Infections with human coronavirus EMC (HCoV-EMC) are associated with severe pneumonia. We demonstrate that HCoV-EMC resembles severe acute respiratory syndrome coronavirus (SARS-CoV) in productively infecting primary and continuous cells of the human airways and in preventing the induction of <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>)-mediated antiviral <em>alpha</em>/beta <em>interferon</em> (IFN-α/β) responses. However, HCoV-EMC was markedly more sensitive to the antiviral state established by ectopic IFN. Thus, HCoV-EMC can utilize a broad range of human cell substrates and suppress IFN induction, but it does not reach the IFN resistance of SARS-CoV.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs), which can differentiate into several lineages, have immunomodulatory properties similar to those of bone marrow-derived MSCs. However, the specific mechanism by which the immunomodulatory effect of MSCs occurs is not clear. In this study, we isolated canine AD-MSCs (cAD-MSCs) and induced their development into adipocyte, osteocyte, and neuron-like cells. We then investigated their phenotype and cytokine expression to determine whether they were able to exert an immunomodulatory effect and what the underlying mechanisms of this effect were. cAD-MSCs expressed CD44, CD90, and MHC class I and were also partially positive for the expression of CD<em>3</em>4; however, they did not express CD14 and CD45. In addition, they expressed the mRNA of transforming growth factor beta (TGF-beta), IL-6, IL-8, CCL2, CCL5, vascular endothelial growth factor, hepatocyte growth factor (HGF), tissue inhibitor metalloproteinase-1/2, and cyclooxygenase-2 but not that of IL-10. Further, leukocyte proliferation induced by mitogens was suppressed when they were cocultured with irradiated cAD-MSCs, as well as with culture supernatants of cAD-MSCs alone. Moreover, TNF-<em>alpha</em> production significantly decreased, whereas TGF-beta, IL-6, and <em>interferon</em>-gamma production significantly increased in cAD-MSCs that were cocultured with leukocytes. Finally, immonomodulatory factors of MSCs, such as TGF-beta, HGF, prostaglandin E2 (PGE2), and indoleamine 2, <em>3</em> dioxygenase (IDO), increased significantly in cAD-MSCs that were cocultured with leukocytes; however, the production of PGE2 and IDO showed different kinetics, and leukocyte proliferation was effectively restored by PGE2 and IDO inhibitors. Taken together, these results indicate that the immunomodulatory effects of cAD-MSCs are associated with soluble factors (TGF-beta, HGF, PGE2, and IDO). Therefore, it is suggested that cAD-MSCs have a potential therapeutic use in the treatment of immune-mediated disease.

Tick-borne encephalitis virus (TBEV) (family Flaviviridae, genus Flavivirus) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia. Here, we investigated the induction of the antiviral type I <em>interferons</em> (IFNs) (<em>alpha</em>/beta IFN [IFN-<em>alpha</em>/beta]) by TBEV. Using strains Neudörfl, Hypr, and Absettarov, we demonstrate that levels of IFN-beta transcripts and viral RNA are strictly correlated. Moreover, IFN induction by TBEV was dependent on the transcription factor IFN regulatory factor <em>3</em> (IRF-<em>3</em>). However, even strain Hypr, which displayed the strongest IFN-inducing activity and the highest RNA levels, substantially delayed the activation of IRF-<em>3</em>. As a consequence, TBEV can keep the level of IFN transcripts below the threshold value that would permit the release of IFN by the cell. Only after 24 h of infection have cells accumulated sufficient IFN transcripts to produce detectable amounts of secreted IFNs. The delay in IFN induction appears not to be caused by a specific viral protein, since the individual expressions of TBEV C, E, NS2A, NS2B, NS<em>3</em>, NS4A, NS4B, NS5, and NS2B-NS<em>3</em>, as well as TBEV infection itself, had no apparent influence on specific IFN-beta induction. We noted, however, that viral double-stranded RNA (dsRNA), an important trigger of the IFN response, is immunodetectable only inside intracellular membrane compartments. Nonetheless, the dependency of IFN induction on IFN promoter stimulator 1 (IPS-1) as well as the phosphorylation of the <em>alpha</em> subunit of eukaryotic initiation factor 2 (eIF2<em>alpha</em>) suggest the cytoplasmic exposure of some viral dsRNA late in infection. Using ultrathin-section electron microscopy, we demonstrate that, similar to other flaviviruses, TBEV rearranges intracellular membranes. Virus particles and membrane-connected vesicles (which most likely represent sites of virus RNA synthesis) were observed inside the endoplasmic reticulum. Thus, apparently, TBEV rearranges internal cell membranes to provide a compartment for its dsRNA, which is largely inaccessible for detection by cytoplasmic pathogen receptors. This delays the onset of IFN induction sufficiently to give progeny particle production a head start of approximately 24 h.

