PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor <em>3</em> (TLR<em>3</em>), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR<em>3</em> expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR<em>3</em> was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (<em>Interferon</em>-<em>alpha</em>/beta receptor) suppressed MV-induced TLR<em>3</em> induction, indicating that type I IFN, IFN-<em>alpha</em>/beta, is critical for MV-mediated TLR<em>3</em> induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR<em>3</em> expression. A virus-responsive element that up-regulates TLR<em>3</em> was identified in the TLR<em>3</em>-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -<em>3</em>0bp of TLR<em>3</em> promoter responded to MV to induce TLR<em>3</em> expression. This further indicates the importance of type I IFN for TLR<em>3</em> up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR<em>3</em> had been induced beforehand. Thus, the MV-induced expression of TLR<em>3</em> may reflect amplified IFN production that plays a part in host defense to viral infection.

OBJECTIVE

Inflammatory factors are key mediators of wound healing processes following injury, and their modulation may improve healing outcomes. The objective of this study was to characterize in vivo inflammatory factor and extracellular matrix (ECM) messenger RNA (mRNA) expression levels 1 hour after vocal fold injury.

METHODS

Five Sprague-Dawley rats were subjected to bilateral vocal fold injury, 5 rats were reserved as uninjured controls, and 1 rat was subjected to unilateral vocal fold injury and reserved for histology. Tissue was harvested 1 hour after injury. Real-time reverse transcription-polymerase chain reaction was performed to examine the mRNA expression profiles of inflammatory factors nuclear factor kappa beta (NF-kappabeta), <em>interferon</em> gamma (IFN-gamma), cyclooxygenase 2 (COX-2), transforming growth factor beta isoform 1 (TGF-beta1), tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), and interleukin 1 beta (IL-1beta), as well as ECM genes hyaluronic acid synthase (HAS) 1, HAS-2, procollagen 1, procollagen <em>3</em>, and elastin, in the injured samples compared with the uninjured controls.

RESULTS

Injury resulted in subepithelial bleeding throughout the vocal fold. The COX-2, TNF-alpha, IL-1beta, and HAS-1 mRNA expression levels were significantly up-regulated 1 hour after injury compared with the uninjured controls.

CONCLUSIONS

Inflammatory factor and ECM gene expression changes occur in vocal fold wound sites as early as 1 hour after injury. These results should inform future efforts to attenuate vocal fold scarring via the modulation of inflammatory factors.

Fifty-six patients with metastatic renal cell carcinoma (RCC) were treated with recombinant DNA-derived <em>interferon</em> <em>alpha</em> (rIFN <em>alpha</em> A). The first <em>3</em>0 patients were randomized between doses of 2 X 10(6) U/m2 and 20 X 10(6) U/m2 intramuscularly daily. No complete (CR) or partial (PR) remissions were achieved in 15 patients receiving the low dose. In contrast, 27% of those receiving the high dose achieved CR or PR. Subsequently, 26 additional patients were given the high dose and achieved a <em>3</em>1% response rate. Remissions lasted from 1 to more than 12 months (median, <em>3</em> months). Responses occurred predominantly in lung parenchyma or mediastinal node metastases. Toxicity of the high dose required dose reduction in 50% of the patients. Neutralizing antibodies to rIFN <em>alpha</em> A developed in seven of 12 responsive (58%) and nine of 29 (<em>3</em>1%) nonresponsive patients (P = greater than .5). The median duration of remission among the antibody-positive and antibody-negative patients were 2 and 10 months, respectively (P = .009). The clinical significance of the antibodies to rIFN <em>alpha</em> A remains unclear, but the coincidence between the detection of antibodies and the early relapse of the disease in some responsive patients suggests that these antibodies may abrogate the biologic activity of rIFN <em>alpha</em> A. This effect, however, was not associated with adverse clinical sequelae.

Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC<em>3</em> (microtubule-associated protein 1 light chain <em>3</em> <em>alpha</em>) was present in most macrophage vacuoles containing C. albicans. In contrast, LC<em>3</em> was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma <em>interferon</em>-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.

