The aim of this randomized controlled trial was to assess the efficacy of <em>interferon</em> alfa-2b (IFN) for the treatment of advanced hepatocellular carcinoma (HCC). Fifty-eight patients with HCC who were not suitable for resection, transplantation, ethanol injection, or arterial embolization were stratified according to their Okuda stage and randomized to receive IFN (<em>3</em> x 10(6), <em>3</em> times a week, for 1 year) (n = <em>3</em>0) or symptomatic treatment (n = 28). Both groups were identical in terms of age, sex, performance status, presence of constitutional syndrome, Child-Pugh class, Okuda stage, multinodularity, portal thrombosis, and extrahepatic spread. Adhesion to IFN treatment was adequate in 27 patients, with a mean duration of treatment of 8 +/- <em>3</em> months. However, IFN treatment was associated with side effects in 2<em>3</em> patients, leading to treatment discontinuation in 1<em>3</em> patients. Two of the <em>3</em>0 patients (6.6%) presented a partial response with greater than 50% size reduction and normalization of <em>alpha</em>-fetoprotein levels. The survival at 1 and 2 years according to intention to treat was not different between the 2 groups (58% and <em>3</em>8% vs. <em>3</em>6% and 12%, respectively, Breslow P =. 19, log rank P =.14) and the absence of difference was maintained when dividing patients according to their Okuda stage. The probability of presenting tumor progression (P =.17), or deterioration of Child-Pugh class (P =.<em>3</em>7), performance status (P =. 07), or Okuda stage (P =.44) was not modified by IFN treatment. These results indicate that IFN is not properly tolerated in patients with cirrhosis and advanced HCC and that its administration prompts no benefit in terms of tumor progression rate and survival.

Natural killer (NK) cell-mediated cytotoxicity is triggered by multiple activating receptors associated with the signaling adaptor protein DNAX activation protein 12/killer cell-activating receptor-associated protein (DAP12/KARAP). Here, we show that one of these receptors, NKp44, is present on a subset of natural <em>interferon</em>-producing cells (IPCs) in tonsils. NKp44 expression can also be induced on blood IPCs after in vitro culture with interleukin <em>3</em> (IL-<em>3</em>). Crosslinking of NKp44 does not trigger IPC-mediated cytotoxicity but, paradoxically, inhibits <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>) production by IPCs in response to cytosine-phosphate-guanosine (CpG) oligonucleotides. We find that IPCs in tonsils are in close contact with CD8+ T cells and demonstrate that a subset of memory CD8+ T cells produces IL-<em>3</em>. Therefore, IL-<em>3</em>-mediated induction of NKp44 on IPCs may be an important component of the ongoing crosstalk between the innate and adaptive immune response that allows memory CD8+ T cells to control the IPC response to virus.

Several cytokines exhibit a high degree of temporal regulation as well as somnogenic potency (e.g., interleukin-1 [IL-1], tumor necrosis factor-<em>alpha</em> [TNF-<em>alpha</em>]). Seeking the underlying cause of obstructive sleep apnea syndrome (OSAS), we investigated whether circadian rhythms of cytokine release were altered in 10 patients with OSAS. Ten healthy volunteers served as the control population. Seven of the 10 OSAS patients were reexamined after <em>3</em> mo of therapy with nasal continuous positive airway pressure (nCPAP) mask ventilation. Circadian cytokine release (IL-1, IL-6, gamma-<em>interferon</em> [gamma-IFN], TNF-<em>alpha</em>) was investigated ex vivo by short-term culture of blood samples. The circadian rhythm of TNF-<em>alpha</em> release was significantly disturbed in OSAS patients: nocturnal physiologic peaks in this cytokine had almost disappeared and an additional daytime peak had developed. Circadian variations in IL-1, IL-6, and gamma-IFN, and in the immunoregulatory hormones melatonin and cortisol, did not differ from those in the controls. Because TNF-<em>alpha</em> is a known modulator of sleep, and nCPAP therapy did not normalize TNF rhythms, we assume that TNF-<em>alpha</em> could well play a pathophysiologic role in OSAS. Further studies should be directed at whether a physiopathologic and/or pathogenic link exists between TNF-<em>alpha</em> and OSAS.