We have previously observed that a 6-day exposure of human pancreatic islets to a combination of cytokines (interleukin-1beta 50 U/ml + tumour necrosis factor-<em>alpha</em> 1000 U/ml + <em>interferon</em>-gamma 1000 U/ml) severely impairs beta-cell functions. In the present study, we examined whether this condition affects DNA integrity and viability of human islet cells. Cells were studied after <em>3</em>, 6, and 9 days of cytokine treatment by both single cell gel electrophoresis (the "comet assay," a sensitive method for detection of DNA strand breaks) and by a cytotoxicity assay using the DNA binding dyes Hoechst <em>3</em><em>3</em><em>3</em>42 and propidium iodide as indices for the number of viable, necrotic, and apoptotic cells. Cytokine treatment for 6 and 9 days resulted in a 50% increase in comet length (P < 0.01 vs. controls), indicating DNA strand breaks, as well as in a significant increase in the number of apoptotic cells (P < 0.02 vs. controls), but not in the number of necrotic cells. The arginine analogs N(G)-nitro-L-arginine and N(G)-monomethyl-L-arginine prevented nitric oxide formation by the cytokines but did not interfere with cytokine-induced DNA strand breaks and apoptosis. The present data suggest that prolonged (6-9 days) exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks and cell death by apoptosis. These deleterious effects of cytokines appear to be independent of nitric oxide generation.

BACKGROUND

Coxsackievirus-induced myocarditis can be a serious cause of heart failure. In the absence of a specific antiviral therapy, modulating the host immune response may be protective. Interferons (IFNs)-alpha and -beta perform a fundamental role in innate and adaptive antiviral responses, thereby presenting as candidate therapeutics for coxsackievirus infections.

RESULTS

To examine the contribution of IFN-beta in protection from coxsackievirus B3 (CVB3) infection, mice lacking the IFN-beta gene were infected with 10(3) plaque-forming units of CVB3. In contrast to wild-type mice that exhibit an intact IFN-beta response, we observed increased susceptibility to infection (70% mortality), a downregulation of IFN-stimulated gene targets (2'-5' oligoadenylate synthetase, serine/threonine protein kinase, the GTPase Mx), and cardiomyocyte breakdown and disruption in the IFN-beta-/- mice.

CONCLUSIONS

Viewed together, these results clearly demonstrate that IFN-beta is important in mediating protection against CVB3-induced myocarditis.

OBJECTIVE

The search for targeted anti-hepatitis C virus (HCV) drugs is driven by the adverse effect profile and limited efficacy of the current standard of care (pegylated interferon-alpha/ribavirin). In a first-in-human trial, we tested the safety, tolerability, and pharmacokinetics of the macrocyclic HCV NS3/4A protease inhibitor TMC435 in healthy volunteers, followed by HCV genotype 1-infected patients to assess antiviral activity.

METHODS

The TMC435350-C101 study was a phase I, randomized, double-blind, placebo-controlled trial in 49 healthy volunteers, followed by an open-label, nonplacebo-controlled panel in 6 genotype 1 hepatitis C patients. Healthy volunteers received oral, single, ascending doses (up to 600 mg) or 5-day multiple ascending doses (200 mg twice daily or 100, 200, or 400 mg once daily). Patients received 200 mg once daily for 5 days. Pharmacokinetics and safety were evaluated for all panels, and plasma HCV-RNA levels were determined in patients.

RESULTS

There were no serious adverse events, no grade 3 reactions, and no treatment-related discontinuations; pharmacokinetics supported a once daily dosing regimen. Plasma HCV-RNA levels dropped rapidly in all patients, with a median maximal reduction of 3.9-log(10) IU/mL and a median of 6 days to maximal reduction. The initial steep reduction of HCV-RNA (median 3.5-log(10) IU/mL at day 3) was followed by a more gradual decline that was maintained over the dosing period. No viral breakthroughs (>1-log(10) IU/mL HCV-RNA increase from nadir) were observed during treatment nor in the 3 days posttreatment; HCV-RNA returned to pretreatment levels by week 4.