We have recently established a murine model of pulmonary and disseminated infection with a highly virulent strain of Cryptococcus neoformans and demonstrated that administration of interleukin-12 (IL-12) protected the animals against infection. In this study, we extended these studies by investigating the host defense mechanisms. In particular, we examined the expression of mRNA for helper T-cell 1 (Th1) cytokines (IL-2, lymphotoxin, and gamma <em>interferon</em> [IFN-gamma]), Th2 cytokines (IL-4, -6, and -10), macrophage-derived cytokines (tumor necrosis factor <em>alpha</em> [TNF-<em>alpha</em>], IL-1beta, transforming growth factor beta [TGF-beta, IL-12p40, and IFN-gamma-inducing factor [IGIF]), and inducible nitric oxide synthase (iNOS) in the lungs on days 1, <em>3</em>, 7, and 14 after infection and following treatment with IL-12. There was little or no expression of mRNAs for Th1 cytokines, TNF-<em>alpha</em>, IL-12p40, IGIF, and iNOS in the infected mice, but expression increased markedly after treatment with IL-12. In contrast, the mRNAs for Th2 cytokines, IL-1beta, and TGF-beta were detected at considerable levels during the early stages of infection, and, interestingly, expression was not suppressed by IL-12 but rather augmented, particularly during the late stage. Similar results were also obtained for IFN-gamma, IL-4, IL-10, and TNF-<em>alpha</em> measured in the lung homogenates by enzyme-linked immunosorbent assay. These results suggest that the predominance of expression of Th2 cytokines and TGF-beta over Th1 cytokines, TNF-<em>alpha</em>, IL-12p40, IGIF, and iNOS is associated with severe lethal infection in mice and that administration of IL-12 protects infected animals by stimulating Th1 cytokines.

The human mucin gene MUC4 encodes a large transmembrane mucin that is thought to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, we describe the structure and functional activity of the 5'-flanking region, including 1.0 kilobase of the promoter. The long 5'-untranslated region (2.7 kilobases) is characterized by a high content of GC in its <em>3</em>'-end. The first TATA box was located at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-responsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5'-flanking region. Transcriptional activity of the promoter was assessed using pGL<em>3</em>-luciferase deletion mutants in two MUC4-expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Gel retardation assays indicated that Sp1 and Sp<em>3</em> bind to cognate cis-elements found in the 5'-flanking region and that Sp1 transactivates, whereas Sp<em>3</em> inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of protein kinase C with phorbol ester and treatment of cells with epidermal growth factor and transforming growth factor-<em>alpha</em> resulted in up-regulation of the promoter. Tumor necrosis factor-<em>alpha</em> and <em>interferon</em> (IFN)-gamma inflammatory cytokines had no or mild effect on MUC4 transcriptional activity when used alone. However, a very strong synergistic effect (10-12-fold activation) between IFN-gamma and tumor necrosis factor-<em>alpha</em> or IFN-gamma and transforming growth factor-<em>alpha</em> was obtained in CAPAN-2 cells. Altogether these results demonstrate that the 5'-flanking region of MUC4 contains epithelial cell-specific, positive, and negative regulatory cis-elements, that Sp1/Sp<em>3</em> are important regulators of MUC4 basal expression, and that its regulation in pancreatic cancer cells involves complex interplay between several signaling pathways.