OBJECTIVE

Synovitis is a common feature of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the pattern of joint involvement differs in each disease. This study was undertaken to investigate the global gene expression profiles in synovial biopsy tissue from the swollen knees of untreated SLE patients (n = 6), RA patients (n = 7), and osteoarthritis (OA) patients (n = 6).

METHODS

Synovial biopsy samples were obtained from the affected knees of patients in the <em>3</em> groups by needle arthroscopy. Half of the material was used for extraction of total RNA, amplification of complementary RNA, and high-density oligonucleotide spotted hybridization arrays. On the remaining tissue samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical experiments were performed to confirm the microarray data.

RESULTS

SLE synovial biopsy tissue displayed a significant down-regulation of genes involved in extracellular matrix (ECM) homeostasis and a significant up-regulation of interferon-inducible (IFI) genes. Real-time RT-PCR experiments confirmed the up-regulation of selected IFI genes (IFI27, IFI44, and IFI44L) in the SLE synovial tissue. Immunohistochemical analyses showed that <em>3</em> molecules involved in ECM regulation, chondroitin sulfate proteoglycan 2, latent transforming growth factor beta binding protein 2, and fibroblast activation protein alpha, were significantly down-regulated in SLE synovium. In contrast, immunostaining for IFI27, Toll-like receptor 4, and STAT-1 resulted in higher quantitative scores in SLE synovial tissue, which could be attributed to the fact that the RA samples had a large population of inflammatory cell infiltrates that were negative for these markers.

CONCLUSIONS

Arthritis in SLE has a very distinct molecular signature as compared with that in OA and RA, characterized by up-regulation of IFI genes and down-regulation of genes involved in ECM homeostasis.

The development of T1-cell-mediated immunity is required to clear a pulmonary Cryptococcus neoformans infection. The objective of these studies was to determine the mechanism by which tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) augments the development of pulmonary T1 immunity to C. neoformans infection. TNF-<em>alpha</em> expression was detected in lavage sample cells at days 2, <em>3</em>, and 7 following C. neoformans infection. The numbers of CFU in the lung were not different between control and anti-TNF-<em>alpha</em>-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-<em>alpha</em> prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-<em>alpha</em> monoclonal antibody (day 0) diminished the lung levels of TNF-<em>alpha</em>, interleukin-12 (IL-12), and gamma <em>interferon</em> (IFN-gamma) induced by C. neoformans at day 7 postinfection. Neutralization of TNF-<em>alpha</em> (day 0) also altered the IFN-gamma/IL-4 ratio in the lung-associated lymph nodes at day 7 following C. neoformans infection. Anti-TNF-<em>alpha</em>-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-<em>alpha</em>-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-gamma in response to the infection. These findings demonstrate that afferent-phase TNF-<em>alpha</em> production is essential for the induction of IL-12 and IFN-gamma and neutralization of early TNF-<em>alpha</em> results in a T2 shift of the T1/T2 balance of antifungal immunity.

A <em>3</em>9-nt DNA sequence, the <em>interferon</em> gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the <em>3</em>' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF<em>3</em>, the IFN-<em>alpha</em>-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-<em>alpha</em> can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-<em>alpha</em> does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Eye muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for <em>interferon</em> gamma (IFNgamma), tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>), interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-<em>3</em>-phosphate dehydrogenase. IFNgamma, TNF<em>alpha</em>, IL-1beta, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and <em>3</em>8% of the patients, respectively, and IFNgamma, IL-1beta, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as assessed by computed tomography correlated significantly with TNF<em>alpha</em> mRNA expression in EM tissue. The orbital volume was positively correlated with IL-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest that T helper (Th) 1-like cytokines predominate in EM tissue in most patients and that the predominant cytokine profile in OF tissue varies from patient to patient. Both Th1-like and Th2-like immune responses may play roles in the development of two components of ophthalmopathy.