CONCLUSIONS

Once daily TMC435 given orally was generally safe and well tolerated and demonstrated potent antiviral activity.

1. Cyclo-oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo-oxygenase-1 (COX-1) is expressed constitutively whereas COX-2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX-1 or COX-2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549). 2. COX-1 or COX-2 protein was measured by western blot analysis using specific antibodies to COX-2 and selective antibodies to COX-1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2 <em>alpha</em>, thromboxane B2 or 6-oxo PGF1 <em>alpha</em> measured by radioimmunoassay. Thus, COX-1 or COX-2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12h before fresh culture medium was added containing exogenous arachidonic acid (<em>3</em>0 microM) for 15 min after which COX metabolites were measured. <em>3</em>. Untreated primary cells or A549 cells contained low amounts of COX-1 or COX-2 protein. Bacterial LPS (1 micro g ml-1 for 24 h) induced COX-2 protein in the primary cells, a process which was enhanced by <em>interferon</em>-gamma, with no further increase in the presence of a mixture of cytokines (interleukin-1 beta, tumour necrosis factor-<em>alpha</em> and <em>interferon</em>-gamma, 10 ng ml-1 for all). In contrast, A549 cells contained only low levels of COX-2 protein after exposure to LPS or LPS plus <em>interferon</em>-y, but contained large amounts of COX-2 protein after exposure to the mixture of cytokines.4. Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin-1 beta , tumour necrosis factors- and <em>interferon</em>-gamma, 10 ng ml-1 for all).PGE2 was the principal COX metabolite released by cytokine-activated epithelial cells. The release of PGE2 induced by cytokines occurred after a lag period of more than 6 h.5. The glucocorticosteroid, dexamethasone (1 micro M; <em>3</em>0 min prior to cytokines) completely suppressed the cytokine-induced expression of COX-2 protein and activity in both primary cells and A549 cells.6. In experiments where COX-2 activity was supported by endogenous stores of arachidonic acid,treatment of A549 cells with interleukin-l beta but not tumour necrosis factor a or <em>interferon</em>-gamma alone caused a similar release of PGE 2 to that seen when the cytokines were given in combination. However, both interleukin-l beta and necrosis factor- alone produced similar increases in COX-2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin-l beta, tumour necrosis factor- <em>alpha</em> and <em>interferon</em>-gamma were used to stimulate the cells.7. These findings show that COX-2 expression correlates with the exaggerated release of prostaglandins from cytokine-activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor <em>alpha</em>, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1<em>alpha</em>, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa <em>interferon</em>-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and IRF-7. Gamma <em>interferon</em> (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-<em>3</em>. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-<em>3</em>, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

Treating chronic hepatitis C virus (HCV) infection using pegylated <em>alpha</em> <em>interferon</em> and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and <em>3</em> and among African-American (AA) patients compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed for a group of <em>3</em><em>3</em> African-American and <em>3</em>6 Caucasian American patients with chronic HCV genotype 1 infection during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from peripheral blood mononuclear cells irrespective of degree of decrease in HCV RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (<1.5 log(10)-IU/ml decrease at 28 days) compared to those in patients with a marked >><em>3</em>.5 log(10)-IU/ml decrease) or intermediate (1.5 to <em>3</em>.5 log(10)-IU/ml decrease) response. The number of genes that were up- or down-regulated by pegylated <em>interferon</em> and ribavirin treatment was fewer in patients with a poor response than in those with an intermediate or marked viral response. However AA patients had a stronger <em>interferon</em> response than CA patients in general. The induced levels of known <em>interferon</em>-stimulated genes such as the 2'5'-oligoadenylate synthetase, MX1, IRF-7, and toll-like receptor TLR-7 genes was lower in poor-response patients than in marked- or intermediate-response patients. Thus, the relative lack of viral response to <em>interferon</em> therapy of hepatitis C virus infection is associated with blunted <em>interferon</em> cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or the racial differences.