Although several virus- and host-related predictive factors for the response to <em>interferon</em> alfa (IFN-<em>alpha</em>) have been defined in patients with chronic hepatitis C, no pretreatment parameter can definitely predict the response to antiviral treatment. Assessment of the initial response by quantification of serum hepatitis C virus RNA before and 4 weeks after initiation of therapy may be a clinically applicable and reliable parameter to predict long-term response. Therefore, the aims of the present study were to test the predictive value of a decline in HCV RNA of at least <em>3</em> log in the first 4 weeks of treatment (deltaHCV RNA) in patients treated with <em>3</em> x 10(6) units of recombinant IFN-<em>alpha</em>2a (rIFN-<em>alpha</em>2a) three times per week subcutaneously and to compare deltaHCV RNA with other established predictive factors, such as HCV genotype and pretreatment viremia. Serum HCV RNA was measured by a validated quantitative reverse transcription-polymerase chain reaction (RT-PCR). Geno/subtyping of HCV was performed by direct sequencing of the nonstructural (NS) 5B region of PCR-amplified isolates and subsequent phylogenetic analysis. Stable HCV RNA levels (deltaHCV RNA < or = 1 log) within the first 4 weeks of IFN-<em>alpha</em> treatment were present in 42 of 70 patients. A decline in HCV RNA levels between 1 to <em>3</em> log and more than <em>3</em> log was observed in 9 (1<em>3</em>%) and 19 patients (27%), respectively. In 21 of 70 patients (<em>3</em>0%), HCV RNA was not detectable at the end of 12 months' treatment. Three of 26 patients (11%) with a pretreatment viremia of < or = 10(6) copies/mL (all HCV subtype <em>3</em>a) and 6 of 44 patients (14%) with a pretreatment viremia of>> 10(6) copies/mL (HCV subtypes 1b, 2a, 2c, <em>3</em>a [two patients], and 4) achieved a virological sustained response to <em>interferon</em>-<em>alpha</em>2a treatment. All patients with a virological sustained response had an initial deltaHCV RNA of more than <em>3</em> log. In a stepwise discriminant-function analysis, the initial deltaHCV RNA was confirmed as the strongest predictor of virological sustained response (P < .0001). In conclusion, the data of the present study suggest that IFN-<em>alpha</em> treatment can be terminated after 4 weeks in patients with a decrease in HCV RNA levels of less than <em>3</em> log, when apparent HCV eradication is considered the therapeutic target. The predictive value of deltaHCV RNA clearly exceeds the significance of HCV genotype and pretreatment viremia as predictors of successful IFN-<em>alpha</em> treatment.

OBJECTIVE

To compare gene expression in normal and osteoarthritic (OA) human chondrocytes using microarray technology. Of the novel genes identified, we selected follistatin, a bone morphogenetic protein (BMP) antagonist, and investigated its expression/regulation as well as that of <em>3</em> other antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts.

METHODS

Basal and induced gene expression were determined using real-time polymerase chain reaction. Gene regulation was monitored following treatment with inflammatory, antiinflammatory, growth, and developmental factors. Follistatin protein production was measured using a specific enzyme-linked immunosorbent assay, and localization of follistatin and gremlin in cartilage was determined by immunohistochemical analysis.

RESULTS

All BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin were significantly up-regulated in OA chondrocytes but not in OA synovial fibroblasts. Chordin was weakly expressed in normal and OA cells. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. Tumor necrosis factor alpha and interferon-gamma significantly stimulated follistatin expression but down-regulated expression of gremlin. Interleukin-1beta (IL-1beta) had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. IL-1<em>3</em>, dexamethasone, transforming growth factor beta1, basic fibroblast growth factor, platelet-derived growth factor type BB, and endothelial cell growth factor down-regulated the expression of both antagonists.

CONCLUSIONS

This study is the first to show the possible involvement of follistatin and gremlin in OA pathophysiology. The increased activin/BMP-binding activities of these antagonists could affect tissue remodeling. The data suggest that follistatin and gremlin might appear at different stages during the OA process, making them interesting targets for the treatment of this disease.

