The present study underlines the importance of gemfibrozil, a lipid-lowering drug and an activator of peroxisome proliferator-activated receptor-<em>alpha</em> (PPAR-<em>alpha</em>), in inhibiting the disease process of adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of relapsing-remitting multiple sclerosis. Clinical symptoms of EAE, infiltration of mononuclear cells, and demyelination were significantly lower in SJL/J female mice receiving gemfibrozil through food chow than those without gemfibrozil. It is noteworthy that the drug was equally effective in treating EAE in PPAR-<em>alpha</em> wild-type as well as knockout mice. Gemfibrozil also inhibited the encephalitogenicity of MBP-primed T cells and switched the immune response from a Th1 to a Th2 profile independent of PPAR-<em>alpha</em>. Gemfibrozil consistently inhibited the expression and DNA-binding activity of T-bet, a key regulator of <em>interferon</em>-gamma (IFN-gamma) expression and stimulated the expression and DNA-binding activity of GATA<em>3</em>, a key regulator of IL-4. Gemfibrozil treatment decreased the number of T-bet-positive T cells and increased the number of GATA<em>3</em>-positive T cells in spleen of donor mice. The histological and immunohistochemical analyses also demonstrate the inhibitory effect of gemfibrozil on the invasion of T-bet-positive T cells into the spinal cord of EAE mice. Furthermore, we demonstrate that the differential effect of gemfibrozil on the expression of T-bet and GATA<em>3</em> was due to its inhibitory effect on NO production. Although excess NO favored the expression of T-bet, scavenging of NO stimulated the expression of GATA-<em>3</em>. Taken together, our results suggest gemfibrozil, an approved drug for hyperlipidemia in humans, may find further therapeutic use in multiple sclerosis.

Hepatitis C virus (HCV) is the leading cause of liver transplantation in Europe and is associated with an increased risk of hepatocellular carcinoma (HCC). Because of the chronic nature of the disease, estimates suggest that the burden on healthcare will increase dramatically for this entity. Clinical care of patients with HCV-related liver disease has advanced considerably in the last two decades, thanks to increasing knowledge about the mechanisms of the disease, development of diagnostic procedures, and advances in therapeutic and preventive approaches. HCV RNA testing, HCV genotyping and staging of liver disease are essential for the diagnosis and the management of HCV therapy. Furthermore, the important role of host polymorphisms of the IL28B gene on virological response to treatment with pegylated <em>interferon</em> (PEG-IFN) <em>alpha</em> and ribavirin (RBV) has recently been clearly demonstrated. In relation to treatment, although numerous drugs for HCV are in various stages of preclinical and clinical development, the current standard of care (SoC) is the combination of PEG-IFN-α and RBV for chronic hepatitis C. With SoC, a sustained viral response (SVR) is achieved in approximately 45% of patients infected with HCV genotype 1 and in approximately 80% of patients infected with HCV genotypes 2 and <em>3</em>. The EASL HCV guidelines recommend treating all naïve patients with compensated disease from HCV without contraindications to treatment and strongly suggest initiating SoC promptly in patients with advanced fibrosis. Further recommendations on monitoring treatment efficacy, treatment duration, dose reduction indications and the role of co-factors are provided.

In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new <em>alpha</em>-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with <em>interferon</em>-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- <em>3</em> microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability.

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor <em>alpha</em>, and IL-6 production. Studies using neutralizing antibodies for type I <em>interferons</em> (IFNs), IFN-<em>alpha</em> and IFN-beta, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-beta. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-<em>alpha</em>/beta receptor knockout mice. Cotransfection of human Toll-like receptors <em>3</em> and 9 (receptors for dsRNA and CpG DNA, respectively) into 29<em>3</em>T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.

We investigated the molecular basis of the synergistic induction by <em>interferon</em>-gamma (IFN-gamma)/tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-<em>alpha</em> action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -7<em>3</em> and -<em>3</em>6, which is the minimal element inducible by LPS or TNF-<em>alpha</em>; 2) an element located between -181 and -7<em>3</em>, which appeared to regulate the response to IFN-gamma and TNF-<em>alpha</em> negatively; and <em>3</em>) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-<em>alpha</em>, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.

