BACKGROUND

Retinoids (vitamin A derivatives) and interferon-alpha (IFN-alpha) are potent regulators of malignant cell differentiation and proliferation, and both have immunomodulatory and antiangiogenesis activity. A large body of preclinical and clinical data supports the use of combination therapy with 13-cis-retinoic acid (13-cRA) and IFN-alpha in patients with squamous cell carcinoma of the skin. This carcinoma is an extremely common and frequently severely disfiguring cancer, for which about 10% of patients remain uncured following standard local therapy.

OBJECTIVE

Our purpose was to test whether a 20% or greater complete response rate could be achieved using a combination of these two agents in patients with advanced squamous cell carcinoma of the skin in whom local therapy had failed or was unfeasible or who had regional and/or distant metastases.

METHODS

Thirty-two patients with heavily pretreated, advanced inoperable cutaneous squamous cell carcinoma of the skin were given a combination of oral 13-cRA (1 mg/kg per day) and subcutaneous recombinant human IFN alpha-2a (3 million units per day) for at least 2 months, unless disease progressed earlier, in a phase II trial.

RESULTS

Nineteen (68%) of the 28 assessable patients responded, seven (25%) of whom had complete responses. Response rates were 93% (13 of 14) in patients with advanced local disease (six complete responses), 67% (four of six) in patients with regional disease (no complete responses), and 25% (two of eight) in patients with distant metastases (one complete response). The relationship between decreased response rate and increased extent of disease was highly statistically significant (P less than .005, chi-square test). The median response duration was greater than 5 months. No life-threatening toxic effects occurred in assessable patients treated with this combination, although dose reductions were required in 18 patients. The major limiting side effect in this elderly patient population (median age, 67 years) was cumulative fatigue.

CONCLUSIONS

These results indicate that combined systemic therapy with 13-cRA and IFN alpha-2a is highly effective in patients with advanced squamous cell carcinoma of the skin.

<em>Alpha</em> <em>interferon</em> is the only drug that has been shown to be effective in the treatment of chronic hepatitis C, but only half of patients respond, either transiently or permanently. Pretreatment features that are associated with a greater likelihood of response to short courses of <em>interferon</em> include low hepatitis C virus (HCV) RNA levels, viral genotypes 2 or <em>3</em>, and the absence of fibrosis or cirrhosis on liver biopsy. Each of these features is more predictive of sustained response (SR) than the end-of-treatment response (ETR). However, the accuracy of these features in predicting response in individual patients is poor. Furthermore, there are several limitations to using these factors in the clinical management of patients. Most importantly, they were identified in 6-month treatment trials. Longer treatment or combination of <em>interferon</em> with ribavirin reduces relapses and will therefore lessen the association of these factors with long-term response. In addition, changes in the definition of treatment end points and the technology used to measure HCV RNA might change the association between these predictive factors and response. The best predictor of a treatment response is the early normalization of the serum alanine aminotransferase (ALT) level during <em>interferon</em> treatment. HCV RNA loss during treatment may also be helpful in predicting response, but it is probably no better than serum ALT levels and is expensive. In summary, several clinical and virological features are associated with higher response rates to <em>interferon</em> treatment. Although pretreatment factors do not accurately predict treatment outcome in individuals, they may be helpful in counseling patients and making treatment decisions.

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and <em>3</em>' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to <em>3</em>6 h after transfection with a signal-to-noise ratio of 2 to <em>3</em> log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and <em>alpha</em> <em>interferon</em> inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

OBJECTIVE

To determine the efficacy of isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF) in combination with interferon-gamma (IFN) and melphalan as induction therapy to render tumors resectable and avoid amputation in patients with nonresectable extremity soft tissue sarcomas (STS).

METHODS

Among 55 patients with 30 primary and 25 recurrent sarcomas, there were 48 high-grade and seven grade 1 sarcomas (very large, recurrent, or multiple). The composition of this series of patients is unusual: 13 patients (24%) had multifocal primary sarcomas or multiple recurrent tumors; tumors were very large (median, 18 cm); and nine patients (16%) had known systemic metastases. IFN was administered subcutaneously on the 2 days before ILP with TNF, IFN, and melphalan. A delayed marginal resection of the tumor remnant was usually performed 2 to 3 months after ILP.