Current regimens of systemic chemotherapy result in only modest lengthening of survival in patients with advanced stage, liver-dominant, metastatic colorectal cancer who have failed first-line chemotherapy. The objective of this study was to investigate the safety and tolerability of NV1020, a replication-competent, attenuated, genetically engineered herpes simplex virus type 1 (HSV-1), in patients with hepatic colorectal metastases refractory to first-line chemotherapy. A phase I, open-label, dose-escalating study of a single 10-min hepatic arterial infusion of NV1020 in four cohorts. Three patients in each cohort received doses of <em>3</em> x 10(6), 1 x 10(7), <em>3</em> x 10(7), and 1 x 10(8) plaque-forming units. Adverse events were either mild or moderate in severity, and self-limiting. Only three serious adverse events (one transient rise in serum y-glutamyltransferase, one diarrhea, and one leukocytosis) experienced by three patients were considered to be possibly or probably related to NV1020. There were no deaths during the study, and there was no evidence of disseminated herpes infection. Viral presence was detected in only one saliva sample and two serum samples from one asymptomatic patient in the highest dose cohort. In the first week after viral administration only rare and minor increases were noted for tumor necrosis factor-<em>alpha</em> (six samples; three patients; peak, 40 pg/ml), interleukin (IL)-1 (two samples; two patients; peak, 28 pg/ml), and <em>interferon</em>-y (four samples; two subjects; peak, 54 pg/ml). No IL-2 was detected. Mild liver enzyme elevations were self-limiting and not associated with clinical symptoms. We conclude that NV1020, a genetically engineered but replication-competent HSV-1 oncolytic virus, can be safely administered into the hepatic artery without significant effects on normal liver function.

The leader protein of Theiler's virus was previously shown to block the production of <em>alpha</em>/beta <em>interferon</em> by infected cells. Here, we observed that expression of the leader protein in infected cells triggered subcellular redistribution of a nucleus-target green fluorescent protein. It enhanced redistribution of the nuclear polypyrimidine tract-binding protein but had no influence on the localization of the nuclear splicing factor SC-<em>3</em>5. The leader protein also interfered with trafficking of the cytoplasmic <em>interferon</em> regulatory factor <em>3</em>, a factor critical for transcriptional activation of <em>alpha</em>/beta <em>interferon</em> genes.

OBJECTIVE

To examine the expression, regulation, and potential roles of bone morphogenetic proteins (BMPs) in arthritic synovium.

METHODS

Expression of BMPs in arthritic synovium from patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) and in noninflamed synovium from patients undergoing diagnostic or therapeutic arthroscopies was studied by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and 2-color immunofluorescence. In vitro regulation of gene expression in fibroblast-like synoviocytes (FLS) was determined by real-time quantitative RT-PCR and immunohistochemistry. We used (<em>3</em>)H-thymidine incorporation after serum deprivation-induced growth arrest to examine effects on FLS proliferation. FLS apoptosis was evaluated by flow cytometry cell cycle analysis. Apoptotic cells in synovial tissue were detected by TUNEL staining.

RESULTS

Transcripts of different BMPs, most strikingly BMP-2 and BMP-6, were detected in synovial tissues. By Western blot, BMP-2 and BMP-6 precursor protein was found in RA and SpA synovial tissue extracts, but not in extracts of noninflamed synovial tissue. By immunohistochemistry, BMP-2 and BMP-6 were detected in the hyperplastic lining and the sublining layer of synovium from RA and SpA patients, both in CD90+ cells (FLS) and in some CD68+ cells (macrophages). Proinflammatory cytokines, such as interleukin-1beta and tumor necrosis factor alpha, but not interferon-gamma, enhanced the expression of BMP-2 and BMP-6 transcripts in FLS in vitro. Neither BMP-2 nor BMP-6 affected FLS proliferation. BMP-2 promoted FLS apoptosis, whereas BMP-6 protected against nitric oxide-induced FLS apoptosis. BMP-2-positive apoptotic cells were found in arthritic synovium.

CONCLUSIONS

BMP-2 and BMP-6 are expressed in arthritic synovium and are strongly up-regulated by proinflammatory cytokines. Although BMP signaling has been proposed to be involved in cartilage and bone repair in arthritis, this pathway may be equally important in modulating FLS cell populations in inflamed synovium.