OBJECTIVE

The natural course of chronic hepatitis C (CHC) in children is not well understood. The aim of this study was to assess the long-term course of CHC in a large sample of otherwise healthy children.

METHODS

From 1990 to 2005, 504 consecutive antihepatitis C virus (HCV)-positive children were enrolled at 12 centers of a national observatory and were followed up retrospectively/prospectively.

RESULTS

Putative exposure was perinatal in 28<em>3</em> (56.2%) cases, parenteral in 158 (<em>3</em>1.<em>3</em>%), and unknown in 6<em>3</em> (12.5%). At baseline, 477 (94.6%) cases were HCV RNA seropositive, 118 (24.7%) of which were treated with standard <em>interferon</em> <em>alpha</em>. Ten years after putative exposure, the outcome in <em>3</em>59 HCV RNA-positive, untreated patients was (1) undetectable viremia in 27 (7.5%) (by Cox regression analysis, spontaneous viral clearance was independently predicted by genotype <em>3</em> [hazard ratio 6.44; 95% confidence interval: 2.7-15.5]) and (2) persistent viremia in <em>3</em><em>3</em>2 (92%) cases. Six of these <em>3</em><em>3</em>2 cases (1.8%) progressed to decompensated cirrhosis (mean age, 9.6 years). This latter group included 5 Italian children perinatally infected with genotype 1a (4 of the mothers were drug users). Thirty-three (27.9%) treated patients achieved a sustained virologic response.

CONCLUSIONS

Over the course of a decade, few children with chronic HCV infection cleared viremia spontaneously, and those who did were more likely to have genotype <em>3</em>. Persistent viral replication led to end-stage liver disease in a small subgroup characterized by perinatal exposure, maternal drug use, and infection with HCV genotype 1a. Children with such features should be considered for early treatment.

OBJECTIVE

To evaluate whether remissions of chronic hepatitis B induced by alpha-interferon therapy are of long duration.

METHODS

Cohort study.

METHODS

Clinical Center of the National Institutes of Health, a tertiary referral center.

METHODS

Sixty-four patients with chronic hepatitis B were treated with alpha-interferon between 1984 and 1986.

METHODS

Patients were followed with frequent examinations and determinations of serum liver biochemical tests and hepatitis B virus (HBV) markers including hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA using blot hybridization and polymerase chain reaction.

RESULTS

Among 64 patients with chronic hepatitis B who were treated with alpha-interferon, 23 (36%) responded to treatment with loss of HBeAg and improvement in serum aminotransferases. All 23 have been followed for 3 to 7 years (mean, 4.3 years). During follow-up, 3 of 23 patients relapsed, with reappearance of HBeAg and abnormal serum aminotransferases, all within 1 year of therapy. The remaining 20 patients continued to have no detectable HBeAg or HBV DNA (using blot hybridization) in serum and to be asymptomatic for liver disease, although 3 had minimal elevations in serum aminotransferases. Thirteen patients (65%) became negative for HBsAg between 0.2 and 6 years (mean, 3 years) after loss of HBeAg. Although no patient had HBV DNA that was detectable by blot hybridization, the 7 patients who remained HBsAg positive all had HBV DNA in serum detected by polymerase chain reaction, but only 2 of 13 HBsAg-negative patients had viral genome using this method. Testing sequential samples indicated that HBV DNA detected by polymerase chain reaction usually disappeared at or around the time that test results for HBsAg became negative.

CONCLUSIONS

Remissions in chronic hepatitis B induced by alpha-interferon are of long duration and are followed, in most patients, by the loss of HBsAg and all evidence of residual virus replication.

Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or <em>interferon</em> alfa (IFN-<em>alpha</em>) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-<em>alpha</em> or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The <em>3</em>-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.