High levels of ambient air pollution are associated with exacerbation of asthma and respiratory morbidity, yet little is known concerning the mechanisms of inflammation and toxicity by components of inhaled particulate matter (PM). Brief inhalation of PM(2.5) (particles of an aerodynamic diameter of < 2.5 microns) (<em>3</em>00 microg/m(<em>3</em>) air for 6 h followed by a period of 24 h in clean air) by either C<em>3</em>H/HeJ or C57/BL6 mice caused significant (P </= 0.05) increases in steady-state messenger RNA (mRNA) levels of a number of nuclear factor (NF)-kappaB-associated and/ or -regulated genes, including tumor necrosis factor-<em>alpha</em> and -beta, interleukin-6, <em>interferon</em>-gamma, and transforming growth factor-beta. Lung mRNA levels of lymphotoxin-beta and macrophage migration inhibitory factor were unchanged. In murine C10 alveolar cells and an NF-kappaB-luciferase reporter cell line, exposure to PM(2.5) at noncytotoxic concentrations resulted in increases in transcriptional activation of NF-kappaB-dependent gene expression which were inhibited in the presence of catalase. Early and persistent increases in intracellular oxidants, as measured by flow cytometry and cell imaging using the oxidant probe 2'-7'-dichlorofluoroscin diacetate, were observed in epithelial cells exposed to PM(2.5) and ultrafine carbon black particles. Studies here are the first to show NF-kappaB-related inflammatory and cytokine gene expression after inhalation of PM(2.5) and oxidant-dependent induction of NF-kappaB activity by PM(2.5) in pulmonary epithelial cells.

The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), IFN-gamma, interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (<em>3</em>0 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = <em>3</em> per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-<em>alpha</em> less than IL-6 less than IFN-gamma less than IL-1 <em>alpha</em> less than IFN-<em>alpha</em> less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-<em>alpha</em> and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)

The enzyme IMP dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, namely, the conversion of IMP to XMP. The major event occurring in cells exposed to competitive IMPDH inhibitors such as ribavirin or uncompetitive inhibitors such as mycophenolic acid (MPA) is a depletion of the intracellular GTP and dGTP pools. Ribavirin is approved as an inhaled antiviral agent for treatment of respiratory syncytial virus (RSV) infection and orally, in combination with <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>), for the treatment of chronic hepatitis C virus (HCV) infection. VX-497 is a potent, reversible uncompetitive IMPDH inhibitor which is structurally unrelated to other known IMPDH inhibitors. Studies were performed to compare VX-497 and ribavirin in terms of their cytotoxicities and their efficacies against a variety of viruses. They included DNA viruses (hepatitis B virus [HBV], human cytomegalovirus [HCMV], and herpes simplex virus type 1 [HSV-1]) and RNA viruses (respiratory syncytial virus [RSV], parainfluenza-<em>3</em> virus, bovine viral diarrhea virus, Venezuelan equine encephalomyelitis virus [VEEV], dengue virus, yellow fever virus, coxsackie B<em>3</em> virus, encephalomyocarditis virus [EMCV], and influenza A virus). VX-497 was 17- to 186-fold more potent than ribavirin against HBV, HCMV, RSV, HSV-1, parainfluenza-<em>3</em> virus, EMCV, and VEEV infections in cultured cells. The therapeutic index of VX-497 was significantly better than that of ribavirin for HBV and HCMV (14- and <em>3</em>9-fold, respectively). Finally, the antiviral effect of VX-497 in combination with IFN-<em>alpha</em> was compared to that of ribavirin with IFN-<em>alpha</em> in the EMCV replication system. Both VX-497 and ribavirin demonstrated additivity when coapplied with IFN-<em>alpha</em>, with VX-497 again being the more potent in this combination. These data are supportive of the hypothesis that VX-497, like ribavirin, is a broad-spectrum antiviral agent.

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19(+) cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) receptor. Despite significant activation in the draining LN at day <em>3</em> after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80(+) or CD169(+) subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-<em>alpha</em>/beta receptor.

