low as 5 nm. None of the antagonists described induced the internalization of CCR<em>3</em>, indicating that they lack agonistic effects and thus qualify as pure antagonists. These results suggest that chemokines that attract Th1 cells via CXCR<em>3</em> can concomitantly block the migration of Th2 cells in response to CCR<em>3</em> ligands, thus enhancing the polarization of T cell recruitment.

Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or <em>interferon</em> (IFN)-gamma/TNF-<em>alpha</em>, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through G<em>alpha</em>(s) protein and prevented serum starvation- and IFN-gamma/TNF-<em>alpha</em>-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol <em>3</em>-kinase (PI<em>3</em>K)/Akt and ERK1/2, whereas PI<em>3</em>K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI<em>3</em>K/Akt.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8(+) T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire <em>alpha</em>-fetoprotein (AFP), glypican-<em>3</em> (GPC-<em>3</em>), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8(+) T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected <em>interferon</em>-γ (IFN-γ)-producing CD8(+) T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8(+) T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8(+) T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8(+) T-cell proliferation but did not restore IFN-γ-production.

CONCLUSIONS

Naturally occurring TAA-specific CD8(+) T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8(+) T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8(+) T-cell responses.

P56 is the most abundant protein induced by <em>interferon</em> (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-<em>alpha</em>/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-<em>3</em> as judged by its translocation to the nucleus from the cytoplasm.

The VP<em>3</em>5 protein of Ebola virus is a viral antagonist of <em>interferon</em>. It acts to block virus or double-stranded RNA-mediated activation of <em>interferon</em> regulatory factor <em>3</em>, a transcription factor that facilitates the expression of <em>interferon</em> and <em>interferon</em>-stimulated genes. In this report, we show that the VP<em>3</em>5 protein is also able to inhibit the antiviral response induced by <em>alpha</em> <em>interferon</em>. This depends on the VP<em>3</em>5 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP<em>3</em>5 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with <em>alpha</em>/beta <em>interferon</em>, displaying an <em>interferon</em>-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2<em>alpha</em> was suppressed in cells expressing the VP<em>3</em>5 protein. This activity of the VP<em>3</em>5 protein was required for efficient viral replication in PKR+/+ but not PKR-/- mouse embryo fibroblasts. Furthermore, VP<em>3</em>5 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R<em>3</em>12A substitution in the RNA binding motif, abolished the ability of the VP<em>3</em>5 protein to confer viral resistance to <em>interferon</em>. However, the R<em>3</em>12A substitution rendered the VP<em>3</em>5 protein unable to inhibit the induction of the beta <em>interferon</em> promoter mediated by virus infection. Together, these results show that the VP<em>3</em>5 protein targets multiple pathways of the <em>interferon</em> system.

Cytokines, signaling molecules of the immune system, have been implicated as a contributing factor for mood disorders such as depression. Several lines of evidence supporting this contention are briefly reviewed and caveats are introduced. Essentially, a relationship between cytokines and depression is based on the findings that: 1) proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor-<em>alpha</em>) and bacterial endotoxins elicit sickness behaviors (e.g., fatigue, soporific effects) and symptoms of anxiety/depression that may be attenuated by chronic antidepressant treatment, 2) cytokines induce neuroendocrine and central neurotransmitter changes reminiscent of those implicated in depression, and these effects are exacerbated by stressors, <em>3</em>) severe depressive illness is accompanied by signs of immune activation and by elevations of cytokine production or levels, and 4) immunotherapy, using interleukin-2 or <em>interferon</em>-<em>alpha</em>, promotes depressive symptoms that are attenuated by antidepressant treatment. It is argued that cytokine synthesis and release, elicited upon activation of the inflammatory response system, provoke neuroendocrine and brain neurotransmitter changes that are interpreted by the brain as being stressors, and contribute to the development of depression. Furthermore, such effects are subject to a sensitization effect so that a history of stressful experiences or cytokine activation augment the response to later challenges and hence the evolution of depression