Human tumors can constitutively express cytokines and growth factors, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, growth factors, and their receptors, we tested 21 melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With <em>3</em>0 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory factor (LIF), IL-7, gro <em>alpha</em>, IL-8 and the p<em>3</em>5 chain of IL-12 was detected in more than 60% of melanomas. Concomitant receptors for IL-6 and IL-7 were also detected. IL-1 <em>alpha</em>, IL-5, Rantes, IL-10, <em>interferon</em> (IFN)-beta, tumor-necrosis factor (TNF)-<em>alpha</em>, G-colony-stimulating factor (CSF) and GM-CSF were expressed at lower levels. Melanocytes showed greatly reduced cytokine RNA transcripts, and only gro <em>alpha</em> was consistently detected. No expression of IL-2, IL-<em>3</em>, IL-4, IL-9, the p40 chain of IL-12, IFN-<em>alpha</em> or IFN-gamma RNA transcripts was detected in melanomas or melanocytes. The growth factors expressed by melanomas and, after further signal amplification, by melanocytes were transforming growth factor (TGF)-<em>alpha</em>, epidermal growth factor (EGF), TGF-beta, endothelial-cell growth factor (ECGF), basic-fibroblast growth factor (bFGF), nerve growth factor (NGF) and steel. The receptors EGFR, FGFR, NGFRp70 and c-kit were also expressed by melanomas and melanocytes. These results point to new possible autocrine and paracrine pathways in melanoma biology.

Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low-dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first <em>3</em> h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5-fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with>> or = 1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 micrograms/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor <em>alpha</em>, anti-interleukin-6, anti-gamma <em>interferon</em>, and anti-LPS-binding protein. LPS-induced tumor necrosis factor <em>alpha</em> production was abolished after a second 4-h challenge. In contrast, the LPS-induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents.

Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-<em>3</em>), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-<em>3</em>, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-<em>alpha</em> or interleukin-1beta, established agonists of MMP expression. <em>Interferon</em> gamma, another T-lymphocyte- derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-<em>3</em> with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I <em>interferons</em> (IFN-<em>alpha</em> and IFN-beta) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5' and <em>3</em>' end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-<em>alpha</em> (CpG-B) gained the potency of CpG-A to induce IFN-<em>alpha</em>. Higher ODN uptake in PDC was not responsible for the higher IFN-<em>alpha</em>-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-<em>alpha</em> in PDC.

OBJECTIVE

Human intestinal epithelial cells inducibly express neutrophil and monocyte chemoattractants, yet little is known about the regulated production of T-cell chemoattractants by the intestinal epithelium. IP-10, Mig, and I-TAC are <em>3</em> CXC chemokines that are known to act as CD4(+) T-cell chemoattractants.

METHODS

We studied constitutive chemokine expression in human colon, and defined the regulated expression of these chemokines by reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistology using cultured human intestinal epithelial cell lines and a novel adaptation of an in vivo human intestinal xenograft model.

RESULTS

IP-10 and Mig were constitutively expressed by normal human colon epithelium, and their cognate receptor, CXCR<em>3</em>, was expressed by mucosal mononuclear cells. Interferon (IFN)-gamma stimulation increased mRNA expression and the polarized basolateral secretion of these chemokines by human colon epithelial cell lines; infection with enteroinvasive bacteria, or stimulation with the proinflammatory cytokines tumor necrosis factor alpha and interleukin 1alpha, strongly potentiated IFN-gamma-induced epithelial cell IP-10, Mig, and I-TAC production. Epithelial cell mRNA and protein expression of IP-10, Mig, and I-TAC were rapidly up-regulated in human intestinal xenografts in response to stimulation with IFN-gamma alone or in combination with IL-1.

CONCLUSIONS

The constitutive and regulated production of the IFN-gamma-inducible chemokines IP-10, Mig, and I-TAC by human intestinal epithelium, and the expression of their cognate receptor, CXCR<em>3</em>, by mucosal mononuclear cells, suggest that the intestinal epithelium can play a role in modulating physiologic and pathologic T cell-mediated mucosal inflammation.