RESULTS

A major tumor response was seen in 87% of patients and rendered the sarcomas resectable in most cases. Clinical response rates were as follows: 10 (18%) completes responses (CRs), 35 (64%) partial responses (PRs), and 10 (18%) no change (NC). Final outcome was defined as follows by clinical and pathologic response: 20 (36%) CRs, 28 (51%) PRs, and seven (13%) NC. Limb salvage was achieved in 84% (follow-up duration, 20+ to 50+ months). In 39 patients, resection of the tumor remnant (n = 31) or of two to eight tumors (n = 8) after ILP was performed; local recurrence developed in five (13%). When no resection was performed (multiple tumors or systemic metastases), local recurrences were frequent (five of 16), but limb salvage was often achieved as patients died of systemic disease. Regional toxicity was limited and systemic toxicity minimal to moderate with no toxic deaths. Histology showed hemorrhagic necrosis; angiographies showed selective destruction of tumor-associated vessels.

CONCLUSIONS

ILP with TNF, IFN, and melphalan is a safe and highly effective induction biochemotherapy procedure that can achieve limb salvage in patients with nonresectable extremity STS. TNF is an active anticancer drug in humans in the setting of ILP.

Mounting evidence suggests that aberrations in immune-inflammatory pathways contribute to the pathophysiology of major depressive disorder (MDD), and individuals with MDD may have elevated levels of predominantly pro-inflammatory cytokines and C-reactive protein. In addition, previous meta-analyses suggest that antidepressant drug treatment may decrease peripheral levels of interleukin-1 beta (IL-1β) and IL-6. Recently, several new studies examining the effect of antidepressants on these cytokines have been published, and so we performed an updated meta-analysis of studies that measured peripheral levels of cytokines and chemokines during antidepressant treatment in patients with MDD. The PubMed/MEDLINE, EMBASE, and PsycInfo databases were searched from inception through March 9, 2017. Forty-five studies met inclusion criteria (N = 1517). Peripheral levels of IL-6, tumor necrosis factor-<em>alpha</em> (TNF-α), IL-1β, IL-10, IL-2, IL-4, <em>interferon</em>-γ, IL-8, the C-C motif ligand 2 chemokine (CCL-2), CCL-<em>3</em>, IL-1 receptor antagonist, IL-1<em>3</em>, IL-17, IL-5, IL-7, and the soluble IL-2 receptor were measured in at least three datasets and thus were meta-analyzed. Antidepressant treatment significantly decreased peripheral levels of IL-6 (Hedges g = -0.454, P <0.001), TNF-α (g = -0.202, P = 0.015), IL-10 (g = -0.566, P = 0.012), and CCL-2 (g = -1.502, P = 0.006). These findings indicate that antidepressants decrease several markers of peripheral inflammation. However, this meta-analysis did not provide evidence that reductions in peripheral inflammation are associated with antidepressant treatment response although few studies provided separate data for treatment responders and non-responders.

OBJECTIVE

To study cytokine expression in muscle tissues of patients with inflammatory myopathies and to compare the profiles of patients with polymyositis (PM), inclusion body myositis (IBM), and dermatomyositis (DM).

METHODS

We performed indirect immunohistochemistry studies of muscle tissue sections with a panel of 16 different cytokine-specific monoclonal antibodies, directed against interleukin-1<em>alpha</em>, (IL-1<em>alpha</em>), IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-2, IL-<em>3</em>, IL-4, IL-6, IL-8, IL-10, IL-1<em>3</em>, <em>interferon</em>-gamma (IFN gamma), tumor necrosis factor <em>alpha</em> (TNF <em>alpha</em>), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor beta1 (TGF beta1), TGF beta2, and TGF beta<em>3</em> in 5 untreated patients each with PM, DM, and IBM and in 4 normal controls. Fresh frozen muscle tissue sections were fixed in formaldehyde before the procedure. The use of saponin as a detergent to permeabilize the cell membranes enabled identification of intracellular cytokine production.