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected by hepatitis B virus. <em>Interferon</em> <em>alpha</em> at high doses, although poorly efficient, is the only treatment reported to provide some benefit in chronic hepatitis delta. Pegylated <em>interferon</em> <em>alpha</em> (PEG-IFN) has not yet been evaluated. Treatment is usually monitored by the qualitative detection of HDV-RNA in serum. In this study, safety and efficacy of PEG-IFN were assessed in chronic hepatitis delta, and serum HDV-RNA kinetics were determined using quantitative RT-PCR. Fourteen patients with chronic hepatitis delta received subcutaneous PEG-IFN <em>alpha</em>-2b during 12 months (1.5 microg/kg per week). Serum HDV-RNA was quantified at initiation and during the course of therapy, and during the posttreatment follow-up period, which ranged from 6 to 42 months (median 16 months). PEG-IFN <em>alpha</em>-2b was well tolerated, inducing no serious adverse effect. Sustained biochemical response was obtained in 8 patients (57%). At the end of treatment, 8 patients (57%) had achieved virological response (undetectable HDV-RNA). Sustained virological response throughout the posttreatment follow-up period was observed in 6 patients (4<em>3</em>%). HDV-RNA kinetics were predictive of the response: after <em>3</em> months of PEG-IFN, HDV-RNA levels were significantly lower in the responders than in the nonresponders group (P=.018). After 6 months of therapy, a negative HDV-RNA was predictive of sustained response (P=.021). In conclusion, this preliminary study indicates that PEG-IFN <em>alpha</em>-2b is safe and efficient for treatment of chronic hepatitis delta. The follow-up of HDV-RNA levels during therapy, which allows the differentiation of various profiles of virological responses, improves treatment monitoring.

Intestinal epithelial barrier function is impaired after the exposure of enterocytes to proinflammatory cytokines. The mechanism(s) responsible for this phenomenon remain incompletely understood. We used cultured monolayers of Caco-2 enterocyte-like cells to characterize the effect of cytomix, a mixture of <em>interferon</em>-gamma, tumor necrosis factor-<em>alpha</em>, and interleukin-1beta, on the expression and localization of several tight junctions proteins. Cells were stimulated with cytomix in the presence or absence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1 -oxyl-<em>3</em>-oxide (cPTIO), an NO* scavenger. Some cells were treated with (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate] (DETA-NONOate), an NO* donor. Tight junction protein expression was measured in cellular extracts by Western blotting and localized in cells using immunofluorescence. Steady-state mRNA levels were determined using semi-quantitative reverse-transcription polymerase chain reaction. Incubation of cells with DETA-NONOate or cytomix decreased epithelial barrier function, decreased expression of ZO-1 mRNA, decreased expression of ZO-1, ZO-<em>3</em>, and occludin protein, and increased expression of claudin-1 protein. The effects of cytomix on barrier function and tight junction protein expression were modulated by cPTIO. Cytomix caused incorrect subcellular localization of ZO-1, occludin, and claudin-1, and this was modulated by co-incubation with cPTIO. DETA-NONOate caused similar protein mislocalization as observed with cytomix. The effectiveness of cPTIO in maintaining tight junction protein expression and correct subcellular localization was less apparent at early time points (12 h) compared with later points, suggesting an NO*-independent effect of cytokines on barrier function. Thus, cytomix appears to increase the permeability of Caco-2 monolayers through NO*-dependent and -independent mechanisms that are associated with changes in the expression and/or targeting of proteins involved in tight junction function.