Type I <em>interferons</em> (IFN-<em>alpha</em> and -beta) play an important role in the innate host defense against viral infection by inducing antiviral responses. In addition to direct antiviral activities, type I IFN serves as an important link between the innate and adaptive immune response through multiple mechanisms. Therefore, the outcome of a viral infection can be affected by IFN induction and the IFN sensitivity of a virus. North American porcine reproductive and respiratory syndrome virus (PRRSV) field isolates were studied with regard to IFN-<em>alpha</em> sensitivity and induction in order to understand the role of type I IFN in PRRSV pathogenesis. PRRSV isolates were differentially sensitive to porcine recombinant IFN-<em>alpha</em> (rIFN-<em>alpha</em>) and varied in their ability to induce IFN-<em>alpha</em> in porcine alveolar macrophages (PAM) cultures as measured by a porcine IFN-<em>alpha</em> specific ELISA on cell culture supernatants. Fifty-two plaques were purified from three PRRSV isolates (numbers <em>3</em>, 7, and 12) and tested for IFN sensitivity and IFN induction. Plaque-derived populations were composed of heterogeneous populations in terms of IFN-inducing capacity and sensitivity to rIFN-<em>alpha</em>. When macrophages infected with isolates <em>3</em>, 7, or 12 were treated with polycytidylic acid (polyI:C), IFN-<em>alpha</em> production was enhanced. Cells infected with isolate <em>3</em> and treated with polyI:C showed the most consistent and strongest enhancement of IFN-<em>alpha</em> production. It was demonstrated that the relatively low concentrations of IFN-<em>alpha</em> produced by isolate <em>3</em> contributed to the enhanced IFN-<em>alpha</em> synthesis in response to polyI:C. Isolates 7 and 12 significantly suppressed the enhanced IFN-<em>alpha</em> production by isolate <em>3</em> in polyI:C treated cells. To determine if suppression was at the level of IFN-<em>alpha</em> transcription, quantitative RT-PCR was performed for IFN-<em>alpha</em> mRNA and compared to GAPDH and cyclophilin mRNA quantification. However, the relative number of IFN-<em>alpha</em> transcript copies did not correlate with IFN-<em>alpha</em> protein levels, suggesting a post-transcriptional mechanism of suppression. In summary, these results demonstrate that PRRSV field isolates differ both in IFN-<em>alpha</em> sensitivity and induction. Furthermore, a PRRSV field isolate strongly enhance polyI:C-induced IFN-<em>alpha</em> production in PAM cultures and this priming effect was suppressed by other PRRSV isolates.

<em>Interferons</em> (IFN) are antiviral proteins that may be important in mediating cellular defenses against Epstein-Barr virus (EBV) infection. However, the means by which IFN-<em>alpha</em>, -beta and -gamma modify EBV infectivity are not clear. We have evaluated the effects of purified recombinant preparations of all three classes of IFN on EBV-induced B lymphocyte proliferation and Ig secretion. When added early after EBV infection, all three recombinant IFN reduced B cell outgrowth and Ig secretion. IFN-gamma exerted a 7-10-fold more potent antiviral effect than IFN-<em>alpha</em> or -beta. All three types of IFN act directly on B cells. Monocytes and natural killer cells are not necessary for the anti-EBV activity. Of the three recombinant IFN, only IFN-gamma reduced EBV-induced proliferation and Ig secretion when added <em>3</em>-4 days after virus infection; IFN-<em>alpha</em>/beta were only effective up to 24 h. B lymphoblastoid lines already transformed by EBV are insensitive to the anti-proliferative actions of all three types of IFN. On the basis of these findings, we propose three phases of regulation during EBV infection. In the early phase, EBV-infected cells can be regulated by all IFN. Subsequently, there is an intermediate period where only IFN-gamma is capable of directly affecting EBV-induced B cell responses. In the third phase, B lymphocytes become insensitive to direct actions of all IFN and are now subject to regulation only by cytotoxic cells.

We report that osteopontin (OPN), a secreted, Arg-Gly-Asp-containing phosphoprotein expressed at high levels in the kidney, suppresses nitric oxide (NO) synthesis induced by the inflammatory mediators gamma-<em>interferon</em> and lipopolysaccharide in primary mouse kidney proximal tubule epithelial cells. Northern blot and immunofluorescence analyses of inducible nitric oxide synthase (iNOS) expression revealed that the inflammatory mediators increased iNOS mRNA and protein levels. Recombinant human OPN (purified from both mammalian cells and from Escherichia coli) inhibited this response by a process that was blocked by anti-OPN antiserum and by the peptide GRGDS, but not GRGES. The data suggest that inhibition of NO synthesis by OPN in these kidney cells is mediated by an integrin, possibly the <em>alpha</em> v beta <em>3</em> integrin, which is known to be an OPN receptor. NO is believed to control blood flow through the glomerulus, regulating salt and water balance, and to be important as a defense against tumor cells and infecting microorganisms. The ability of OPN to inhibit the induction of iNOS suggests that OPN may be an important regulator of the NO signaling pathway and NO-mediated cytotoxic processes.