RESULTS

The most prominent finding was the expression of IL-1<em>alpha</em> observed in all patients but in none of the normal controls. In all patients with PM, DM, and IBM, IL-1<em>alpha</em> was expressed in endothelial cells of capillaries, arterioles, and venules in areas surrounded by inflammatory cells, and also in areas with no or scarce inflammatory cells in both endomysium and perimysium. Furthermore, IL-1<em>alpha</em> was also expressed in mononuclear inflammatory cells in all 15 cases. IL-1beta was observed in inflammatory cells in 10 of the 15 patients but, in contrast to IL-1<em>alpha</em>, it was not expressed in blood vessel walls. TGF beta1, TGF beta2, and TGF beta<em>3</em> were strongly positive in all 15 patients, but only scattered cells were positive in the normal controls. The remaining cytokines were observed only in relatively few cells and only in occasional patients (although the patients were selected for the presence of large infiltrates), and in none of the controls. The patterns were similar in PM, DM, and IBM.

CONCLUSIONS

Cytokine expression in muscle tissue of patients with inflammatory myopathy is dominated by IL-1<em>alpha</em>, IL-1beta, and TGF beta1-<em>3</em>. The predominant IL-1<em>alpha</em> expression in the blood vessels indicates an importance of the endothelial cells in the inflammatory process in PM, IBM, and DM. A sustained, local release of T cell-derived cytokines may not be a requirement for tissue injury in the inflammatory myopathies. There does not appear to be a qualitative difference in cytokine expression patterns in PM, IBM, and DM.

Obesity and related metabolic conditions are of epidemic proportions in most of the world, affecting both adults and children. The accumulation of lipids in the body in the form of white adipose tissue in the abdomen is now known to activate innate immune mechanisms. Lipid accumulation causes adipocytes to directly secrete the cytokines interleukin (IL) 6 and tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>), but also monocyte chemoattractant protein 1 (MCP-1), which results in the accumulation of leukocytes in fat tissue. This sets up a chronic inflammatory state which is known to mediate the association between obesity and conditions such as cardiovascular disease, type 2 diabetes, and cancer. There is also a substantial literature linking inflammation with risk for depression. This includes the observations that: (1) people with inflammatory diseases such as multiple sclerosis, cardiovascular disease, and psoriasis have elevated rates of depression; (2) many people administered inflammatory cytokines such as <em>interferon</em> <em>alpha</em> develop depression that is indistinguishable from depression in non-medically ill populations; (<em>3</em>) a significant proportion of depressed persons show upregulation of inflammatory factors such as IL-6, C-reactive protein, and TNF<em>alpha</em>; (4) inflammatory cytokines can interact with virtually every pathophysiologic domain relevant to depression, including neurotransmitter metabolism, neuroendocrine function, and synaptic plasticity. While many factors may contribute to the association between inflammatory mediators and depression, we hypothesize that increased adiposity may be one causal pathway. Mediational analysis suggests a bi-directional association between adiposity and depression, with inflammation possibly playing an intermediary role.

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor <em>alpha</em> (ER<em>alpha</em>) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ER<em>alpha</em>-, or ERbeta-deficient mice, and each was treated with the highly selective ER<em>alpha</em> agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ER<em>alpha</em> ligand treatment ameliorated clinical disease in both wild-type and ERbeta knock-out mice, but not in ER<em>alpha</em> knock-out mice, thereby demonstrating that the ER<em>alpha</em> ligand maintained ER<em>alpha</em> selectivity in vivo during disease. ER<em>alpha</em> ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNF<em>alpha</em>, <em>interferon</em>-gamma, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/beta<em>3</em>-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac <em>3</em>+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1-2 d after the onset of clinical signs. Treatment with either estradiol or the ER<em>alpha</em> ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ER<em>alpha</em> ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.