Prospective identification of patients with stage IV renal cell carcinoma more likely to benefit from cytokine therapy could be used as a stratification factor in Phase III trials and in risk-directed therapy. The relationship between pretreatment clinical features and survival was evaluated in patients treated in Phase II and III clinical trials for metastatic renal cell carcinoma at the Memorial Sloan-Kettering Cancer Center. The first analysis was performed in 670 patients treated with cytokines or chemotherapy, and a multivariate model was created to predict survival. Studies that followed addressed four topics: (1) the survival of patients given <em>interferon</em> <em>alpha</em> as first-line therapy, (2) a comparison of survival for patients treated with chemotherapy versus cytokine therapy, (<em>3</em>) survival of patients with nonclear cell histologic features, and (4) survival of patients treated with a second-line therapy. Prognostic models based on pretreatment clinical and laboratory variables can help identify patients more likely to benefit from standard therapies, as well as assist in the interpretation of drug effectiveness in Phase II clinical trials. Investigations into new prognostic factors based on tumor biology are needed and of high priority.

Immunotherapy with <em>interferon</em>-<em>alpha</em> (IFN<em>alpha</em>) may induce depressive symptoms, anxiety and major depression when administered for at least 1-<em>3</em> months at a dose of <em>3</em>-10 MUI daily, twice or three times a week. Previously, it has been shown that immunotherapy with interleukin-2 (IL-2) significantly induces the cytokine network, as measured by increases in serum IL-6, IL-10 and the IL-2 receptor (IL-2R), and that the immunotherapy-induced changes in the cytokine network are significantly correlated with the increases in depression ratings. The main aim of this study was to examine the effects of immunotherapy with IFN<em>alpha</em> on the cytokine network in relation to changes in depression and anxiety ratings. Fourteen patients, affected by chronic active C-hepatitis, were treated with IFN<em>alpha</em> (<em>3</em>-6 MUI s.c. three/six times a week for 6 months) and had measurements of serum IFN-gamma (IFNgamma), IL-2, IL-6, IL-6R, IL-8 and IL-10 before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFN<em>alpha</em>. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Repeated measure (RM) design ANOVAs showed significantly higher MADRS and HAM-A scores 2-4 weeks and 4-6 months after starting IFN<em>alpha</em>-based immunotherapy than at baseline. RM design ANOVAs showed significantly higher serum IL-6 and IL-8 levels 2-4 weeks after starting IFN<em>alpha</em>-based immunotherapy and higher serum IL-10 levels 2-4 weeks and 4-6 months after starting therapy than at baseline. There were significant relationships between the IFN<em>alpha</em>-induced changes in serum IL-6 or IL-8 and the depression and anxiety scores. The findings show that IFN<em>alpha</em>-based immunotherapy induces the cytokine network and that IFN<em>alpha</em>-induced increases in IL-6 predicts the development of depressive symptoms. Depressive symptoms following IFN<em>alpha</em> treatment may be secondary to cytokine induction, including that of IL-6.

Viral infection of mammalian cells triggers the synthesis and secretion of type I <em>interferons</em> (i.e. IFN-<em>alpha</em>/beta), which induce the transcription of genes that cause cells to adopt an antiviral state. Many viruses have adapted mechanisms to evade IFN-<em>alpha</em>/beta-mediated responses. The leader protein of mengovirus, a picornavirus, has been implicated as an IFN-<em>alpha</em>/beta antagonist. Here, we show that the leader inhibits the transcription of IFN-<em>alpha</em>/beta and that both the presence of a zinc finger motif in its N-terminus and phosphorylation of threonine-47 are required for this function. Transcription of IFN-<em>alpha</em>/beta genes relies on the activity of a number of transcription factors, including <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>). We show that the leader interferes with the transactivation activity of IRF-<em>3</em> by interfering with its dimerization. Accordingly, mutant viruses with a disturbed leader function were impaired in their ability to suppress IFN-<em>alpha</em>/beta transcription in vivo. By consequence, the leader mutant viruses had an impaired ability to replicate and spread in normal mice but not in IFNAR-KO mice, which are incapable of mounting an IFN-<em>alpha</em>/beta-dependent antiviral response. These results suggest that the leader, by suppressing IRF<em>3</em>-mediated IFN-<em>alpha</em>/beta production, plays an important role in replication and dissemination of mengovirus in its host.