10 patients with borderline and lepromatous leprosy were selected for a prolonged trial with recombinant <em>interferon</em> gamma (rIFN-gamma). Patients received <em>3</em>0 micrograms intradermally for six injections over a 9-d period, and then either 100 micrograms intradermally every 1 mo for 10 mo or every 2 wk for 5 mo (total, 1.2 mg). Erythema nodosum leprosum (ENL) was induced in 60% of the patients within 6-7 mo, as compared with an incidence of 15% per year with multiple drug therapy alone. The mean whole-body reduction in bacterial index over the first 6 mo was 0.9 log units. Cutaneous induration at the intradermal injection sites of greater than or equal to 15 mm predicted the development of a subsequent reactional state. Monocytes obtained from patients receiving the lymphokine demonstrated an increased respiratory burst and a 2.5-5.1-fold increase in tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) secretion in response to agonists. Patients in ENL had an even higher release of TNF-<em>alpha</em> from monocytes as well as high levels of TNF-<em>alpha</em> in the plasma (mean, 2,000 pg/ml). Thalidomide therapy was required to treat the systemic manifestations of ENL. Control of toxic symptoms with thalidomide was associated with a 50-80% reduction in agonist-stimulated monocyte TNF-<em>alpha</em> secretion. IFN-gamma enhanced the monocyte release of TNF-<em>alpha</em> by <em>3</em>-7.5-fold (agonist dependent) when added to patient's cells in vitro, and this could be suppressed by the in vitro addition of 10 micrograms/ml of thalidomide.

Studies were carried out on some biological activities of Helicobacter pylori porins in vitro. We extracted and purified a porin with an apparent molecular mass of <em>3</em>0 kDa. Human polymorphonuclear leukocytes preincubated with H. pylori porins showed a decrease of chemotaxis, of adherence to nylon wool, and of chemiluminescence. Used as chemotaxins in place of zymosan-activated serum or as chemotaxinogens in place of zymosan, the porins induced polymorphonuclear leukocyte migration. Human monocytes and lymphocytes cultivated in the presence of H. pylori porins released cytokines. Release of the various cytokines studied was obtained with differentiated kinetics and at various porin concentrations. Starting only <em>3</em> h after culture, tumor necrosis factor <em>alpha</em> is released quickly, reaching a peak at 18 h, at a porin concentration of 1 microgram/ml/10(6) cells. Interleukin-6 (IL-6) appears later, with a peak at 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells. Lymphocytes stimulated by H. pylori porins release gamma <em>interferon</em> after 18 h of culture at higher concentrations of porins (20 micrograms/ml/10(6) cells). Granulocyte macrophage colony-stimulating factor is released from 6 to 48 h at a concentration of 1 microgram/ml/10(6) cells, while both IL-<em>3</em> and IL-4 are released after 18 h of culture at different porin concentrations (0.1 and 1 microgram/ml/10(6) cells, respectively). Our results lead us to think that during H. pylori infection, surface components, porins in particular, are able to induce a series of chain reactions ranging from the inflammatory to the immunological responses.

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 <em>alpha</em> (rIL 1 <em>alpha</em>) and recombinant tumor necrosis factor <em>alpha</em> (rTNF <em>alpha</em>), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 <em>alpha</em> or rTNF <em>alpha</em> gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within <em>3</em> hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 <em>alpha</em> and rTNF <em>alpha</em> were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced <em>interferon</em> in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.