The antiproliferative effects of <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>) and <em>interferon</em> gamma (IFN-gamma) were found to be cell-dependent. Among the human cell lines examined, IFN-gamma had a greater antiproliferative effect against cell lines that exhibited induction of indoleamine 2,<em>3</em>-dioxygenase, such as the KB oral carcinoma or WiDr colon adenocarcinoma, than against those that lacked the enzyme activity, such as the SW480 colon adenocarcinoma or NCI-H128 small-cell lung carcinoma. Induction of this dioxygenase showed a clear temporal relationship with increased metabolism of L-tryptophan and the depletion of this amino acid in the culture medium. While 70-80% of L-tryptophan remained in the medium of IFN-<em>alpha</em>- or vehicle-treated cells, virtually all of this amino acid was depleted in the medium of the IFN-gamma-treated group following 2-<em>3</em> days of culture. Supplementing the growth medium with additional L-tryptophan reversed the antiproliferative effect of IFN-gamma against KB cells in a dose- and time-dependent manner. The antiproliferative effects of IFN-<em>alpha</em> and IFN-gamma on SW480 and NCI-H128 cells, which are independent of the dioxygenase activity, and the inability of added L-tryptophan to reverse the effects of IFN-gamma in WiDr cells suggest multiple mechanisms of action of the IFNs. The data show that the antiproliferative effect of IFN-gamma through induction of indoleamine 2,<em>3</em>-dioxygenase, with a consequent L-tryptophan deprivation, is an effective means of regulating cell growth.

BACKGROUND

The prognosis of hepatocellular carcinoma (HCC) invading into the major branches of the portal vein (Vp<em>3</em>) is extremely poor.

METHODS

Eleven consecutive patients with HCC and Vp<em>3</em> were treated with 2-6 cycles of a "basic" combination therapy consisting of continuous arterial infusion of 5-fluorouracil (450-500 mg/day, for the initial 2 weeks) and subcutaneous injection of <em>interferon</em>-<em>alpha</em> (5 million international units, <em>3</em> times/week, 4 weeks). In the first <em>3</em> patients, methotrexate (90 mg/day 1 of every week), cisplatin (10 mg/day), and leucovorin (<em>3</em>0 mg/days 2 and <em>3</em> of every week) also were administered for the initial 2 weeks ("full" regimen).

RESULTS

In 8 (7<em>3</em>%) of 11 patients, an objective response (complete response [CR] or partial response [PR]) was observed with marked regression of tumor and decrease in tumor markers. The use of the full regimen was associated with objective response in all patients; instead, they developed thrombocytopenia or leukopenia. In the subsequent 8 patients with basic regimen, 5 patients showed CR (2 cases) or PR (<em>3</em> cases; objective response rate, 6<em>3</em>%), and leukopenia was observed only in 1 patient.

CONCLUSIONS

Simple combination therapy with subcutaneous <em>interferon</em>-<em>alpha</em> and intraarterial 5-fluorouracil therefore is a promising treatment modality for intractable HCC with Vp<em>3</em>.

The nature of the host cellular immune response largely determines the expression of disease following infection with the intracellular protozoans Leishmania spp. In experimental animals control and resolution of infection are mediated by gamma <em>interferon</em> and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), whereas disease progression is associated with the production of interleukin 4 (IL-4), IL-5, IL-10, and transforming growth factor beta (TGF-beta). We have analyzed the profile of cytokine gene expression directly in the lesions of 1<em>3</em> patients with localized cutaneous leishmaniasis due to Leishmania mexicana. All but one patient had a single lesion, and the time of evolution ranged from 8 days to 18 months. Cytokine gene expression was quantitated by reverse transcriptase PCR and interpolation from a standard curve. Gamma <em>interferon</em>, TNF-<em>alpha</em>, IL-1 <em>alpha</em>, IL-6, IL-10, and TGF-beta gene expression was present in all samples. IL-<em>3</em> and IL-4 gene expression was barely detectable in 1 and <em>3</em> of 1<em>3</em> samples, respectively. IL-2 and IL-5 mRNAs were not found. A significant increase in the expression of IL-1 <em>alpha</em>, TNF-<em>alpha</em>, IL-10, and TGF-beta was observed in late lesions >> or = 4 months) compared with that in early lesions (< or = 2 months). Because of their inhibitory effects on macrophage function, the expression of IL-10 and TGF-beta may play a role in the immunopathogenesis of chronic cutaneous leishmaniasis.