The in vitro regulation of adult human monocyte DR antigen expression was studied. Normally about 75% of freshly obtained human peripheral blood monocytes express DR antigens as determined by anti-DR and complement-mediated cytotoxicity assays. DR expression on monocytes in unfractionated peripheral blood mononuclear cell cultures persisted to variable degrees for up to 5 d of incubation. However, when the mononuclear cells were thoroughly depleted of nonadherent cells, cultured monocytes consistently exhibited progressively decreased DR expression over 2-5 d of incubation. Readdition of nonadherent cells to the adherent cell population prevented or delayed this decrease in monocyte DR antigen expression. Thus, monocyte DR expression diminished markedly during in vitro incubation; however, the presence of nonadherent cells somehow interfered with this process. In other experiments, peripheral adherent monocytes, which had been cultured for 2-<em>3</em> d to reduce their DR expression, could be induced to reexpress DR antigens after 2 d of incubation with unpurified lymphokine-containing culture supernatants, recombinant human <em>interferon</em>-<em>alpha</em>, or recombinant human gamma <em>interferon</em> (IFN-gamma). The reinduction of DR expression on human monocytes by lymphokines was abrogated by an antiserum produced to the synthetic N-terminal amino acids of human IFN-gamma, indicating that IFN-gamma is the active mediator in the lymphokine-containing preparations. Monocytes cultured with lymphokines or recombinant <em>interferons</em> also could initiate a significantly greater mixed lymphocyte response than control monocytes. Thus, IFN-gamma-containing lymphokines and recombinant <em>interferons</em> are required to induce human monocyte DR expression and accessory cell capacity in vitro, since in their absence monocytes become DR antigen-deficient. Finally, incubation of unfractionated human mononuclear cells with anti-human IFN-gamma also promoted the loss of monocyte DR expression. These findings suggest that resting lymphocytes are probably capable of producing sufficient IFN-gamma in vitro to result in the maintenance of the monocyte DR phenotype.

This study illustrates the use and efficacy of a combination of pegylated <em>interferon</em>-<em>alpha</em> (Peg-IFN-<em>alpha</em>) and ribavirin (RBV), with or without rituximab (RTX), in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC). Twenty-two patients with HCV-related MC received Peg-IFN-<em>alpha</em> (2a: 180 mug or 2b: 1.5 mug/kg) weekly plus RBV (1000 or 1200 mg) daily for 48 weeks, and RTX (<em>3</em>75 mg/m(2)) once a week for 1 month followed by two 5-monthly infusions (termed PIRR). Fifteen additional patients received Peg-IFN-<em>alpha</em>/RBV with the same modalities as the PIRR schedule. Complete response was achieved in 54.5% (12/22) and in <em>3</em><em>3</em>.<em>3</em>% (5/15) of patients who received PIRR and Peg-IFN-<em>alpha</em>/RBV, respectively (P < .05). Clearance of HCV RNA and conversion of B-cell populations from oligoclonal to polyclonal in liver, bone marrow, and peripheral blood was maintained for up to <em>3</em> years in 10 of 12 (8<em>3</em>.<em>3</em>%) and in 2 of 5 (40%) patients receiving PIRR and Peg-IFN-<em>alpha</em>/RBV, respectively (P < .01). Cryoproteins in 22.7% (5/22) of patients with PIRR and in <em>3</em><em>3</em>.<em>3</em>% (5/15) with Peg-IFN-<em>alpha</em>/RBV persisted despite sustained HCV RNA clearance. No response occurred in remaining 5 patients of both groups. PIRR therapy is well tolerated and more effective than Peg-IFN-<em>alpha</em>/RBV combination in HCV-related MC. Its effect may last for more than <em>3</em> years.

We compared thalidomide-dexamethasone (TD) with melphalan-prednisolone (MP) in 289 elderly patients with multiple myeloma (MM). Patients received either thalidomide 200 mg plus dexamethasone 40 mg, days 1 to 4 and 15 to 18 on even cycles and days 1 to 4 on odd cycles, during a 28-day cycle or to melphalan 0.25 mg/kg and prednisolone 2 mg/kg orally on days 1 to 4 during a 28- to 42-day cycle. Patients achieving stable disease or better were randomly assigned to maintenance therapy with either thalidomide 100 mg daily and <em>3</em> MU <em>interferon</em> <em>alpha</em>-2b thrice weekly or to <em>3</em> MU <em>interferon</em> <em>alpha</em>-2b thrice weekly only. TD resulted in a higher proportion of complete and very good remissions (26% vs 1<em>3</em>%; P= .006) and overall responses (68% vs 50%; P= .002) compared with MP. Time to progression (21.2 vs 29.1 months; P= .2), and progression-free survival was similar (16.7 vs 20.7 months; P= .1), but overall survival was significantly shorter in the TD group (41.5 vs 49.4 months; P= .024). Toxicity was higher with TD, particularly in patients older than 75 years with poor performance status. The study was registered at ClinicalTrials.gov as NCT00205751.