We evaluated oral ribavirin as therapy for chronic hepatitis C infection in a pilot study including 10 patients. Patients (7 men, <em>3</em> women; mean age 40 years, range 2<em>3</em>-54) all had biopsy-proven chronic non-A, non-B hepatitis and were repeatedly positive for antibodies to hepatitis C virus. Treatment was with oral ribavirin 1000-1200 mg per day in two divided doses for 12 weeks. The median serum alanine aminotransferase concentration for all patients at enrollment was <em>3</em>.15 mu kat/l (range 1.22-7.79) and decreased significantly (p less than 0.005) to 1.25 mu kat/l (0.78-2.04) after 12 weeks of treatment. Within 6 weeks of the end of treatment the median serum alanine aminotransferase concentration was not significantly different from that before treatment. Side-effects were mild and fully reversible after cessation of therapy. We conclude that ribavirin is the first drug to offer a potentially effective oral treatment for chronic hepatitis C. It should be further evaluated in controlled trials, possibly in combination with <em>interferon</em> <em>alpha</em>.

To develop a resource for the identification and isolation of genes expressed in the early mammalian embryo, large and representative cDNA libraries were constructed from unfertilized eggs, and two-cell, eight-cell, and blastocyst-stage mouse embryos. Using these libraries, we now report the first stages at which the cytokines interleukin (IL)-6, IL-1 beta, and <em>interferon</em> (IFN)-gamma are transcribed in the developing embryo and the presence of IL-7 transcripts in the unfertilized egg. Transcripts for IL-1 <em>alpha</em>, -2, -<em>3</em>, -4, or -5 were not detected at these stages. To identify novel genes expressed on activation of the embryonic genome, the egg and eight-cell stage-specific cDNA libraries were subtracted from the two-cell library, yielding a specialized cDNA library enriched for transcripts expressed at the two-cell stage. Sequence and Southern blot analysis of several of these cDNAs expressed predominantly at the two-cell stage of embryogenesis revealed them to be from novel genes, thereby providing the first molecular tools with which to approach the study of gene expression in the early mammalian embryo.

OBJECTIVE

The primary objective of this study was to determine whether pharmacokinetic interactions occurred between interferon alpha-2b (IFN) and ribavirin in patients with chronic hepatitis C infections. Additionally this study assessed the single and multiple-dose pharmacokinetics of ribavirin and IFN, and compared the safety, tolerability and antiviral pharmacodynamics of IFN plus ribavirin compared with either drug alone.

METHODS

In this open label parallel group study, patients with chronic hepatitis C were randomized to receive IFN 3 million IU thrice weekly s.c. alone, ribavirin 600 mg twice daily p.o. alone or both drugs in combination over 6 weeks. Single and multiple dose pharmacokinetics and indices of antiviral pharmacodynamics were assessed during weeks 1 and 6, along with safety assessments during the study.

RESULTS

The range of mean ribavirin terminal phase half-lives after single doses was 44-49 h. Comparison of week 1 and week 6 AUC(0,12h) values showed accumulation in plasma of approximately 6-fold. The range of mean washout half-lives after week 6 was 274-298 h, reflecting release of ribavirin from deep compartment stores. The range of single and multiple dose IFN terminal phase half-lives was 5-7 h. IFN demonstrated an increase in bioavailability (approximately 2-fold) upon multiple dose administration. Ribavirin and IFN pharmacokinetic parameters for combined ribavirin and IFN were similar to those during monotherapy with either compound, although the power of this study to detect differences was low. Serum HCV-RNA titers and ALT concentrations were reduced by IFN alone, ribavirin alone reduced ALT concentrations only, and combined IFN plus ribavirin produced numerically greater falls in both measurements than either treatment alone. Serum concentrations of neopterin and activity of 2',5'-oligoadenylate synthetase (2'5'-OAS) were increased by IFN alone and in combination with ribavirin, whereas serum 2'5'-OAS activity was decreased and neopterin concentrations unaltered by ribavirin monotherapy. IFN and ribavirin monotherapy produced characteristic changes in safety laboratory tests (IFN--reductions in white cells, neutrophils and platelets; ribavirin--reduced haemoglobin) and characteristic adverse event profiles (IFN--headache, flu-like symptoms, fatigue, anorexia, nausea, myalgia, and insomnia; ribavirin--headache, fatigue, myalgia, and pruritus). There was no additive effect of combination therapy on safety laboratory tests or reported adverse events. All changes were fully reversible upon treatment cessation.