Both type I <em>interferon</em> (IFN-<em>alpha</em>/beta) and type II IFN (IFN-gamma) exert many functions that are restricted to immune cells. Thus, they play critical roles in innate and adaptive immunity. IFN regulatory factor-4 (IRF-4) and IRF-8 (formerly PU.1 interaction partner [Pip] and IFN consensus sequence binding domain [ICSBP], respectively) are immune cell-specific members of the IRF family that regulate the development of myeloid, lymphoid, and dendritic cells. They form a heterodimeric complex with another immune cell-specific transcription factor PU.1-Spi-1 and regulate transcription of genes in the immune system. This review describes the role of the IRF-8-PU.1 complex in modulating IFN signaling in an immune cell-specific manner. Our studies revealed that some but not all IFN-gamma-inducible genes carry an IFN-gamma activation site (GAS) element that contains a binding site for the IRF- 8-PU.1 complex. The IRF-8-PU.1 complex can take part in GAS-mediated transcription and amplify expression of IFN-gamma-responsive genes initiated by Stat1 in macrophages. Similarly, some but not all IFN-<em>alpha</em>/beta-responsive genes are shown to carry an IFN-stimulated response element (ISRE) that contains an IRF-8-PU.1 binding site. The participation of IRF-8-PU.1 in ISRE-mediated transcription results in the augmentation of IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>)-induced transcription in macrophages. Thus, GAS and ISRE elements, classically defined as universal IFN-<em>alpha</em>/beta and IFN-gamma response sequences, are not the same, and some harbor an embedded motif for IRF- 8-PU.1 binding that functions only in immune cells. Accordingly, the IRF-8-PU.1complex provides secondary IFN signaling pathways unique to the immune system. Collectively, the contribution of IRF-8 and PU.1 to IFN-regulated gene expression may in part account for immune cell-specific functions of IFNs.

The vitamin D receptor (VDR) is a nuclear receptor expressed in a number of different cells of the immune system. This study was performed to determine the effect of VDR deficiency on immune function and inflammation of the gastrointestinal tract in a model of inflammatory bowel disease, namely interleukin-10 (IL-10) knockout mice. IL-10 knockout mice were generated which either could or could not respond to vitamin D (double IL-10/VDR knockout; DKO). The distribution and function of lymphocytes in both the primary and secondary lymphoid organs were compared and determined as a function of the severity of intestinal inflammation. DKO mice had normal thymic development and peripheral T-cell numbers at <em>3</em> weeks of age, but a week after intestinal disease was detected the thymus was dysplastic with a reduction in cellularity. The atrophy was coupled with increased apoptosis. The spleen weight of DKO mice increased as a result of the accumulation of red blood cells; however, there was a 50% reduction in the numbers of T and B cells. Conversely, the mesenteric lymph nodes were enlarged and contained increased numbers of lymphocytes. The T cells from DKO mice were of a memory phenotype and were hyporesponsive to T-cell receptor stimulation. Colitis in the DKO mice was associated with local and high expression of IL-2, <em>interferon</em>-gamma, IL-1beta, tumour necrosis factor-<em>alpha</em> and IL-12. The primary and secondary lymphoid organs in DKO mice are profoundly altered as a consequence of the fulminating inflammation in the gastrointestinal tract. VDR expression is required for the T cells and other immune cells to control inflammation in the IL-10 KO mice.

In the multinational IRIS study comparing imatinib with interferon plus cytarabine (IFN/Ara-C) in patients with newly diagnosed chronic-phase chronic myelogenous leukemia (CP CML), imatinib demonstrated significantly higher rates of complete cytogenetic responses (CCyRs) and improved progression-free survival (PFS). However, because of a high early crossover rate to imatinib, survival benefit was not assessable. Here, we report the result of a study comparing long-term outcome of patients included in 2 prospective randomized trials: 551 patients assigned to imatinib in the IRIS trial from 2000 to 2001 and 325 patients who received the combination IFN/Ara-C in the CML91 trial between 1991 and 1996 before imatinib was available. With a follow-up of 42 months for both groups of patients, estimated CCyR, survival free of transformation, and overall survival were significantly higher with imatinib compared with IFN/Ara-C (P < .001, P = .004, and P < .001, respectively). Improved overall survival was also confirmed within different Sokal prognostic risk groups. Of interest, among all patients who achieved major cytogenetic response or CCyR at 12 months, the survival rate was similar irrespective of their treatment. In conclusion, within the limitation of this historical comparison, there is a survival advantage from first-line therapy with imatinib over IFN/Ara-C.