Insulin resistance is the major feature of the metabolic syndrome and depends on insulin secretion and insulin sensitivity. In chronic hepatitis C, insulin resistance and type 2 diabetes mellitus are more often seen than in healthy controls or chronic hepatitis B patients. Hepatitis C virus (HCV) infection promotes insulin resistance, mainly by increased TNF production together with enhancement of suppressor of cytokine (SOC-<em>3</em>); both events block PI<em>3</em>K and Akt phosphorylation. Two types of insulin resistance could be found in chronic hepatitis C patients: "viral" and "metabolic" insulin resistance. Insulin resistance in chronic hepatitis C is relevant because it promotes steatosis and fibrosis. The mechanisms by which insulin resistance promotes fibrosis progression include: (1) steatosis, (2) hyperleptinemia, (<em>3</em>) increased TNF production, (4) impaired expression of PPARgamma receptors. Lastly, insulin resistance has been found as a common denominator in patients difficult-to-treat like cirrhotics, overweight, HIV coinfected and Afro-American. Insulin resistance together with fibrosis and genotype has been found to be independently associated with impaired response rate to peg<em>interferon</em> plus ribavirin. Indeed, in genotype 1, the sustained response rate was twice (60%) in patients with HOMA < or = 2 than patients with HOMA>> 2. In experiments carried out on Huh-7 cells transfected by full length HCVRNA, <em>interferon</em> <em>alpha</em> blocks HCV replication. However, when insulin (at doses of 128 microU/mL, similar that seen in the hyperinsulinemic state) was added to <em>interferon</em>, the ability to block HCV replication disappeared, and the PKR synthesis was abolished. In summary, hepatitis C promotes insulin resistance and insulin resistance induces <em>interferon</em> resistance, steatosis and fibrosis progression.

BACKGROUND

Infection with Helicobacter species has been associated with the development of mucosal inflammation and inflammatory bowel disease (IBD) in several mouse models. However, consensus regarding the role of Helicobacter as a model organism to study microbial-induced IBD is confounded by the presence of a complex colonic microbiota.

OBJECTIVE

To investigate the kinetics and inflammatory effects of immune system activation to commensal bacteria following H bilis colonisation in gnotobiotic mice.

METHODS

C<em>3</em>H/HeN mice harbouring an altered Schaedler flora (ASF) were selectively colonised with H bilis and host responses were investigated over a 10-week period. Control mice were colonised only with the defined flora (DF). Tissues were analysed for gross/histopathological lesions, and bacterial antigen-specific antibody and T-cell responses.

RESULTS

Gnotobiotic mice colonised with H bilis developed mild macroscopic and microscopic lesions of typhlocolitis beginning <em>3</em> weeks postinfection. ASF-specific IgG responses were demonstrable within <em>3</em> weeks, persisted throughout the 10-week study, and presented as a mixed IgG1:IgG2a profile. Lymphocytes recovered from the mesenteric lymph node of H bilis-colonised mice produced increased levels of <em>interferon</em> gamma, tumour necrosis factor <em>alpha</em> (TNF<em>alpha</em>), interleukin 6 (IL6) and IL12 in response to stimulation with commensal- or H bilis-specific bacterial lysates. In contrast, DF mice not colonised with H bilis did not develop immune responses to their resident flora and remained disease free.

CONCLUSIONS

Colonisation of gnotobiotic C<em>3</em>H/HeN mice with H bilis perturbs the host's response to its resident flora and induces progressive immune reactivity to commensal bacteria that contributes to the development of immune-mediated intestinal inflammation.