CONCLUSIONS

There was no evidence of pharmacokinetic interactions between IFN and ribavirin in this study. There were numerical trends indicating that the combination of IFN and ribavirin reduced titers of HCV-RNA to a greater extent than did either treatment alone, and the safety profile of combination therapy was similar to those of both monotherapy treatments.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by <em>interferon</em>-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), was intermediately depressed by tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-<em>alpha</em>, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 <em>alpha</em>. The ratio between the maximal stimulation and depression observed was around <em>3</em>0-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-<em>alpha</em>, IL-2, IL-<em>3</em>, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-<em>alpha</em>, IL-1 <em>alpha</em>, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.

Recently, we have shown that SOCS-1/<em>3</em> overexpression in hepatic cells abrogates signaling of type I <em>interferons</em> (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-<em>alpha</em> because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT<em>3</em> phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/<em>3</em> phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-<em>alpha</em> due to inhibition by SOCS proteins.

BACKGROUND

To our knowledge, changes in the patterns of whole-transcriptome gene expression that occur during the induction and resolution of experimental gingivitis in humans were not previously explored using bioinformatic tools.

METHODS

Gingival biopsy samples collected from 14 subjects during a 28-day stent-induced experimental gingivitis model, followed by treatment, and resolution at days 28 through <em>3</em>5 were analyzed using gene-expression arrays. Biopsy samples were collected at different sites within each subject at baseline (day 0), at the peak of gingivitis (day 28), and at resolution (day <em>3</em>5) and processed using whole-transcriptome gene-expression arrays. Gene-expression data were analyzed to identify biologic themes and pathways associated with changes in gene-expression profiles that occur during the induction and resolution of experimental gingivitis using bioinformatic tools.

RESULTS

During disease induction and resolution, the dominant expression pathway was the immune response, with 1<em>3</em>1 immune response genes significantly up- or downregulated during induction, during resolution, or during both at P <0.05. During induction, there was significant transient increase in the expression of inflammatory and oxidative stress mediators, including interleukin (IL)-1 <em>alpha</em> (IL1A), IL-1 beta (IL1B), IL8, RANTES, colony stimulating factor <em>3</em> (CSF<em>3</em>), and superoxide dismutase 2 (SOD2), and a decreased expression of IP10, <em>interferon</em> inducible T-cell <em>alpha</em> chemoattractant (ITAC), matrix metalloproteinase 10 (MMP10), and beta 4 defensin (DEFB4). These genes reversed expression patterns upon resolution in parallel with the reversal of gingival inflammation.

CONCLUSIONS

A relatively small subset (11.9%) of the immune response genes analyzed by array was transiently activated in response to biofilm overgrowth, suggesting a degree of specificity in the transcriptome-expression response. The fact that this same subset demonstrates a reversal in expression patterns during clinical resolution implicates these genes as being critical for maintaining tissue homeostasis at the biofilm-gingival interface. In addition to the immune response pathway as the dominant response theme, new candidate genes and pathways were identified as being selectively modulated in experimental gingivitis, including neural processes, epithelial defenses, angiogenesis, and wound healing.

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-<em>alpha</em> is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine <em>interferon</em> (IFN)-gamma remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-gamma transgenic mice, which constitutively express IFN-gamma in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-gamma mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4(+) and CD8(+) T cells but also Mac2(+) (galectin <em>3</em>(+)) macrophages and CD11c(+) dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-gamma overexpression. IFN-gamma-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-<em>alpha</em> and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-<em>alpha</em>. Myotoxic IFN-gamma effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-gamma effect. The precise mechanism of IFN-gamma cardiotoxicity remains to be elucidated.

Pharmaceutical preparations of normal human immunoglobulin (IgG) are known to contain high-avidity and neutralizing antibodies (Ab) to the cytokines interleukin (IL)-1<em>alpha</em>, IL-6, and <em>interferon</em> (IFN)<em>alpha</em>. To test for other cytokine Ab, 2<em>3</em> batches of IgG were tested for saturable binding to eight 125I-labeled recombinant cytokines. All batches bound granulocyte-macrophage colony-stimulating factor (GM-CSF) with high avidity (Kav approximately 10 pmol/L) and capacities of up to 5 mumol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also with high avidity, whereas 1<em>3</em> of 15 batches bound to IL-10 but with lower capacities and avidities. None of the IgG preparations bound IL-1 receptor antagonist (IL-1ra), IL-2, IL-<em>3</em>, IL-4, or G-CSF. Cross-binding and absorption analyses revealed identical or slightly stronger binding of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts. GM-CSF-IgG complexes did not bind to cellular GM-CSF receptors, but Fc-dependent binding occurred to blood polymorphonuclear cells. Increased binding of GM-CSF to patient sera correlated positively with the binding capacities of infused IgG preparations. Patient and normal sera did not interfere with the binding of Ab to GM-CSF. From these and previous experiments, we conclude that pools of normal human IgG contain variable amounts of specific and high-avidity Ab to some cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reaction in vivo following IgG therapy.

The cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF), induce a dose-dependent production of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) in cultured human synovial cells, as measured by immunoassay. With IL-1, significant levels of both CSFs were first detected within 6 to 12 hours, with a maximum reached 24 to 48 hours after commencement of stimulation. A synergistic effect was detected between IL-1 and TNF in production of both CSFs in these cells. No evidence was obtained for the IL-1-induced effect to be mediated by induction of endogenous TNF nor for the TNF-induced stimulation to involve IL-1. IL-1-stimulated synovial cells were shown to secrete biologically active GM-CSF and G-CSF, which were specifically inhibited by their respective monoclonal antibodies. The transcription inhibitor, actinomycin D, and protein synthesis inhibitor, cycloheximide, inhibited the increase in GM-CSF and G-CSF production induced by IL-1 and TNF. Finally, other cytokines, IL-<em>3</em>, <em>interferon</em> gamma (IFN gamma), IL-2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and transforming growth factor <em>alpha</em> (TGF <em>alpha</em>), failed to stimulate either GM-CSF or G-CSF production, whether alone or in the presence of IL-1. These results suggest that cytokine-stimulated synovial fibroblasts may be a major source of intraarticular CSF production in the joints of patients with inflammatory arthritis; as a result, monocyte/macrophages and granulocytes may be activated, leading to perpetuation of the inflammation and destructive events occurring in these lesions.

The selective destruction of the pancreatic islet beta cells in type 1 diabetes mellitus is thought to be mediated by a cellular autoimmune process, possibly triggered by virus infection in genetically susceptible individuals. Because of the potentially important role of cell-cell adhesion in the immune response, we investigated whether cytokine products of mononuclear cells, or virus infection, induced the expression of intercellular adhesion molecule 1 (ICAM-1) on human endocrine islet cells. By flow cytofluorimetry, control islet cells did not express detectable ICAM-1. However, after a 72-hr exposure of islets to <em>interferon</em> gamma (IFN-gamma) and/or tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) (each at 250 units/ml), ICAM-1 was induced on greater than 85% of islet cells. IFN-gamma was 50% more potent than TNF-<em>alpha</em>; together, their effects were additive. Class I major histocompatibility complex (MHC) protein expression, detected on control islet cells, was also stimulated by IFN-gamma and/or TNF-<em>alpha</em>. In contrast, infection with reovirus type <em>3</em> did not induce ICAM-1 on islet cells, although it stimulated the expression of class I MHC proteins. By double-label indirect immunofluorescence microscopy, ICAM-1 expression was identified on both beta (insulin-secreting) and delta (somatostatin-secreting) islet cells. Monoclonal antibody to ICAM-1 precipitated protein of Mr 97,000 from [<em>3</em>5S]methionine-labeled islets exposed to IFN-gamma and TNF-<em>alpha</em>, but not from control islets. RNA blot analysis revealed a major species of <em>3</em>.<em>3</em> kilobases and a minor species of 2.2 kilobases induced in islets exposed to the cytokines. These findings have implications for the molecular mechanisms of beta-cell destruction in type 1 diabetes, in that expression of ICAM-1 by beta cells may facilitate adhesion of antigen-targeted immune cells.