Mouse hepatitis virus (MHV) was used as a model to study the interaction of coronaviruses with the <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) response. While MHV strain A59 appeared to induce IFN-beta gene transcription and low levels of nuclear translocation of the IFN-beta transcription factor <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>), MHV did not induce IFN-beta protein production during the course of infection in L2 mouse fibroblast cells. In addition, MHV was able to significantly decrease the level of IFN-beta protein induced by both Newcastle disease virus (NDV) and Sendai virus infections, without targeting it for proteasomal degradation and without altering the nuclear translocation of IRF-<em>3</em> or IFN-beta mRNA production or stability. These results indicate that MHV infection causes an inhibition of IFN-beta production at a posttranscriptional level, without altering RNA or protein stability. In contrast, MHV induced IFN-beta mRNA and protein production in the brains of infected animals, suggesting that the inhibitory mechanisms observed in vitro are not enough to prevent IFN-<em>alpha</em>/beta production in vivo. Furthermore, MHV replication is highly resistant to IFN-<em>alpha</em>/beta action, as indicated by unimpaired MHV replication in L2 cells pretreated with IFN-beta. However, when L2 cells were coinfected with MHV and NDV in the presence of IFN-beta, NDV, but not MHV, replication was inhibited. Thus, rather than disarming the antiviral activity induced by IFN-beta pretreatment completely, MHV may be inherently resistant to some aspects of the antiviral state induced by IFN-beta. These findings show that MHV employs unique strategies to circumvent the IFN-<em>alpha</em>/beta response at multiple steps.

We have recently described a panel of monoclonal antibodies (mAb), that recognize two novel leukocyte surface antigens, BDCA-2 and BDCA-4. BDCA-2 is a novel type II C-type lectin specifically expressed by plasmacytoid dendritic cells (PDCs) that can internalize antigen for presentation to T cells. Furthermore, signaling via BDCA-2 may play a role in switching from <em>interferon</em> (IFN)-<em>alpha</em>/beta-controlled to interleukin (IL)-12-controlled immune response pathways, as triggering of BDCA-2 potently inhibits secretion of IFN-<em>alpha</em>/beta by PDCs and thereby promotes IL-12 p70 production in PDCs and other cells. Viruses may exploit this switch to escape innate antiviral immunity, but it may be beneficial for patients with systemic lupus erythematosus (SLE) if induced, for instance by anti BDCA-2 mAb treatment. BDCA-4 is shown here to be identical to neuropilin-1 (NP-1), a neuronal receptor for the axon guidance factors belonging to the class-<em>3</em> semaphorin subfamily, and a receptor on endothelial and tumor cells for vascular endothelial growth factor (VEGF-A). In blood and bone marrow, BDCA-4/NP-1 is exclusively expressed on PDCs, but in tonsils also on a few other cells, primarily follicular B helper memory T cells (T(FH)).