A systematic review of radiation therapy trials in several tumour types was performed by The Swedish Council of Technology Assessment in Health Care (SBU). The procedures for evaluation of the scientific literature are described separately (Acta Oncol 200<em>3</em>; 42: <em>3</em>57-<em>3</em>65). This synthesis of the literature on radiation therapy for soft tissue sarcomas (STS) is based on data from five randomized trials. Moreover, data from 6 prospective studies, 25 retrospective studies and <em>3</em> other articles were used. In total, <em>3</em>9 scientific articles are included, involving 4 579 patients. The results were compared with those of a similar overview from 1996 which included <em>3</em> <em>3</em>44 patients. The conclusions reached can be summarized as follows: The well-established prognostic factors for tumour-related death from STS-histological grade, tumour size and age-are well documented. The importance of superficial versus deep site as well as the anatomic site is also reaffirmed to some extent. There is strong evidence that adjuvant radiotherapy improves the local control rate in combination with conservative surgery in the treatment of STS of extremities and trunk in patients with negative, marginal or minimal microscopic positive surgical margins. A local control rate of 90% has been achieved. Improvement is obtained with radiotherapy added in the case of intralesional surgery, but the local control rate is somewhat lower. More studies are needed on this issue. For STS in other anatomic sites, retroperitoneum, head and neck, breast and uterus, there is only weak indication of a benefit for the local control rate, with the use of adjuvant radiotherapy. There is still insufficient data to establish that preoperative radiotherapy is favourable compared to postoperative radiotherapy for local control in patients presenting primarily with large tumours. One small study has shown a possible survival benefit for preoperative radiotherapy. There is fairly good evidence to suggest that the preoperative setting results in more wound complications. There is no randomized study comparing external beam radiotherapy and brachytherapy. The data suggest that external beam radiotherapy and low dose rate brachytherapy result in comparable local control for high-grade tumours. Some patients with low-grade soft tissue sarcomas benefit from external beam radiotherapy in terms of local control. Brachytherapy with low dose rate for low-grade tumours seems to be of no benefit, but data are sparse. The available data are inconclusive concerning the effect of intraoperative high dose rate radiotherapy for retroperitoneal STS. Further studies are needed. Neutron radiotherapy might be beneficial for patients with low- and intermediate-grade tumours considered inoperable and for those operated with intralesional margins. More severe side effects for neutrons have been registered. In two small studies investigating hyperfractionation schedules there was no indication of improvements compared to daily fractions of 2 Gy. Further studies should be encouraged. One small study using preoperative limb perfusion with TNF <em>alpha</em> melphalan and +/- <em>interferon</em> gamma combined with postoperative radiotherapy in the case of marginal or positive surgical margin has shown excellent local control without enhanced morbidity.

<em>Interferon</em>-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by <em>interferon</em>-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 4<em>3</em>1, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 1<em>3</em>8 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and <em>3</em>-hydroxyanthranilic acid). <em>Interferon</em>-gamma regulated only the activity of indoleamine 2,<em>3</em>-dioxygenase. All other enzyme activities detected were independent of <em>interferon</em>-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 4<em>3</em>1 and A 549 and 10-fold with U 1<em>3</em>8 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though <em>interferon</em>-gamma was the most potent inducer of tryptophan metabolism, <em>interferon</em>-<em>alpha</em> and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to <em>interferon</em>-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,<em>3</em>-dioxygenase is a common feature of <em>interferon</em>-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,<em>3</em>-dioxygenase is the only enzyme in the formation of <em>3</em>-hydroxyanthranilic acid from tryptophan which is regulated by <em>interferon</em>-gamma.