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKK<em>alpha</em> subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKK<em>alpha</em>'s physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKK<em>alpha</em> is also a global regulator of tumor necrosis factor <em>alpha</em>- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A<em>3</em>, C<em>3</em>, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx<em>3</em>, beta(2)-microglobulin, IL-1<em>alpha</em>, Mcp-1 and -<em>3</em>, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKK<em>alpha</em> or IKKbeta. Constitutive expression of a trans-dominant IkappaB<em>alpha</em> superrepressor (IkappaB<em>alpha</em>SR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 1<em>3</em>, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, <em>interferon</em> (<em>alpha</em> and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and <em>3</em>, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-<em>3</em>, a negative effector of STAT<em>3</em> signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta <em>interferon</em> (IFN-beta) promoter and the IFN regulatory factor <em>3</em> (IRF<em>3</em>)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor <em>3</em> (TLR<em>3</em>) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 4<em>3</em>9, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin-<em>alpha</em> <em>3</em> and karyopherin-<em>alpha</em> 4. Nuclear localization of W protein therefore enables it to target both virus and TLR<em>3</em> pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKKepsilon. We demonstrate that, when the TLR<em>3</em> pathway is stimulated, the levels of phosphorylated IRF<em>3</em> are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF<em>3</em>.

Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately <em>3</em> months. In contrast, the initial onset of virus-specific <em>interferon</em> (IFN)-gamma-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-gamma SC (comprising predominantly CD4/CD8 <em>alpha</em> double positive memory T cells with a minority being typical CD4(-)/CD8 <em>alpha</em> beta(+) T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-gamma response and preferentially stimulates the synthesis of antibodies incapable of neutralization.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor <em>alpha</em>, interleukin 1 beta, and <em>interferon</em> gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first <em>3</em>.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, <em>3</em>- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -<em>3</em>.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and <em>interferon</em> gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

The purpose of the study was to identify a comprehensive prognostic system of pretreatment clinical parameters in 425 patients (pts) with metastatic renal-cell carcinoma treated with different subcutaneous (s.c.) recombinant cytokine-based home therapies in consecutive trials. Treatment consisted of (A) s.c. <em>interferon</em>-<em>alpha</em> 2a (INF-<em>alpha</em>), s.c. interleukin-2 (IL-2) (n=102 pts), (B) s.c. IFN-<em>alpha</em> 2a, s.c. IL-2, and i.v. 5-fluorouracil (5-FU) (n=2<em>3</em>5 pts) or (C) s.c. IFN-<em>alpha</em> 2a, s.c. IL-2, and i.v. 5-FU combined with p.o. 1<em>3</em>-cis-retinoic acid (1<em>3</em>cRA) (n=88 pts). Kaplan-Meier survival analysis, log-rank statistics, and Cox regression analysis were employed to identify risk factors and to create a multiple risk factor model. The following pretreatment risk factors were identified by univariate analysis: (1) three and more metastatic sites, (2) presence of liver, lymph node or bone metastases, (<em>3</em>) neutrophil count>> or = 6500 cells microl(-1), (4) serum lactate dehydrogenase level (LDH)>> or = 220 U l(-1), and (5) serum C-reactive protein level (CRP)>> or = 11 mg l(-1). Cox regression analysis with forward stepwise variable selection identified neutrophil count as the major prognostic factor (hazard ratio=1.9, P<0.001), while serum levels of LDH and CRP, time between diagnosis of tumour and onset of metastatic disease, number of metastatic sites, and bone metastases were significant but somewhat less important prognostic variables within the multiple risk factor model (hazard ratio < or = 1.5). Patients were assigned to one of the three risk groups according to cumulative risk defined as the sum of simplified risk s.c.ores for six pretreatment variables. Low-, intermediate-, and high-risk patients achieved a median overall survival of <em>3</em>2+ months (95% CI 24, 4<em>3</em>; 5-year survival of 27%), 18+ months (95% CI 15, 20; 5-year survival of 11%), and 8+ months (95% CI 6, 10; 5-year survival of 5%), respectively. These prognostic categories are helpful both in individual patient care and in the assessment of patients entering prospective clinical trials.

The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 10(<em>3</em>) to 10(4) CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day <em>3</em> postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma <em>interferon</em> and tumor necrosis factor <em>alpha</em>.