Alcoholic liver disease (ALD) features increased hepatic exposure to bacterial lipopolysaccharide (LPS). Toll-like receptor-4 (TLR4) recognizes LPS and activates signaling pathways depending on MyD88 or TRIF adaptors. We previously showed that MyD88 is dispensable in ALD. TLR4 induces Type I <em>interferons</em> (IFNs) in an MyD88-independent manner that involves <em>interferon</em> regulatory factor-<em>3</em> (IRF<em>3</em>). We fed alcohol or control diets to wild-type (WT) and IRF<em>3</em> knock-out (KO) mice, and to mice with selective IRF<em>3</em> deficiency in liver parenchymal and bone marrow-derived cells. Whole-body IRF<em>3</em>-KO mice were protected from alcohol-induced liver injury, steatosis, and inflammation. In contrast to WT or bone marrow-specific IRF<em>3</em>-KO mice, deficiency of IRF<em>3</em> only in parenchymal cells aggravated alcohol-induced liver injury, associated with increased proinflammatory cytokines, lower antiinflammatory cytokine interleukin 10 (IL-10), and lower Type I IFNs compared to WT mice. Coculture of WT primary murine hepatocytes with liver mononuclear cells (LMNC) resulted in higher LPS-induced IL-10 and IFN-β, and lower tumor necrosis factor <em>alpha</em> (TNF-α) levels compared to LMNC alone. Type I IFN was important because cocultures of hepatocytes with LMNC from Type I IFN receptor KO mice showed attenuated IL-10 levels compared to control cocultures from WT mice. We further identified that Type I IFNs potentiated LPS-induced IL-10 and inhibited inflammatory cytokine production in both murine macrophages and human leukocytes, indicating preserved cross-species effects. These findings suggest that liver parenchymal cells are the dominant source of Type I IFN in a TLR4/IRF<em>3</em>-dependent manner. Further, parenchymal cell-derived Type I IFNs increase antiinflammatory and suppress proinflammatory cytokines production by LMNC in paracrine manner.

CONCLUSIONS

Our results indicate that IRF<em>3</em> activation in parenchymal cells and resulting type I IFNs have protective effects in ALD by way of modulation of inflammatory functions in macrophages. These results suggest potential therapeutic targets in ALD.

C<em>3</em>H/HeN mice infected intravenously with a dose of Rickettsia conorii (Malish 7 strain) that is sublethal for immunocompetent animals (1.1 x 10(<em>3</em>) PFU) developed disseminated infection of endothelial cells of the brain, lungs, heart, liver, kidney, testis, and testicular adnexa. In R. conorii-infected mice depleted of gamma <em>interferon</em> (IFN-gamma) and/or tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) by intravenous administration of neutralizing monoclonal antibodies on days 0, 2, and 4, the mortality rate was 100%. Death of the cytokine-depleted animals on days 5 and 6 was associated with overwhelming rickettsial infection documented by titration of rickettsial content in the brain and liver and by immunohistologic demonstration of massive quantities of R. conorii in endothelial cells of all organs examined, in macrophages of the liver and spleen, and in hepatocytes. Nondepleted, immunocompetent animals showed markedly reduced rickettsial content in the tissues on day 6, with rickettsial destruction in phagolysosomes not only in macrophages but also in endothelial cells and hepatocytes. All nondepleted, infected mice recovered and appeared completely healthy by day 9. Assay of liver infiltrated by lymphocytes and macrophages revealed mRNA of IFN-gamma and TNF-<em>alpha</em>, indicating that the host defenses were activated at the site of infection. Treatment of mice with an analog of L-arginine reduced the synthesis of nitric oxide and impaired rickettsial killing. Nitric oxide production was also impaired in cytokine-depleted infected mice. These observations support the hypothesis that IFN-gamma secreted by T lymphocytes and natural killer cells and TNF-<em>alpha</em> secreted by macrophages act in a synergistic, paracrine fashion on adjacent rickettsia-infected endothelial cells, hepatocytes, and macrophages to stimulate synthesis of nitric oxide, which kills intracellular R. conorii.

Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,<em>3</em>-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating <em>interferon</em> (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-<em>alpha</em> and interleukin (IL)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-<em>alpha</em> and IL-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-<em>3</em>-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and 20-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