Proinflammatory cytokines including <em>interferon</em>-gamma (IFNgamma), interleukin-6 (IL-6), and tumor necrosis factor-<em>alpha</em> (TNF<em>alpha</em>) are implicated in the pathogenesis of acute graft-versus-host disease (aGVHD). Cytokine gene polymorphism is associated with functional differences in cytokine regulation and altered clinical performance in a variety of diseases. Polymorphism in the IFNgammaIntron1 microsatellite (CA)n repeat has been linked with in vitro IFNgamma production and renal transplant rejection. The IL-6(-174)(G/C) single nucleotide polymorphism has been linked to in vitro and in vivo IL-6 production, juvenile chronic arthritis, and renal transplant rejection. This study examined the potential association of GVHD with IFNgamma and IL-6 polymorphisms in 80 sibling bone marrow transplant (BMT) donor/recipient pairs. Patients homozygous for the IFNgammaIntron1 allele <em>3</em> had more severe (grade III-IV) aGVHD. Patients possessing the IL-6(-174)G allele had a trend toward higher grades of aGVHD, and those homozygous for the IL-6(-174)G allele were more likely to develop chronic GVHD (cGVHD). The associations of previously identified aGVHD severity-associated cytokine gene polymorphisms (TNFd and IL-10(-1064)) with severe aGVHD were reconfirmed. Logistic regression analysis confirmed the association of severe aGVHD with recipient genotype at IFNgammaIntron1 (odds ratio [OR] <em>3</em>.92; P =.02), IL-10(-1064) (OR 4.61; P =.026) and TNFd (OR <em>3</em>.29; P =.0<em>3</em>9), and that of cGVHD with recipient IL-6(-174) genotype (OR 4.25; P =.007), in addition to age, gender mismatch, and underlying disease. Assessment of cytokine genotype may potentially allow more accurate prediction of GVHD and appropriate adjustment of GVHD prophylaxis, as well as indicating novel areas for future studies of GVHD pathogenesis.

<em>Interferon</em> regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 11<em>3</em> kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-<em>3</em> (IL-<em>3</em>). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma <em>interferon</em> (IFN-gamma)-activated sequence of the <em>interferon</em> regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-<em>alpha</em> nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-<em>3</em>, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

Hepatitis B virus-associated polyarteritis nodosa (HBV-PAN) is a typical form of classic PAN whose pathogenesis has been attributed to immune-complex deposition with antigen excess. We conducted the current study to 1) analyze the frequency of HBV infection in patients with PAN, in light of the classification systems described since 1990; 2) describe the clinical characteristics of HBV-PAN; <em>3</em>) compare the evolution according to conventional or antiviral treatment; and 4) evaluate long-term outcome. One hundred fifteen patients were included in therapeutic trials organized by the French Vasculitis Study Group and/or referred to our department for HBV-PAN between 1972 and 2002. To determine the frequency of HBV-PAN during the <em>3</em>0-year period, we analyzed a control group of patients with PAN without HBV infection, followed during the same period and diagnosed on the same bases. Depending on the year of diagnosis, different treatments were prescribed. Before the antiviral strategy was established, some patients were given corticosteroids (CS) with or without cyclophosphamide (CY). Since 198<em>3</em>, treatment for patients with HBV markers has combined 2 weeks of CS followed by an antiviral agent (successively, vidarabine, <em>interferon</em>-<em>alpha</em>, and lamivudine) combined with plasma exchanges (PE).Ninety-three (80.9%) patients entered remission during this period and 9 (9.7%) of them relapsed; 41 (<em>3</em>5.7%) patients died. For the 80 patients given the antiviral strategy as intention-to-treat, 4 (5%) relapsed and 24 (<em>3</em>0%) died vs 5 (14.<em>3</em>%) relapses (not significant [NS]) and 17 (48.6%) deaths (NS) among the <em>3</em>5 patients treated with CS alone or with CY or PE. HBe-anti-HBe seroconversion rates for the 2 groups, respectively, were: 49.<em>3</em>% vs 14.7% (p < 0.001). Patients who seroconverted obtained complete remission and did not relapse.Thus, HBV-PAN, a typical form of classic PAN, can be characterized as follows: when renal involvement is present, so is renal vasculitis; glomerulonephritis due to vasculitis is never found; antineutrophil cytoplasmic antibodies (ANCA) are not detected; relapses are rare, and never occur once viral replication has stopped and seroconversion has been obtained. Combining an antiviral drug with PE facilitates seroconversion and prevents the development of long-term hepatic complications of HBV infection. The major cause of death is gastrointestinal tract involvement. Importantly, the frequency of HBV-PAN has decreased in relation to improved blood safety and vaccination campaigns.