Osteopontin (OPN) is a multifunctional cytokine involved in long bone remodeling and immune system signaling. Additionally, OPN is critical for <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) production in murine plasmacytoid dendritic cells. We have previously shown that IFN-<em>alpha</em> is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variants of OPN have been associated with SLE susceptibility, and one study suggests that this association is particular to men. In this study, the <em>3</em>' UTR SLE-risk variant of OPN (rs91<em>3</em>8C) was associated with higher serum OPN and IFN-<em>alpha</em> in men (P=0.0062 and P=0.0087, respectively). In women, the association between rs91<em>3</em>8 C and higher serum OPN and IFN-<em>alpha</em> was restricted to younger subjects, and risk allele carriers showed a strong age-related genetic effect of rs91<em>3</em>8 genotype on both serum OPN and IFN-<em>alpha</em> (P<0.0001). In African-American subjects, the 5' region single nucleotide polymorphisms, rs117<em>3</em>0582 and rs28<em>3</em>57094, were associated with anti-RNP antibodies (odds ratio (OR)=2.9, P=0.00<em>3</em>8 and OR=<em>3</em>.9, P=0.021, respectively). Thus, we demonstrate two distinct genetic influences of OPN on serum protein traits in SLE patients, which correspond to previously reported SLE-risk variants. This study provides a biologic relevance for OPN variants at the protein level, and suggests an influence of this gene on the IFN-<em>alpha</em> pathway in SLE.

Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 10(6) copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 10(<em>3</em>) copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma <em>interferon</em> (IFN-gamma) and tumor necrosis factor <em>alpha</em> responses in the previously infected animal, responses that were <em>3</em>0-fold greater than baseline responses at week 4 p.i. for IFN-gamma in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.

In Echinococcus infection, at the metacestode stage, studies of the immune responses in the experimental murine model as well as in humans have shown that (1) cellular immunity induced by a Th1-type cytokine secretion was able to successfully kill the metacestode at the initial stages of development; (2) antigenic proteins and carbohydrates (and perhaps non-antigenic, mitogenic components) of the oncosphere/metacestode were able to interfere with antigen presentation and cell activation so that host lymphocytes and other immune cells could produce cytokines (especially IL-10) and other mediators able to inhibit the effector phase of cellular immune reaction; and (<em>3</em>) immunogenetic characteristics of the host were essential to this parasite-induced deviation of the immune response. In E. multilocularis infection, a combined Th1 and Th2 cytokine profile appears crucial for prolonged metacestode growth and survival. It may be hypothesized that Th1 cytokines promote the initial cell recruitment around the metacestode and are involved in the chronicity of the cell infiltrate leading to a fully organized periparasitic granuloma and its consequences, fibrosis and necrosis. The Th2 cytokines, on the other hand, could be responsible for the inhibition of a successful parasite killing especially because of the 'anti-inflammatory' potency of IL-10. This combination of various arms of the immune response results in a partial protection of both Echinococcus metacestode and host. However, it may also be considered responsible for several complications of the disease. The Th2-related IgE synthesis and mast cell activation, well known to be responsible for anaphylactic reactions in cystic echinococcosis, are more rarely involved in 'allergic' complications in alveolar echinococcosis (AE). However, the partial but chronic effects of the efficient Th1-related cellular immune response are responsible for cytotoxic events which both help metacestode growth and dissemination and lead to the central necrosis of the lesions and clinical complications of AE. Moreover, the Th-1 response is responsible for the major and irreversible fibrosis which leads to bile duct and vessel obstruction. In addition, the peri-parasitic fibrosis may be one of the reasons for the relative lack of efficacy of antiparasitic drugs. Modulation of the host immune response, by using <em>Interferon</em> <em>alpha</em> for instance, may be a new tool to generate an effective immune response against the parasite and to prevent AE and its complications.

OBJECTIVE

To investigate the in vivo acute phase molecular response of the brain to ionizing radiation.

METHODS

C<em>3</em>Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 <em>alpha</em>, IL-1 beta, IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6), <em>interferon</em> (IFN-gamma), tumor necrosis factors (TNF-<em>alpha</em> and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), <em>alpha</em> 1-antichymotrypsin (EB22/5.<em>3</em>), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined.

RESULTS

Levels of TNF-<em>alpha</em>, IL-1 beta, ICAM-1, EB22/5.<em>3</em> and to a lesser extent IL-1 <em>alpha</em> and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen.

CONCLUSIONS

The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances.