Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E)-4-hydroxy-<em>3</em>-methyl-but-2-enyl pyrophosphate (HMB-PP), an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL)-6, <em>interferon</em> (IFN)-gamma, tumor necrosis factor (TNF)-<em>alpha</em>, and oncostatin M (OSM); the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL). Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs) with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan) induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+) effector <em>alpha</em>beta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD) patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe-responsive gammadelta T cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity.

We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP<em>3</em>5 protein. Cross-linking studies demonstrated the ability of VP<em>3</em>5 to form trimers, consistent with the presence of a functional coiled-coil motif. VP<em>3</em>5 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP<em>3</em>5 inhibits signaling that activates <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and inhibits (IFN)-<em>alpha</em>/beta production. Experiments comparing the ability of VP<em>3</em>5 mutants to block IFN responses demonstrated that the VP<em>3</em>5 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP<em>3</em>5 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP<em>3</em>5, suggesting that an oligomerization function at the amino-terminus facilitates an "IFN-antagonist" function exerted by the carboxy-terminal half of VP<em>3</em>5.

This study determined the presence of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor <em>alpha</em> (TNF <em>alpha</em>), tumour necrosis factor beta (TNF beta), <em>interferon</em> gamma (IFN gamma), transforming growth factor beta 2 (TGF beta 2) and fibroblast proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that IL-1 and IL-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of IL-6 greater than 20 pg/ml were more frequently found in PVR than in RD (p = 0.0<em>3</em>1) or control specimens (p = 0.006). Low levels of TNF <em>alpha</em> were observed in 4/18 eyes with PVR, 1/15 eyes with RD and 1/15 control vitreous, and small concentrations of TNF <em>alpha</em> were seen in <em>3</em>/18 eyes with PVR, 1/15 eyes with RD and 2/15 control vitreous. IFN gamma was detected in 12/18 eyes with PVR, but only in 5/15 eyes with RD (p = 0.048) and 6/15 control specimens. TGF beta 2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.0<em>3</em>1) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of IL-1, IL-6 and IFN gamma in cellular interactions leading to chronicity.

The expression of intercellular adhesion molecule-1 (ICAM-1) by human cerebral endothelium was studied in primary cultures of human brain microvessel endothelial cells following treatment with bacterial lipopolysaccharide (LPS), tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), interleukin-1 beta (IL-1 beta) and <em>interferon</em>-gamma (IFN-gamma). Surface expression of ICAM-1 was examined with the immunogold silver staining technique. Intact cerebral endothelial cells constitutively express low levels of ICAM-1. Stimulation with LPS and cytokines induces upregulation of ICAM-1 which is minimal with IFN-gamma and maximal with LPS or a combination of IFN-gamma and TNF-<em>alpha</em>. Upregulation of ICAM-1 expression is concentration- and time-dependent, is observed as early as 4 h following incubation and persists for up to 72 h in the continuous presence of LPS or cytokines. The ICAM-1 expression is not reversed by <em>3</em> days after removal of the LPS or cytokines. These findings may be relevant to the interactions between leukocytes and brain microvessel endothelial cells in inflammatory and demyelinating diseases of the CNS.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 <em>alpha</em>/beta, TNF-<em>alpha</em>/beta, IL-<em>3</em>, IL-6, <em>interferon</em>-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 <em>alpha</em>/beta and TNF-<em>alpha</em>/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-<em>3</em>, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.

Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and <em>interferon</em>-gamma (IFN-gamma) are capable of decreasing the levels of <em>alpha</em> 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of <em>alpha</em> 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (<em>3</em>) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+<em>3</em>428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.

We have documented the key role of toll-like receptor 4 (TLR4) activation and its signaling pathway mediated by <em>interferon</em> (IFN) regulatory factor <em>3</em>, in the induction of inflammation leading to the hepatocellular damage during liver ischemia/reperfusion injury (IRI). Because type I IFN is the major downstream activation product of that pathway, we studied its role in comparison with IFN-gamma. Groups of type I (IFNAR), type II (IFNGR) IFN receptor-deficient mice, along with wild-type (WT) controls were subjected to partial liver warm ischemia (90 minutes) followed by reperfusion (1-6 hours). Interestingly, IFNAR knockout (KO) but not IFNGR KO mice were protected from IR-induced liver damage, as evidenced by decreased serum alanine aminotransferase and preservation of tissue architecture. IR-triggered intrahepatic pro-inflammatory response, assessed by tumor necrosis factor (TNF-<em>alpha</em>), interleukin 6 (IL-6), and chemokine (C-X-C motif) ligand 10 (CXCL-10) expression, was diminished selectively in IFNAR KO mice. Consistent with these findings, our in vitro cell culture studies have shown that: (1) although hepatocytes alone failed to respond to lipopolysaccharide (LPS), when co-cultured with macrophages they did respond to LPS via macrophage-derived IFN-beta; (2) macrophages required type I IFN to sustain CXCL10 production in response to LPS. This study documents that type I, but not type II, IFN pathway is required for IR-triggered liver inflammation/damage. Type I IFN mediates potential synergy between nonparenchyma and parenchyma cells in response to TLR4 activation.

<em>Interferon</em> (IFN)-gamma induces the expression of the indoleamine 2, <em>3</em>-dioxygenase (INDO) gene in human cells, which plays a role in the inhibitory effect of IFN-gamma on intracellular pathogens and on cell proliferation. Earlier studies established that the IFN-gamma-inducible expression of the INDO gene was dependent on two upstream elements: (i) a 14-base pair sequence homologous to an <em>interferon</em>-stimulated response element (ISRE) sequence found in IFN-<em>alpha</em>-inducible genes and (ii) a 9-base pair palindromic sequence (palindromic element (PE) II) homologous to an <em>interferon</em>-gamma-activated site (GAS) element found in IFN-gamma-inducible genes. A second GAS element (PE I), between ISRE and PE II, was ineffective in supporting a response to IFN-gamma. Studies were carried out to determine the distinction between the two GAS elements and the relative role of the two elements (ISRE and PE II) required for a response to IFN-gamma. The PE I element was able to form a complex with IFN-gamma-activated p91 (STAT1) factor but with lower efficiency than the complex formed with PE II sequence. However, switching the positions of PE I and II sequences in reporter plasmid constructs (containing chloramphenicol acetyltransferase gene) showed that both PE I and PE II were able to support a response to IFN-gamma if located at the position of PE II but not at the position of PE I. Increasing the distance between the ISRE and PE II also affected the level of response, suggesting that the relative position of the two elements is important for optimal stimulus. To explore whether an interaction between the IFN-gamma-regulated factors (IRF-1 and p91) binding to the ISRE and PE II might be important, we tested whether the ISRE sequence could be replaced by another response element, NF-kappaB. The plasmid construct with NF-kappaB element in place of the ISRE was responsive to IFN-gamma, indicating that an interaction between the IRF-1 and p91 factors was not required. The results indicate that the response of INDO gene to IFN-gamma depends on a cooperative role of IFN-gamma-responsive factors binding to the ISRE and GAS elements.

Plasmacytoid dendritic cells (pDCs) represent a DC subtype that exerts divergent functions in innate and adoptive immunity including the immediate reaction to microbial factors and the induction of immunoregulatory responses. It is thought that different DC subtypes may be critically involved in the pathogenesis of multiple sclerosis (MS). In our study we assessed the phenotype, maturation and functional properties of peripheral blood pDCs from <em>3</em>5 clinically stable, untreated multiple sclerosis patients, <em>3</em>0 healthy controls and 9 patients with pneumonia, which was used as a non-specific inflammatory condition (NIC). Ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from multiple sclerosis patients than from controls and patients with NIC (22 versus 47 versus 41% and 12 versus <em>3</em>5 versus <em>3</em>2%, respectively). When stimulated with IL-<em>3</em> and CD40L, pDCs of multiple sclerosis patients showed inefficient maturation as demonstrated by significantly lower or delayed upregulation of CD86, 4-1BBL, CD40 and CD8<em>3</em>. Additionally, in multiple sclerosis, stimulation of pDCs by unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) resulted in a significantly lower <em>interferon</em> (IFN) <em>alpha</em> secretion than in controls. In multiple sclerosis, but not in controls, pDCs failed to upregulate proliferative responses and IFN-gamma secretion of autologous peripheral blood mononuclear cells (PBMC) in a co-culture system. Moreover, depletion of pDCs in multiple sclerosis patients, but not in controls, had no effect on generation of CD4+Foxp<em>3</em>+ regulatory T cells. We also provide data showing that glatiramer acetate (GA) treatment partially restores phenotype and function of pDCs in multiple sclerosis patients. These findings suggest functional abnormalities of pDCs in these patients, which might be of importance in the understanding of the development of immune dysregulation in this disease.

The occurrence of thyroid abnormalities and the appearance of organ- and non-organ-specific autoantibodies during long-term recombinant <em>interferon</em> <em>alpha</em>-2a (IFN-<em>alpha</em>) therapy were studied in 86 and 51 consecutive outpatients with hepatitis C and B virus-related chronic active hepatitis (CAH-HCV and CAH-HBV), respectively. Most patients had longstanding community-acquired hepatitis. At baseline, 9.<em>3</em>% of CAH-HCV and <em>3</em>.9% of CAH-HBV patients showed clinical and/or biochemical signs of thyroid dysfunction. The remaining patients were euthyroid, although anti-thyroid autoantibodies were found in <em>3</em><em>3</em>/78 (42.<em>3</em>%) of CAH-HCV and in 5/49 (10.2%) of CAH-HBV patients. During IFN-<em>alpha</em> treatment, increased anti-thyroid autoantibody levels were seen in 40% of CAH-HCV initially negative patients, while they became detectable in no more than 10% of CAH-HBV patients. <em>Interferon</em>-<em>alpha</em>-induced hypo- or hyperthyroidism was recorded in 12 of <em>3</em>5 CAH-HCV patients treated for 12 months (<em>3</em>4.<em>3</em>%). Only one CAH-HBV patient developed hyperthyroidism. High titers of anti-nuclear autoantibodies (ANA) were recorded at enrollment in 5/<em>3</em>6 (1<em>3</em>.8%) of CAH-HCV and in <em>3</em>/16 (18.7%) of CAH-HBV patients. Only one CAH-HCV patient displayed anti-parietal cell antibodies (PCA). After IFN-<em>alpha</em> treatment, ANA were found in 10/28 (<em>3</em>5.7%) and PCA in 2/28 (7.1%) of CAH-HCV patients, while an additional CAH-HBV patient developed PCA, but not ANA. However, no signs of systemic autoimmune disease were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by <em>interferon</em>-gamma (IFN-gamma) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-gamma induced the expression of indoleamine 2,<em>3</em>-dioxygenase (IDO) activity. Whereas tumor necrosis factor-<em>alpha</em> did not affect enzyme induction by IFN-gamma, lipopolysaccharide modulated IDO activity differently in the two IFN-gamma-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine <em>3</em>-hydroxylase, and <em>3</em>-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine <em>3</em>-hydroxylase activity was stimulated by IFN-gamma. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-gamma markedly stimulated the activity of this enzyme only in MT2 cells. IFN-gamma-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled <em>3</em>-hydroxyanthranilic and quinolinic acids from L-[5-<em>3</em>H] tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.

OBJECTIVE

Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes.

METHODS

The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses.

RESULTS

Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation.

CONCLUSIONS

IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.

A recent nonrandomized pilot trial showed that hepatitis C virus (HCV) patients with genotype 2/<em>3</em> and rapid virological response (RVR) had a 90% sustained virological response (SVR) rate after 14 weeks of treatment. We aimed to assess this concept in a randomized controlled trial. In the trial, 428 treatment-naïve HCV RNA-positive patients with genotype 2 or <em>3</em> were enrolled. Patients with RVR were randomized to 14 (group A) or 24 (group B) weeks of treatment. Patients were treated with pegylated <em>interferon</em> <em>alpha</em>-2b (1.5 microg/kg) subcutaneously weekly and ribavirin (800-1400 mg) orally daily. The noninferiority margin was set to be 10% between the two groups with a one-sided 2.5% significance level. RVR was obtained in <em>3</em>02 of 428 (71%), and 298 of these were randomized to group A (n = 148) or group B (n = 150). In the intention-to-treat analysis, SVR rates were 120 of 148 (81.1%) in group A and 1<em>3</em>6 of 150 (90.7%) in group B (difference, 9.6%; 95% confidence interval, 1.7-17.7). Among patients with an HCV RNA test 24 weeks after the end of treatment, 120 of 1<em>3</em>9 (86.<em>3</em>%) patients in group A achieved SVR compared with 1<em>3</em>6 of 146 (9<em>3</em>.2%) in group B (difference, 6.9%; 95% confidence interval, -0.1 to +1<em>3</em>.9).

CONCLUSIONS

We cannot formally claim that 14 weeks of treatment is noninferior to 24 weeks of treatment. However, the SVR rate after 14 weeks of treatment is high, and although longer treatment may give slightly better SVR, we believe economical savings and fewer side effects make it rational to treat patients with genotype 2 or <em>3</em> and RVR for only 14 weeks.

Bladder cancer is the fourth leading cause of cancer in American men, accounting for more than 12,000 deaths annually. It was one of the first malignancies in which carcinogens were recognized as an important factor in its cause. Currently, cigarette smoking is by far the most common cause of bladder cancer, although occupational exposure to arylamines has been implicated in the past. Gross or microscopic hematuria is the most common sign at presentation. Initial radiologic evaluation usually includes the excretory urography (intravenous pyelography), although further evaluation of the renal parenchyma with ultrasound or computed tomography scanning has been advocated by some. These radiologic studies are unable to provide adequate bladder imaging, and thus cystoscopy is required for the diagnosis of bladder cancer. Most bladder cancers present as "superficial" disease, confined to the bladder mucosa or submucosal layer, without muscle invasion. Superficial tumors consist of papillary tumors that are mucosally confined (Ta), papillary or sessile tumors extending into the lamina propria (T1), and carcinoma in situ, which occurs as "flat" mucosal dysplasia, which can be focal, diffuse, or associated with a papillary or sessile tumor. The natural history of these pathologic subtypes differ significantly. Most superficial tumors (60% to 70%) have a propensity for recurrence after transurethral resection. Some (15% to 25%) are at high risk for progression to muscle invasion. Most superficial tumors can be stratified into high- or low-risk groups depending on tumor stage, grade, size, number, and recurrence pattern. It is important to identify those tumors at risk for recurrence or progression so that adjuvant intravesical therapies can be instituted. Many intravesical chemotherapeutic agents have been shown to reduce tumor recurrence when used in conjunction with transurethral tumor resection. Unfortunately, however, none of these agents have proved to be of benefit in preventing disease progression. Most are given intravesically on a weekly basis, although many studies suggest that a single instillation immediately after transurethral resection may be as good as a longer course of therapy. Although all of these drugs have toxicity, they usually are well tolerated. Intravesical bacille Calmette-Guérin (BCG) is an immunotherapeutic agent that when given intravesically is very effective in the treatment of superficial transitional cell carcinoma. Compared with controls, BCG has a 4<em>3</em>% advantage in preventing tumor recurrence, a significantly better rate than the 16% to 21% advantage of intravesical chemotherapy. In addition, BCG is particularly effective in the treatment of carcinoma in situ, eradicating it in more than 80% of cases. In contrast to intravesical chemotherapy, BCG has also been shown to decrease the risk of tumor progression. The optimal course of BCG appears to be a 6-week course of weekly instillations, followed by a <em>3</em>-week course at <em>3</em> months in those tumors that do not respond. In high-risk cancers, maintenance BCG administered for <em>3</em> weeks every 6 months may be optimal in limiting recurrence and preventing progression. Unfortunately, adverse effects associated with this prolonged therapy may limit its widespread applicability. In those patients at high risk in whom BCG therapy fails, intravesical <em>interferon</em>-<em>alpha</em> with or without BCG may be beneficial in some. Photodynamic therapy has also been used but is limited by its toxicity. In patients who progress or do not respond to intravesical therapies, cystectomy should be considered. With the development of orthotopic lower urinary tract reconstruction to the native urethra, the quality of life impact of radical cystectomy has been lessened.

Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-<em>alpha</em>. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), <em>interferon</em>-gamma (IFN-gamma), and TNF-<em>alpha</em> were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-<em>alpha</em> on CYP24A1, chorionic gonadotropin (hCG), <em>3</em>beta-hydroxysteroid dehydrogenase (HSD<em>3</em>B1) and P(450)-aromatase (CYP19) mRNA expression were also studied. TNF-<em>alpha</em> stimulated IL-6, IFN-gamma and its own expression more than <em>3</em>-fold over controls (P<0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose-response manner (P<0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-<em>alpha</em> also significantly inhibited expression of hCG, HSD<em>3</em>B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-<em>alpha</em> induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-<em>alpha</em> inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

The serious and characteristic side effects of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) therapy on the central nervous system, resulting in such problems as affective disorders or parkinsonism, have led us to investigate the biochemical mechanism of the effects of IFN-<em>alpha</em> on the monoaminergic neurotransmitter system using an animal model (rats). We first examined the concentrations of tetrahydrobiopterin (BH(4)) and monoamines in several regions of the brain after the intramuscular injection of IFN-<em>alpha</em> into rats; the levels of BH(4) and dopamine significantly decreased in the amygdala and raphe areas as compared with those of the controls. Based on these results, we further examined the concentrations of BH(4) and nitrite (NO(2)(-)) plus nitrate (NO(<em>3</em>)(-)), metabolites of nitric oxide (NO), in the amygdala and raphe areas after the intramuscular injection of IFN-<em>alpha</em>; the concentrations of both BH(4) and NO(2)(-)+NO(<em>3</em>)(-) significantly decreased as compared with the control. Furthermore, the addition of N(G)-monomethyl L-arginine, an inhibitor of NO synthase, after the injection of IFN-<em>alpha</em> restored the decreased levels of both NO(2)(-)+NO(<em>3</em>)(-) and BH(4) to control levels. As a result, nitric oxide induced by the intramuscular injection of IFN-<em>alpha</em> was found to cross the blood-brain barrier and suppress both tetrahydrobiopterin biosynthesis and dopamine production in the amygdala and raphe areas.

Astrocyte inflammation, reactive oxygen species (ROS) formation, and dysfunction form a common denominator shared by all the major neurodegenerative disorders. Viral infections are emerging as important events in the etiology of CNS damage involving astrocytes, but molecular understanding is incomplete. Double-stranded RNA (dsRNA) is a byproduct of viral replication and serves as the signature molecule for viral infection via Toll-like receptor <em>3</em> (TLR<em>3</em>) largely restricted to circulating peripheral dendritic cells. However, astrocytes are strategically located at the blood-brain barrier (BBB) and throughout brain tissues, making these cells ideal candidates as innate immunity sentinels within the CNS. We hypothesized that extracellular dsRNA, mimicked by polyinosinic-polycytidylic acid (Poly(I:C); PIC), initiates signaling of the double-edged sword of antiviral plus pathophysiological events in astrocytes. Using Western blot analysis and real-time qPCR, we determined that neonatal rat astrocyte cultures constitutively express TLR<em>3</em> mRNA and protein, and that PIC dsRNA induced phosphorylation of eIF2<em>alpha</em>, as well as mRNA type I <em>interferon</em> (<em>alpha</em>/beta IFN)-response genes Mx1, PKR, and TLR<em>3</em>. Astrocyte TLR<em>3</em> protein was downregulated after PIC treatment, however. PIC signaled degradation of IkappaB<em>alpha</em> with the consequence of upregulating iNOS, TNF-<em>alpha</em>, and IL-1beta mRNAs and proteins. In addition to antiviral protection events, dsRNA induced astrocyte dysfunction, evidenced by inhibiting EAAT1/GLAST transporter gene expression and attenuating L-glutamate uptake via sodium-dependent transport system X(AG)-, as well as inducing cytotoxicity. Anti-TLR<em>3</em> blocking antibody attenuated PIC upregulation of TNF-<em>alpha</em> mRNA and iNOS activity. Extracellular PIC-induced events were prevented by 2-aminopurine, implicating PKR as an important downstream player in astrocyte dsRNA sensing pathways. The effects of plasma membrane impermeable poly(I:C) were dose-dependent (0-50 microM). In concert, these data provide evidence that dsRNA/TLR<em>3</em>-activated astrocytes initiate a battery of rapid innate pathogen-associated molecular pattern (PAMP) immune responses that are important for mounting antiviral defense in the CNS, yet also lead to pathophysiological events associated with the glutamate neurotoxicity of neurodegenerative diseases.

OBJECTIVE

From in vitro studies using cultures of orbital fibroblasts, it has become clear that cytokines play an important role in the orbital inflammation in Graves' ophthalmopathy (GO). Orbital fibroblasts seem to be the key target cells of the autoimmune attack, and they are able to express the TSH receptor (TSH-R). In vivo data on the presence of cytokines in orbital tissues are sparse, and mostly limited to samples obtained from patients with endstage, inactive GO; the same holds true for the presence of the TSH-R. The aim of the present study was to determine whether the cytokine profile and TSH-R expression differ in the active vs. the inactive stage of GO.

METHODS

Orbital fat/connective tissue was obtained from six patients with active, untreated GO undergoing emergency orbital decompression, and from 11 patients with inactive GO subjected to rehabilitative decompressive surgery. The mRNA levels of various cytokines and the TSH-R were assessed by real-time polymerase chain reaction (PCR) using the LightCycler. Data are expressed as ratios (unknown mRNA/beta-actin mRNA).

RESULTS

Active GO patients had much higher TSH-R expression than inactive patients: 4/0-24 (median value/range) vs. 0/0-9, P = 0.01. TSH-R expression was related to the Clinical Activity Score (r = 0.595, P = 0.015). Patients with active GO compared to those with inactive GO had higher mRNA levels of the proinflammatory cytokines interleukin-1beta (IL-1beta) (445/15<em>3</em>-877 vs. 0/0-455, P = 0.001), IL-6 (158<em>3</em>/968-18825 vs. 559/0-7181, P = 0.01), IL-8 (1422/<em>3</em>8-7579 vs. <em>3</em>2/0-1081, P = 0.046) and IL-10 (145/58-<em>3</em>18 vs. 27/0-189, P = 0.002). In active GO there also existed a trend towards a predominance of T helper 1 (Th1)-derived cytokines as evident from higher IL-2 (<em>3</em>7/0-158 vs. 0/0-68, P = 0.04<em>3</em>), <em>interferon</em>-gamma (IFN-gamma) (20/0-79 vs. 0/0-16, P = 0.12) and IL-12 (2.<em>3</em>/0-14.8 vs. 0/0-1.6, P = 0.10) mRNAs. IL-1 receptor agonist (IL-1RA), IL-2 receptor (IL-2R), IL-<em>3</em>, IL-4, IL-5, IL-1<em>3</em>, IL-18 and tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) mRNAs were similar in both groups.

CONCLUSIONS

These data show that at the mRNA level, TSH-R expression is largely present only during the active stages of GO. The active phase is characterized by the presence of proinflammatory and Th1-derived cytokines, whereas other cytokines, among them Th2-derived cytokines, do not seem to be linked to a specific stage of GO.

BACKGROUND

Until the development of novel targeted agents directed against angiogenesis and tumour growth, few treatment options have been available for the treatment of metastatic renal-cell carcinoma (mRCC).

OBJECTIVE

This review discusses current targeted therapies for mRCC and provides consensus statements regarding treatment algorithms.

METHODS

Medical literature was retrieved from PubMed up to April 2011. Additional relevant articles and abstract reviews were included from the bibliographies of the retrieved literature.

RESULTS

Targeted treatment for mRCC can be categorized for the following patient groups: previously untreated patients, those refractory to immunotherapy, and those refractory to vascular endothelial growth factor (VEGF)-targeted therapy. Sunitinib and bevacizumab combined with interferon alpha are generally considered first-line treatment options in patients with favourable or intermediate prognoses. Temsirolimus is considered a first-line treatment option for poor-risk patients. Either sorafenib or sunitinib may be valid second-line treatments for patients who have failed prior cytokine-based therapies. For patients refractory to treatment with VEGF-targeted therapy, everolimus is now recommended. Pazopanib is a new treatment option in the first- and second-line setting (after cytokine failure). Sequential and combination approaches, and the roles of nephrectomy and tumour metastasectomy will also be discussed.

CONCLUSIONS

Increasing clinical evidence is clarifying appropriate first- and second-line treatments with targeted agents for patients with mRCC. Based on phase 2 and 3 trials, a sequential approach is most promising, while combination therapy is still investigational. The role of nephrectomy in mRCC is being evaluated in ongoing phase 3 clinical trials.

We have shown that activation of toll-like receptor 4 (TLR4) and its <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>)-dependent downstream signaling pathway are required for the development of liver ischemia/reperfusion injury (IRI). This study focused on the role of TLR4-IRF<em>3</em> activation pathway products, in particular, chemokine (C-X-C motif) ligand 10 (CXCL10). The induction of CXCL10 by liver IR was rapid (1 hour postreperfusion), restricted (ischemic lobes), and specific (no CXCL9 and CXCL11 induction). Functionally, CXCL10 was critical for IR-induced liver inflammation and hepatocellular injury. CXCL10 knockout (KO) mice were protected from IRI, as evidenced by reduced serum alanine aminotransferase (sALT) levels and preserved liver histological detail. The induction of pro-inflammatory genes, such as tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), interleukin 1beta (IL-1beta), IL-6, and IL-12beta was diminished, whereas the induction of the IL-10 gene remained intact in CXCL10 KO mice, indicating an altered liver response against IR. This was accompanied by selective down-regulation of extracellular signal-regulated kinase (ERK), but intact Jun N-terminal kinase (JNK), activation in the KO IR livers. This altered liver inflammation response was (1) specific to IR, because lipopolysaccharide (LPS) induced a comparable pro-inflammatory response in CXCL10 KO and wild-type (WT) mice; and (2) responsible for liver cytoprotection from IR, because neutralization of IL-10 restored local inflammation and hepatocellular damage.

CONCLUSIONS

CXCL10 regulates liver inflammation response against IRI, and its deficiency protected livers from IRI by local IL-10-mediated cytoprotection. Targeting CXCL10 may provide a novel therapeutic means to ameliorate liver IRI in clinics.

We have conducted a multicenter randomized controlled trial comparing two doses of recombinant human <em>alpha</em>-<em>interferon</em> for efficacy in 60 patients with chronic non-A, non-B hepatitis. The source of infection appeared to be transfusion in <em>3</em>0 patients, intravenous drug abuse in 16 patients and was unknown in 14 patients. Patients were randomly assigned to no treatment or to treatment with either 1 or <em>3</em> MU of <em>alpha</em>-<em>interferon</em> given three times a week for 24 wk. Forty-five patients (75%) were positive for antibody to hepatitis C virus. During the 24-wk treatment period, mean serum ALT levels decreased in both treatment groups, but the decrease was statistically significant only in the <em>3</em> MU group. However, at 24 wk, the proportion of patients with normal ALT levels was similar in the <em>3</em> MU group (<em>3</em>9%) and the 1 MU group (45%), and both were significantly higher than in controls (0%). Repeat liver biopsy specimens showed a significant decrease in the severity of histological changes in the <em>3</em> MU group but not in the 1 MU group or in controls. Responses to <em>alpha</em>-<em>interferon</em> did not correlate with patient's age, gender, source of infection, pretreatment serum ALT, presence of anti-hepatitis C virus or cirrhosis. After treatment, the mean ALT levels rose in both treated groups. The proportion of patients with normal ALT levels at wk 48 was 28% in the <em>3</em> MU group and 20% in the 1 MU group. In conclusion, a dose of <em>3</em> MU was superior to 1 MU of <em>alpha</em>-<em>interferon</em> given three times weekly for 24 wk in inducing improvements in serum ALT levels and liver histological examinations.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and <em>interferon</em>-gamma (IFN-gamma) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF-<em>alpha</em> or anti-IFN-gamma antibodies ("serotherapy") prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF-<em>alpha</em> treatment. The later treatment with anti-TNF-<em>alpha</em> or anti-IFN-gamma antibodies on day <em>3</em> post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF-<em>alpha</em> and IFN-gamma is required for the restriction of a primary Y. enterocolitica infection in mice.

Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) plus actinomycin D (TNF-<em>alpha</em>/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-<em>alpha</em>/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8-like (IETDase) and caspase-<em>3</em>-like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c-effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid(+/+) cytosol, but not with Bid(-/-) cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8-dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1beta (IL-1beta) plus <em>interferon</em> gamma (IFN-gamma), which induced iNOS expression and NO production, suppressed TNF-<em>alpha</em>/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor N(G)-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1beta and IFN-gamma. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-<em>alpha</em> plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-<em>alpha</em>/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.

The recent demonstration of the ability of human polymorphonuclear neutrophils (PMN) to secrete various cytokines in response to the granulocyte activator granulocyte-macrophage colony-stimulating factor (GM-CSF) but not to other cytokines, has led to the identification of PMN as biosynthetically active cells. In this study we have investigated the ability of PMN to secrete interleukin-6 (IL-6), a molecule known to be involved in inflammatory reactions. Using RNA blotting analysis and bioassays, we show that PMN could be induced to synthesize transcripts specific for IL-6, indistinguishable in size from IL-6 mRNA produced by activated human macrophages. Consequently, PMN released IL-6-like activity into their culture supernatants that could be neutralized by monospecific anti-IL-6 antibody. Interleukin-6 secretion by PMN, however, required previous stimulation with GM-CSF or tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), whereas other cytokines, including interleukin-<em>3</em> (IL-<em>3</em>), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), <em>interferon</em> gamma (IFN-gamma), and lymphotoxin (LT), failed to induce IL-6 mRNA accumulation and protein secretion by PMN. Similar to GM-CSF and TNF-<em>alpha</em>, other compounds, including the inhibitor of protein synthesis cyclohexemide (CHX), endotoxin (Escherichia coli-derived lipopolysaccharide), and phorbol myristate acetate (PMA) (but not the chemoattractant N-formyl-methionyl-leucyl-phenylalanine [FMLP]), induced detectable levels of IL-6 transcripts in PMN.