A brief account of how I became involved in <em>interferon</em> research is followed by recollections of key experiments that led to the discovery of the roles of the JAKs and STATs in <em>interferon</em>-dependent signaling. I then outline the complex responses of cells to <em>interferons</em>, including the roles of kinases other than JAKs and transcription factors other than STATs, differential responses to <em>interferons</em> <em>alpha</em> and beta, modulation of response by prior exposure to other cytokines ("priming"), cytokine-dependent induction of high level expression of STATs 1 and <em>3</em> and the activation of a new set of genes by these unphosphorylated STATs, and diverse patterns of STAT activation in different cell types in response to a single <em>interferon</em>.

In clinical research, the definition of the upper limit of normal (ULN) alanine transaminase (ALT) is never detailed. However, such a definition can vary and may have practical consequences. Our aim was to assess factors associated with serum ALT activity in apparently healthy subjects and then to apply seven different definitions of ULN in three different populations so as to assess the prevalence of subjects with normal ALT among blood donors and among hepatitis C patients before (normal ALT hepatitis C patients) and after treatment (<em>interferon</em> [IFN] responders). ALT measurements were performed in the same laboratory using the same technique; 1,0<em>3</em><em>3</em> donors were prospectively investigated, 186 patients with hepatitis C never treated and 40 patients treated with <em>3</em> MU three times per week of IFN-<em>alpha</em> for at least 6 months. The seven definitions (D) of ULN were: D1: 95th percentile of ALT; D2: 95th percentile after separating males and females; D<em>3</em>: males and females separately, ULN=10 (mean of log10 ALT + 1.96 SD); D4: ULN=45 IU/L given by the manufacturer; D5: mean + 1 SD after exclusion of the 5% extreme values; D6: 95th percentile after separating subjects with body mass index (BMI) under or equal to the median (2<em>3</em>); and D7: 95th percentile after separating subjects according to BMI and sex. BMI and male sex were independently associated (P < .0001; logistic regression) with ALT, without an association with alcohol. The range of ULN varied from 26 IU/L in females (D5) to 66 IU/L in males with BMI >2<em>3</em> (D7). Depending on the definition, the prevalence of blood donors with normal ALT varied from 82% to 96%, i.e., a range of 14%; that of hepatitis C patients with normal ALT varied from 16% to 27%, i.e., a range of 11%; the prevalence of IFN responders varied from 25% to 42%, i.e., a range of 17%. Definitions of normal ALT values should be adjusted for sex and BMI to reduce artificial heterogeneity in blood donor selection and in hepatitis C clinical studies.

The mechanisms by which administration of <em>interferon</em>-<em>alpha</em> induces neuropsychiatric side effects, such as depressive symptoms and changes in cognitive function, are not clear as yet. Direct influence on serotonergic neurotransmission may contribute to these side effects. In addition, the enzyme indoleamine 2,<em>3</em>-dioxygenase (IDO), which converts tryptophan into kynurenine, may play an important role, first, because IDO activation leads to reduced levels of tryptophan, the precursor of serotonin (5-HT), and thus to reduced central 5-HT synthesis. Second, kynurenine metabolites such as <em>3</em>-hydroxy-kynurenine (<em>3</em>-OH-KYN) and quinolinic acid (QUIN) have toxic effects on brain function. <em>3</em>-OH-KYN is able to produce oxidative stress by increasing the production of reactive oxygen species (ROS), and QUIN may produce overstimulation of hippocampal N-methyl-D-aspartate (NMDA) receptors, which leads to apoptosis and hippocampal atrophy. Both ROS overproduction and hippocampal atrophy caused by NMDA overstimulation have been associated with depression.

BACKGROUND

Interferon (IFN)-alpha is a pluripotent inflammatory cytokine typically induced by viral infections. In rupture-prone atherosclerotic plaques, plasmacytoid dendritic cells produce IFN-alpha. In the present study we explored the contribution of IFN-alpha to inflammation and tissue injury in the plaque microenvironment.

RESULTS

In 53% of carotid plaques (n=30), CD123+ plasmacytoid dendritic cells clustered together with CD11c+ myeloid dendritic cells, a distinct dendritic cell subset specialized in sensing danger signals from bacteria and tissue breakdown. Tissue concentrations of IFN-alpha and tumor necrosis factor (TNF)-alpha transcripts were tightly correlated (r=0.76, P<0.001), suggesting a regulatory role of IFN-alpha in TNF-alpha production. Plaque tissue stimulation with CpG ODN, a Toll-like receptor (TLR) 9 ligand, increased IFN-alpha production (57.8+/-23.7 versus 25.9+/-8.6 pg/mL; P<0.001), whereas the TLR4 ligand lipopolysaccharide induced TNF-alpha secretion (225.1+/-3.0 versus 0.7+/-0.2 pg/mL; P<0.001). Treating plaque tissue with IFN-alpha markedly enhanced lipopolysaccharide-triggered TNF-alpha secretion (559.0+/-25.9 versus 225.1+/-3.0 pg/mL; P<0.001). IFN-alpha pretreatment also amplified the effects of lipopolysaccharide on interleukin-12, interleukin-23, and matrix metalloproteinase-9, suggesting that the antiviral cytokine sensitized myeloid dendritic cells and macrophages toward TLR4 ligands. Mechanistic studies demonstrated that IFN-alpha modulated the myeloid dendritic cell response pattern by upregulating TLR4 expression (P<0.001) involving both the STAT (signal transducer and activator of transcription) and the PI(3)K pathway.

CONCLUSIONS

In the atherosclerotic plaque, IFN-alpha functions as an inflammatory amplifier. It sensitizes antigen-presenting cells toward pathogen-derived TLR4 ligands by upregulating TLR4 expression and intensifies TNF-alpha, interleukin-12, and matrix metalloproteinase-9 production, all implicated in plaque destabilization. Thus, IFN-alpha-inducing pathogens, even when colonizing distant tissue sites, threaten the stability of inflamed atherosclerotic plaque.

Achieving a complete cytogenetic response (CCgR) is a major target in the treatment of chronic myeloid leukemia (CML) with <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), but CCgRs are rare. The mean CCgR rate is 1<em>3</em>%, in a range of 5% to <em>3</em><em>3</em>%. A collaborative study of 9 European Union countries has led to the collection of data on <em>3</em>17 patients who were first seen between 198<em>3</em> and 1997 and achieved CCgRs with IFN-<em>alpha</em> alone or in combination with hydroxyurea. The median time to first CCgR was 19 months (95% CI, 17-21; range, <em>3</em>-84 months). At last contact, 212 patients were still alive and in continuous CCgR; 105 patients had lost CCgR, but 5<em>3</em>% of them were still alive and in chronic phase. IFN-<em>alpha</em> treatment was discontinued permanently in 2<em>3</em> cases for response loss, in <em>3</em>6 cases for chronic toxicity (15 are still in unmaintained continuous CCgR), and in 8 cases because it was believed that treatment was no longer necessary (7 of these 8 patients are still in unmaintained continuous CCgR). The 10-year survival rate from first CCgR is 72% (95% CI, 62%-82%) and is related to the risk profile. High-risk patients lost CCgR more frequently and more rapidly and none survived more than 10 years. Low-risk patients survived much longer (10-year survival probability 89% for Sokal low risk and 81% for Euro low risk). These data point out that a substantial long-term survival in CCgRs is restricted mainly to low-risk and possibly intermediate-risk patients and occurs significantly less often in high-risk patients.

Production of type I <em>interferon</em> (IFN-<em>alpha</em>/beta) by virus-infected cells is the central event in their antiviral immune responses. In mammalian cells, IFN-<em>alpha</em>/beta gene transcription is induced through distinct signaling pathways by viral infection or by treatment with double-stranded (ds) RNA, which is an intermediate of virus replication. Toll-like receptor <em>3</em> (TLR<em>3</em>) was found to recognize dsRNA and transmit signals to activate NF-kappaB and the IFN-beta promoter. Recent identification of the TLR<em>3</em>-adaptor protein and its downstream signaling molecules, which are involved in IFN-<em>alpha</em>/beta production, revealed a novel IFN-inducing pathway for an anti-viral immune response. Here, we summarize the current knowledge of TLR<em>3</em>-mediated immune responses.

Pure, E. coli-derived recombinant murine interleukin 1 <em>alpha</em> (IL 1 <em>alpha</em>) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than <em>3</em> h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas <em>interferon</em>-<em>alpha</em> A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 <em>alpha</em> and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 <em>alpha</em> is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 <em>alpha</em>. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.

To evaluate the direct effect of adenosine on cytokine-polarized effector T cells, murine type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1) and Th2/Tc2 cells were generated using an antigen-presenting cell (APC)-free method. Tc1 and Tc2 cells had similar adenosine signaling, as measured by intracellular cyclic AMP (cAMP) increase upon adenosine A(2A) receptor agonism by CGS21680 (CGS). CGS greatly reduced Tc1 and Tc2 cell interleukin 2 (IL-2) and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) secretion, with nominal effect on <em>interferon</em> gamma (IFN-gamma) secretion. Tc2 cell IL-4 and IL-5 secretion was not reduced by CGS, and IL-10 secretion was moderately reduced. Agonist-mediated inhibition of IL-2 and TNF-<em>alpha</em> secretion occurred via A(2A) receptors, with no involvement of A(1), A(2B), or A(<em>3</em>) receptors. Adenosine agonist concentrations that abrogated cytokine secretion did not inhibit Tc1 or Tc2 cell cytolytic function. Adenosine modulated effector T cells in vivo, as CGS administration reduced CD4(+)Th1 and CD8(+)Tc1 cell expansion to alloantigen and, in a separate model, reduced antigen-specific CD4(+) Th1 cell numbers. Remarkably, agonist-mediated T-cell inhibition was abrogated by in vivo IL-2 therapy. Adenosine receptor activation therefore preferentially inhibits type I cytokine secretion, most notably IL-2. Modulation of adenosine receptors may thus represent a suitable target primarily for inflammatory conditions mediated by Th1 and Tc1 cells.

Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-<em>alpha</em>/beta induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein N(pro). Central players in the IFN-<em>alpha</em>/beta induction cascade are <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF<em>3</em> by inhibiting the transcription of IRF<em>3</em> mRNA. In the present study, we show that endogenous IRF<em>3</em> and IRF<em>3</em> expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of N(pro), while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF<em>3</em> promoter and does not affect the stability of IRF<em>3</em> mRNA. In fact, CSFV N(pro) induces proteasomal degradation of IRF<em>3</em>, as demonstrated by proteasome inhibition studies. Furthermore, N(pro) coprecipitates with IRF<em>3</em>, suggesting that the proteasomal degradation of IRF<em>3</em> is induced by a direct or indirect interaction with N(pro). Finally, we show that N(pro) does not downregulate IRF7 expression.

Genetic studies have implicated the early involvement of a gene on chromosome arm 9p in the development of cutaneous melanoma. We have performed loss-of-heterozygosity studies to confirm these original findings and identify the most frequently rearranged or deleted region of 9p. Eight markers were analyzed, including (from 9pter to proximal 9q) D9S<em>3</em><em>3</em>, the beta-<em>interferon</em> (IFNB1) locus, the <em>alpha</em>-<em>interferon</em> (IFNA) gene cluster, D9S126, D9S<em>3</em>, D9S19, the glycoprotein 4 beta-galactosyltransferase (GGTB2) gene, and the argininosuccinate synthetase pseudogene <em>3</em> (ASSP<em>3</em>). Two or more of these loci were found to be hemizygously reduced in 12 of 14 (86%) informative metastatic melanoma tumor and cell line DNAs, and homozygous deletions of the marker D9S126 were observed in 2 of 20 (10%) melanoma cell lines. These findings have resulted in the identification of a small critical region of 2-<em>3</em> megabases on 9p21 in which a putative melanoma tumor-suppressor gene appears likely to reside. Several 9p candidate genes, including IFNB1, the IFNA gene cluster, GGTB2, and the tyrosinase-related protein (TYRP) locus, have all been eliminated as potential targets because they are located outside of the homozygously deleted regions.