Using flow cytometric and RNase protection assays, this study examined the expression of chemokine receptors in nonactivated natural killer (NK) cells and compared this expression with NK cells activated with interleukin (IL)-2, which either adhered to plastic flasks (AD) or did not adhere (NA). None of the NK cell subsets expressed CXCR2, CXCR5, or CCR5. The major differences between these cells include increased expression of CXCR1, CCR1, CCR2, CCR4, CCR8, and CX(<em>3</em>)CR1 in AD when compared to NA or nonactivated NK cells. The chemotactic response to the CXC and CC chemokines correlated with the receptor expression except that all <em>3</em> populations responded to GRO-<em>alpha</em>, despite their lack of CXCR2 expression. Pretreatment of these cells with anti-CXCR2 did not inhibit the chemotactic response to GRO-<em>alpha</em>. In addition, nonactivated and NA cells responded to fractalkine, although they lack the expression of CX(<em>3</em>)CR1. This activity was not inhibited by anti-CX(<em>3</em>)CR1. Viral macrophage inflammatory protein (vMIP)-I, I-<em>3</em>09, and TARC competed with the binding of (125)I-<em>3</em>09 to AD cells with varying affinities. Transforming growth factor (TGF)-beta1 but not any other cytokine or chemokine examined including <em>interferon</em> (IFN)-gamma, MIP-<em>3</em>beta, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) or I-<em>3</em>09, up-regulated the expression of CXCR<em>3</em> and CXCR4 on NK cell surface. This is correlated with increased chemotaxis of NK cells treated with TGF-beta1 toward stromal cell-derived factor (SDF)-1<em>alpha</em> and <em>interferon</em>-inducible protein-10 (IP-10). Messenger RNA for lymphotactin, RANTES, MIP-1<em>alpha</em>, and MIP-1beta, but not IP-10, monocyte chemotactic protein (MCP)-1, IL-8, or I-<em>3</em>09 was expressed in all <em>3</em> NK cell subsets. Our results may have implications for the dissemination of NK cells at the sites of tumor growth or viral replication. (Blood. 2001;97:<em>3</em>67-<em>3</em>75)

Herpes simplex virus type 1 (HSV-1) is resistant to the antiviral effects of <em>interferon</em> (IFN)-<em>alpha</em>, -beta, or -gamma. The fact that ICP0(-) mutants replicate like wild-type virus in IFN-<em>alpha</em>/beta receptor knockout mice (Leib et al., 1999, J. Exp. Med. 189, 66<em>3</em>) suggested that ICP0 may serve a direct role in the resistance of HSV-1 to IFN. To test this hypothesis, the effects of IFN-<em>alpha</em>, -beta, and -gamma were compared against wild-type HSV-1 and an ICP0(-) mutant virus, 71<em>3</em>4. In Vero cells, 71<em>3</em>4 was more sensitive to inhibition by low doses of type I IFN (-<em>alpha</em>/beta) or type II IFN (-gamma) than vesicular stomatitis virus, a well-studied IFN-sensitive virus. At a concentration of 100 U/ml, IFN-<em>alpha</em>, -beta, or -gamma reduced the efficiency of 71<em>3</em>4 plaque formation by 120-, 560-, and 45-fold, respectively. In contrast, none of the IFNs reduced wild-type HSV-1 plaque formation by more than <em>3</em>-fold. Even when Vero cells were infected with 10 pfu per cell, IFN-<em>alpha</em> and -beta inhibited 71<em>3</em>4 replication by over 100-fold, but inhibition by IFN-gamma decreased to less than 10-fold. While IFN-beta efficiently inhibited 71<em>3</em>4 replication in primary mouse kidney and SK-N-SH cells, IFN-gamma did not inhibit 71<em>3</em>4 to a comparable extent in these cells. ICP0 provided in trans from an adenovirus vector allowed 71<em>3</em>4 to replicate efficiently in Vero cells in the presence of IFN-<em>alpha</em>, -beta, or -gamma. While IFN-beta or -gamma efficiently repressed the ICP0 promoter-lacZ reporter gene in 71<em>3</em>4 (i.e., approximately 60-fold reduction in beta-galactosidase activity), ICP0 provided in trans almost completely reversed IFN-mediated repression of the lacZ gene in 71<em>3</em>4. The results suggest that the rate of ICP0 expression in infected cells in vivo may be critical in determining whether host IFNs repress the HSV-1 genome. This concept is discussed in light of its potential relevance to the establishment of latent HSV-1 infections.