The neutrophil-attracting chemokine interleukin 8 (IL-8) is stored in the Weibel-Palade body (WPB) of endothelial cells (ECs) from which it can be rapidly released after exposure to the secretagogues histamine or thrombin. In this manner, IL-8 may enable rapid recruitment of leukocytes to inflammatory sites. To explore the possible storage of EC-derived chemokines that may attract other subsets of leukocytes, we examined the intracellular localization and secretagogue responsiveness of growth-related oncogene <em>alpha</em> (GRO<em>alpha</em>), monocyte chemoattractant protein-1 (MCP-1), eotaxin-<em>3</em>, <em>interferon</em>-gamma-inducible protein 10 (IP-10), and regulated on activation, normal T-cell expressed and secreted (RANTES). While eotaxin-<em>3</em>, GRO<em>alpha</em>, and MCP-1 were rapidly released from ECs, the release of the T-cell attractors RANTES and IP-10 was not sensitive to the secretagogues. Moreover, of the <em>3</em> former chemokines, only eotaxin-<em>3</em> was stored in WPBs. GRO<em>alpha</em> and MCP-1 resided mainly in smaller vesicles compatible with sorting to a different, histamine-responsive compartment, which has been described in ECs although not reported to contain chemokines. In conclusion, we propose that rapid release of chemokines is restricted to those primarily recruiting leukocytes of the innate immune system, and that their storage in ECs is not restricted to the WPB compartment.

It has been reported that <em>interferons</em> (IFNs) may have antitumor activity in multiple myeloma (MM). The mechanism for their effect on MM, however, remains elusive. This study shows that IFN-<em>alpha</em> and -beta, but not -gamma, induce apoptosis characterized by Annexin V positivity, nuclear fragmentation and condensation, and loss of clonogenicity in <em>3</em> MM cell lines (U266, RPMI-8266, and NCI-H929), and in plasma cells from 10 patients with MM. Apo2 ligand (Apo2L, also TRAIL) induction was one of the earliest events following IFN administration in U266 cells. Treatment of these cells with TRAIL, but not with Fas agonistic antibodies, induces apoptosis. Cell death induced by IFNs and Apo2L in U266 cells was partially blocked by a dominant-negative Apo2L receptor, DR5, demonstrating the functional significance of Apo2L induction. This study shows that IFNs activate caspases and the mitochondrial-dependent apoptotic pathway, possibly mediated by Apo2L production. Thus, IFN-<em>alpha</em> and -beta induce cytochrome c release from mitochondria starting at 12 hours, with an amplified release seen at 48 hours. Moreover, Bid cleavage precedes the initial cytochrome c release, whereas the late, amplified cytochrome c release coincides with changes in levels of Bcl-2, Bcl-X(L), and reduction of mitochondrial membrane potential. These results link the Apo2L induction and modulation of Bcl-2 family proteins to mitochondrial dysfunction. Furthermore, IFNs and Apo2L induce cell death of CD<em>3</em>8(+)/CD45(-/dim) plasma cells, without significant effect on nonplasma blood cells, in a caspase and Bcl-2 cleavage-dependent manner. These results warrant further clinical studies with IFNs and Apo2L in MM.

We compared the ability of recombinant human tumor necrosis factor-<em>alpha</em> (rHuTNF-<em>alpha</em>) and tumor necrosis factor-beta (rHuTNF-beta) to stimulate polymorphonuclear neutrophil (PMN) migration and superoxide production. Significant PMN migration occurred across polycarbonate filters after stimulation with rHuTNF-<em>alpha</em> at concentrations ranging from 10(-7) to 10(-10) mol/L and at 10(-7) to 10(-8) mol/L for rHuTNF-beta and N-formylmethionyl-leucyl phenylalanine (FMLP), whereas recombinant human <em>interferon</em>-gamma was only minimally active at 10(-7) mol/L and recombinant human interleukin-1 <em>alpha</em> was inactive at the doses tested. In addition, antibodies to rHuTNF-<em>alpha</em> completely inhibited rHuTNF-<em>alpha</em> but not rHuTNF-beta or FMLP-induced PMN migration. Combinations of rHuTNF-<em>alpha</em> and rHuTNF-beta (at similar molar concentrations) stimulated PMN migration levels comparable to that obtained with rHuTNF-<em>alpha</em> alone. Checkerboard analyses performed by placing different concentrations of rHuTNF-<em>alpha</em> and rHuTNF-beta above and below polycarbonate filters of microchemotaxis chambers demonstrated that rHuTNF-<em>alpha</em> and rHuTNF-beta stimulated both chemotactic and chemokinetic responses by PMN. Additional studies demonstrated that 1 X 10(-8) mol/L rHuTNF-<em>alpha</em> and <em>3</em> X 10(-9) mol/L rHuTNF-beta (which represents 10(4) U/mL of each cytokine) were similar in their ability to induce superoxide production by PMNs; however, at ten- to 100-fold lower molar concentrations (10(<em>3</em>) and 10(2) units), rHuTNF-<em>alpha</em> was significantly more active than rHuTNF-beta. At the doses tested, both cytokines were less active than phorbol myristate acetate at stimulating O2- release. The results demonstrate that rHuTNF-<em>alpha</em> and rHuTNF-beta differ quantitatively but not qualitatively in their effects on PMN functions in vitro and suggest that rHuTNF-beta may be less toxic than rHuTNF-<em>alpha</em> in vivo.