<em>Alpha</em> <em>interferon</em> (IFN-<em>alpha</em>)-induced transcriptional activation requires the induction of a complex of DNA-binding proteins, including tyrosine-phosphorylated Stat1 and Stat2, and of p48, a protein which is not phosphorylated on tyrosine and which comes from a separate family of DNA-binding proteins. The isolation and characterization of U6A cells, which lack Stat2, have allowed the introduction of normal and mutant forms of Stat2 so that various functions of the Stat2 protein can be examined. As reported earlier, Stat1, which is the second target of tyrosine phosphorylation in IFN-<em>alpha</em>-treated cells, is not phosphorylated in the absence of Stat2. We show that all mutations that block Stat2 phosphorylation also block Stat1 phosphorylation. These include not only the mutations of Y-690 and SH2 domain residues that are involved in tyrosine phosphorylation but also short deletions at the amino terminus of the protein. Two mutants of Stat2 that are not phosphorylated on tyrosine can act as dominant negative proteins in suppressing wild-type Stat2 phosphorylation, most likely by competition at the receptor-kinase interaction site(s). We also show that the COOH-terminal 50 amino acids are required for transcriptional activation in response to IFN-<em>alpha</em>. Mutants lacking these amino acids can be phosphorylated, form IFN-stimulated gene factor <em>3</em>, and translocate to the nucleus but cannot stimulate IFN-<em>alpha</em>-dependent transcription. Seven acidic residues are present in the deleted COOH-terminal residues, but 24 acidic residues still remain in the 100 carboxy-terminal amino acids after deletion. Thus, transcriptional activation is unlikely to depend on acidic amino acids alone.

We performed an escalating dose study of the combined administration of interleukin-2 (IL-2) and <em>alpha</em>-<em>interferon</em> (<em>alpha</em>-IFN) in 94 patients with metastatic cancer. Patients received <em>alpha</em>-IFN at a dose of <em>3</em> x 10(6) U/m2 in conjunction with IL-2 at doses of either 1 x 10(6) U/m2 (six patients), <em>3</em> x 10(6) U/m2 (<em>3</em>2 patients), or 4.5 x 10(6) U/m2 (26 patients). Thirty patients received <em>alpha</em>-IFN at 6 x 10(6) U/m2 plus IL-2 at 4.5 x 10(6) U/m2. Patients each received cytokine as an intravenous bolus infusion every 8 hours for up to 5 consecutive days and after a 10-day rest received a second cycle of combination cytokines. Of the 91 patients evaluable for response, seven patients had a complete regression of cancer, and 18 had a partial regression. At the four increasing dose levels used in patients with renal cell cancer (<em>3</em>5 patients) or melanoma (<em>3</em>9 patients), objective responses were seen in 17% (of six patients), 24% (of 25 patients), <em>3</em>8% (of 16 patients), and 41% (of 27 patients), respectively. Of the 25 total responding patients, 16 are still responding 5 to 14 months after treatment. The toxicities associated with the combined administration of IL-2 and <em>alpha</em>-IFN were similar to those expected from each agent alone. There was one treatment-related death in the 94 patients treated in this study. Thus, using increasing doses of the combination of IL-2 and <em>alpha</em>-IFN, it appears that response rates may be related to the doses of the cytokines used, and that at the highest doses of these combination cytokines, response rates may be higher than those for either cytokine alone. A prospective randomized trial comparing the cytokine combinations with each cytokine administered alone is necessary as is the extension of this combination cytokine treatment to patients with other types of solid cancer.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor <em>alpha</em> (IL-2R <em>alpha</em>) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 1<em>3</em>-acetate and ionomycin, the levels of IL-<em>3</em>, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), and gamma <em>interferon</em> (IFN-gamma) were only 2- to <em>3</em>-fold lower compared with normal T cells. In contrast, anti-CD<em>3</em> and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD<em>3</em>- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-<em>alpha</em>, and IFN-gamma, but not IL-<em>3</em> and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-<em>3</em> and GM-CSF.