OBJECTIVE

To elucidate extracellular matrix (ECM) changes underlying intestinal fibrosis, a frequent complication of inflammatory bowel disease, we developed a murine model of chronic colitis associated with intestinal fibrosis.

METHODS

Chronic inflammation was established by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS). In 2 variations of the model an antisense oligonucleotide for nuclear factor kappa B (NF-kappa B) p65 was given prophylactically or therapeutically to block chronic inflammation-associated fibrosis. Colonic inflammation and fibrosis were determined by histology. Total collagen level was estimated by hydroxyproline quantification. Colonic expression of collagens (Col1a2, Col<em>3</em>a2), ECM remodeling genes (matrix metalloproteinase [MMP]-1, -<em>3</em>, and tissue inhibitor of matrix metalloproteinase [TIMP]-1), and inflammation-modulating cytokines (tumor necrosis factor <em>alpha</em> [TNF-<em>alpha</em>], <em>interferon</em> gamma [IFN-gamma], transforming growth factor beta 1 [TGF-beta 1], and insulin-like growth factor 1 [IGF-1]) were assessed by semiquantitative reverse-transcription polymerase chain reaction. Control and TNBS-treated colonic mesenchymal cells were characterized by morphology, phenotype, and functional response to TNF-<em>alpha</em> and IFN-gamma.

RESULTS

Colons of TNBS-treated mice contained acute and chronic inflammatory infiltrates, increased collagen, fibrogenic tissue architecture, and increased expression of TNF-<em>alpha</em>, TGF-beta 1, IGF-1, Col1a2, MMP-1, and TIMP-1. Colonic mesenchymal cells from TNBS-treated mice were also morphologically distinct from those of the control mice, with increased TIMP-1 expression in response to IFN-gamma treatment. Fibrosis persisted for 2-4 weeks after cessation of the TNBS treatment. In mice given NF-kappa B antisense prophylactically, 67% were fibrosis-free, whereas of those treated after establishing chronic inflammation, 4<em>3</em>% were free of fibrosis.

CONCLUSIONS

Extended TNBS treatment of mice yielded chronic intestinal inflammation-associated fibrosis with extensive fibrogenic ECM changes that could be counteracted by specific blockade of NF-kappa B.

The double-stranded RNA (dsRNA) analogue poly(I:C) is a promising adjuvant for cancer vaccines because it activates both dendritic cells (DCs) and natural killer (NK) cells, concurrently promoting adaptive and innate anticancer responses. Poly(I:C) acts through two dsRNA sensors, Toll-like receptor <em>3</em> (TLR<em>3</em>) and melanoma differentiation-associated protein-5 (MDA5). Here, we investigated the relative contributions of MDA5 and TLR<em>3</em> to poly(I:C)-mediated NK cell activation using MDA5(-/-), TLR<em>3</em>(-/-), and MDA5(-/-)TLR<em>3</em>(-/-) mice. MDA5 was crucial for NK cell activation, whereas TLR<em>3</em> had a minor impact most evident in the absence of MDA5. MDA5 and TLR<em>3</em> activated NK cells indirectly through accessory cells and induced the distinct stimulatory cytokines <em>interferon</em>-<em>alpha</em> and interleukin-12, respectively. To identify the relevant accessory cells in vivo, we generated bone marrow chimeras between either wild-type (WT) and MDA5(-/-) or WT and TLR<em>3</em>(-/-) mice. Interestingly, multiple accessory cells were implicated, with MDA5 acting primarily in stromal cells and TLR<em>3</em> predominantly in hematopoietic cells. Furthermore, poly(I:C)-mediated NK cell activation was not notably impaired in mice lacking CD8<em>alpha</em> DCs, providing further evidence that poly(I:C) acts through diverse accessory cells rather than solely through DCs. These results demonstrate distinct yet complementary roles for MDA5 and TLR<em>3</em> in poly(I:C)-mediated NK cell activation.