The <em>interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>) transcription factor has been extensively studied in the context of the type I <em>interferon</em> (IFN-<em>alpha</em>/beta)-mediated antiviral response; it consists of the major DNA-binding component p48, and the signal transducers and activators of transcription (Stat)1 and Stat2. We show here that type II IFN (IFN-gamma) can also invoke the activation of ISGF<em>3</em> in mouse primary embryonic fibroblasts. In fact, the two Stat proteins were tyrosine phosphorylated in IFN-gamma stimulated cells. Our present findings reveal an additional mechanism by which these two distinct types of cytokines, IFN-<em>alpha</em>/beta and -gamma, can commonly elicit antiviral activities.

OBJECTIVE

To evaluate the frequency and the course of complete cytogenetic responses in interferon-alpha (IFN-alpha)-treated patients with chronic myelogenous leukemia.

METHODS

Two prospective trials in consecutive patients.

METHODS

A major tertiary cancer center.

METHODS

Ninety-six consecutive patients with chronic myelogenous leukemia with disease duration of less than 1 year.

METHODS

Patients received partially pure IFN-alpha intramuscularly, from 3 to 9 million U/d (51 patients) or recombinant IFN-alpha 2a (Roferon, Hoffmann-LaRoche, Inc., Nutley, New Jersey), 5 million U/m2 body surface area daily (45 patients).

METHODS

Hematologic and cytogenetic tests were administered.

RESULTS

Seventy of the patients (73%) achieved hematologic remission (95% CI, 63% to 81%), and 18 (19%) had complete suppression of the Philadelphia chromosome on at least one cytogenetic test. A complete cytogenetic response was induced in 7 of 51 or 14% (CI, 6% to 26%) of the patients treated with the partially pure IFN-alpha and in 11 of 45 or 24% (CI, 13% to 40%) of the patients treated with recombinant IFN-alpha 2a. The difference in complete cytogenetic response between the two groups was 10.7% (CI, - 5% to 26%; P greater than 0.2). Eleven patients had durable, ongoing, complete cytogenetic responses from 6 to more than 45 months (median, more than 30 months).

CONCLUSIONS

This study was the first to show sustained, complete cytogenetic responses in a subset of patients with chronic myelogenous leukemia treated with single-agent therapy. The nature of this remission, that is, whether it depends on continuous therapy, requires further study.

21 patients with AIDS and Kaposi's sarcoma were enrolled in an open therapeutic trial to determine the in vivo anti-retroviral activity of recombinant <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>). 8 (<em>3</em>8%) showed a complete or partial anti-tumour response. The mean pretreatment CD4 count for the responders was <em>3</em>99 cells/microliter vs 154 cells/microliter for the non-responders. All 5 of the patients with more than 400 CD4 cells/microliter pretreatment showed a significant reduction in tumour, whereas none of the 7 patients with under 150 CD4 cells/microliter had any response. 5 of the 6 complete or partial responders with greater than 50 pg/ml of human immunodeficiency virus (HIV) p24 before IFN therapy showed a 75% or greater reduction by 12 weeks of therapy, with <em>3</em> patients having persistently negative HIV cultures. The anti-viral effects were also most pronounced in the patients with the highest CD4 counts. These data demonstrate the potential benefits, both anti-tumour and anti-retroviral, of treatment with IFN-<em>alpha</em> in the early stages of HIV infection and Kaposi's sarcoma.