Iron chelators inhibit endotoxin-induced NF-kappaB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-kappaB activation. The present study tested this hypothesis. Analysis of Fe(59)-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW).Fe(59) complex ([LMW.Fe](i)) at </=2 min returning to the basal level within 15 min. The [LMW.Fe](i) response preceded IkappaB kinase (IKK) >>/=15 min) and NF-kappaB >>/=<em>3</em>0 min) activation. Iron chelators (1,2-dimethyl-<em>3</em>-hydroxypyridin-4-one and N,N'-bis-2-hydroxybenzylethylenediamine-N,N'-diacetic acid) abrogated the [LMW.Fe](i) response and IKK and NF-kappaB activation. The [LMW.Fe](i) response was also observed in tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>)-stimulated HMs and RAW264.7 cells treated with LPS and <em>interferon</em>-gamma but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNF<em>alpha</em>. Both [LMW.Fe](i) response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases), l-N(6)-(1-iminoethyl)lysine (selective iNOS inhibitor), and adenoviral-mediated expression of a dominant negative mutant of Rac1 or Cu,Zn-superoxide dismutase, suggesting the role of (.)NO and O(2)() in mediating the iron signaling. In fact, this inhibition was recapitulated by a cell-permeable scavenger of ONOO(-), 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride. Conversely, ONOO(-) alone induced both [LMW.Fe](i) response and IKK activation. Finally, direct addition of ferrous iron to cultured HMs activated IKK and NF-kappaB. These results support a novel signaling role for [LMW.Fe](i) in IKK activation, which appears to be induced by ONOO(-) and selectively operative in macrophages.

<em>Interferon</em> regulatory factor-<em>3</em> (IRF-<em>3</em>) activation directs <em>alpha</em>/beta <em>interferon</em> production and <em>interferon</em>-stimulated gene (ISG) expression, which limits virus infection. Here, we examined the distribution of hepatitis C virus (HCV) nonstructural <em>3</em> protein, the status of IRF-<em>3</em> activation, and expression of IRF-<em>3</em> target genes and ISGs during asynchronous HCV infection in vitro and in liver biopsies from patients with chronic HCV infection, using confocal microscopy and functional genomics approaches. In general, asynchronous infection with HCV stimulated a low-frequency and transient IRF-<em>3</em> activation within responsive cells in vitro that was associated with cell-to-cell virus spread. Similarly, a subset of HCV patients exhibited the nuclear, active form of IRF-<em>3</em> in hepatocytes and an associated increase in IRF-<em>3</em> target gene expression in hepatic tissue. Moreover, ISG expression profiles formed disease-specific clusters for HCV and control nonalcoholic fatty liver disease patients, with increased ISG expression among the HCV patients. We identified the presence of T cell and plasmacytoid dendritic cell infiltrates within all biopsy specimens, suggesting they could be a source of hepatic <em>interferon</em> in the setting of hepatitis C and chronic inflammatory condition.

CONCLUSIONS

These results indicate that HCV can transiently trigger IRF-<em>3</em> activation during virus spread and that in chronic HCV, IRF-<em>3</em> activation within infected hepatocytes occurs but is limited.

OBJECTIVE

Little is known about the long-term effects of interferon alpha on clinical outcome and survival of patients with chronic hepatitis D.

METHODS

Thirty-six patients with chronic hepatitis D who participated in a randomized controlled trial of a 48-week course of high (9 million units) or low (3 million units) doses of interferon alpha or no treatment were followed for an additional 2 to 14 years.

RESULTS

Long-term survival was significantly longer in the high-dose group than in untreated controls (P = 0.003) or in the low-dose group (P = 0.019) but did not differ between patients treated with 3 million units and controls. Among surviving patients at 12 years of follow-up, a biochemical response was present in 7 of 12 treated with 9 million units, in 2 of 4 who received 3 million units, and in none of 3 controls. Long-term alanine aminotransferase (ALT) normalization correlated with improved hepatic function and loss of IgM antibody to hepatitis delta antigen (anti-HD). Patients in the high-dose group had a sustained decrease in HDV replication (P = 0.008), leading to clearance of HDV RNA and, eventually, hepatitis B virus (HBV) in some patients, as well as a dramatic improvement in liver histology with respect to activity grade (P = 0.0004) and fibrosis stage (P = 0.007). Strikingly, we documented an absence of fibrosis in the final biopsy of 4 patients with a long-term biochemical response and an initial diagnosis of active cirrhosis.

CONCLUSIONS

High doses of interferon alpha-2a significantly improved the long-term clinical outcome and survival of patients with chronic hepatitis D, even though the majority had active cirrhosis before the onset of therapy.