Human lungs bearing cancer (n = 27) exhibited up to an approximately 20-fold [on average approximately 5-fold (P less than 0.005)] increase in the enzyme activity that degrades tryptophan to form formylkynurenine, in comparison with lungs with benign lesions (blebs) (n = 7) taken as controls. On the basis of molecular and kinetic properties, this activity was ascribed to indoleamine 2,<em>3</em>-dioxygenase (IDO) [indoleamine:oxygen 2,<em>3</em>-oxidoreductase (decyclizing)]. In vitro studies with human lung slices revealed that human <em>interferon</em> gamma (IFN-gamma) induced the de novo synthesis of IDO dose dependently (10-10(4) units/ml), and at maximum the activity reached nearly 100 times that in the control lungs described above. Human IFN-<em>alpha</em> also served as an inducer, but it was two to three orders of magnitude less potent than IFN-gamma relative to the antiviral titers, suggesting that IFN-gamma is the main mediator of the IDO induction. IDO thus induced in slices avidly metabolized tryptophan in situ: Upon a 24-hr incubation of lung slices pretreated with varied doses of IFN-gamma (10-10(<em>3</em>) units/ml), up to 96% of the tryptophan in the slices was depleted and up to 70% of the tryptophan in the medium was converted, mainly to formylkynurenine, kynurenine, or both. The foregoing results suggest that an IFN-mediated induction of IDO also takes place in vivo in human lungs as a response to cancer, leading to metabolic consequences such as depletion of tryptophan and accumulation of (formyl)kynurenine, which may provide a unique host defense mechanism.

OBJECTIVE

The effect of octreotide plus interferon-alpha versus octreotide monotherapy on the primary study end points of time to treatment failure (progression, death, stop of study treatment) and long-term survival was investigated in patients with progressive metastatic neuroendocrine foregut (mainly pancreatic) and midgut tumors.

METHODS

One hundred nine of 125 registered patients were randomized starting in January 1995, and 105 patients (51 monotherapy, 54 combination treatment) were finally analyzed in March 2000. Tumor growth was assessed at 3-month intervals by computed tomography or magnetic resonance imaging. Long-term survival was studied up to April 2004 in all analyzed patients and in 9 patients not randomized because of stable disease.

RESULTS

Partial tumor regression occurred in 2.9%, 1.9%, and 5.7% and stabilization of tumor growth in 44.8%, 27.6%, and 15.2% at 3, 6, and 12 months, respectively, with no significant differences between both treatment arms. In March 2000, 9.5% of patients were in treatment. Time to treatment failure and long-term survival did not differ significantly between the 2 groups, with a median survival of 32 and 54 months for the octreotide and the combination groups, respectively. Survival was longer in patients not randomized because of stable disease (median, 68 months) and in those with low nuclear Ki-67. A trend toward longer survival was shown for patients with slow spontaneous tumor growth before randomization. Patients responding to treatment lived longer than unresponsive patients.

CONCLUSIONS

Combination treatment was not superior to monotherapy concerning progression-free and long-term survival. Patients responding to treatment and those with slow spontaneous tumor growth had a survival advantage.

BACKGROUND

Increased expression of proinflammatory cytokines, including tumour necrosis factor alpha, interleukin 6, and interferon gamma, as well as activation of proinflammatory signalling molecules such as nuclear factor kappa B, is characteristic of inflammatory bowel disease (IBD).

OBJECTIVE

To investigate expression and activation of signal transducer and activator of transcription (STAT) 1 in patients with IBD.

METHODS

Patients with active IBD (n=42), disease specificity controls (n=8), and normal controls (n=12) were investigated.

METHODS

Expression and activation of STAT1 were assessed by western blotting and electrophoretic mobility shift assays in extracts of endoscopic colonic biopsies. Cellular localisation was determined by immunohistochemistry.

RESULTS

Western blots and immunohistochemical staining revealed an increase in STAT1 expression and activation in mucosal samples from ulcerative colitis and to a lesser extend in Crohn's disease patients. High levels of suppressor of cytokine signalling (SOCS)-3 expression, an inhibitor of STAT activation, were observed in Crohn's disease patients and normal controls in western blot experiments whereas no differences were observed for SOCS-1 expression. Phosphorylated (p) STAT1 was mainly detected in monocytic cells and neutrophils in the inflamed mucosa. Induction of remission by systemic glucocorticoids led to a decrease in levels of pSTAT1. In vitro studies indicated a direct effect of steroid treatment on STAT1 activation.

CONCLUSIONS

Expression and activation of STAT1 are predominantly heightened in ulcerative colitis and may therefore play an important role in the pathophysiology of colonic inflammation.

BACKGROUND

Systemic lupus erythematosus (SLE) is a highly heterogeneous disorder, characterized by differences in autoantibody profile, serum cytokines, and clinical manifestations. SLE-associated autoantibodies and high serum interferon alpha (IFN-α) are important heritable phenotypes in SLE which are correlated with each other, and play a role in disease pathogenesis. These two heritable risk factors are shared between ancestral backgrounds. The aim of the study was to detect genetic factors associated with autoantibody profiles and serum IFN-α in SLE.

METHODS

We undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serology and serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci were selected for follow-up in a large independent cohort of 538 SLE patients and 522 controls using a multi-step screening approach based on novel metrics and expert database review. The seven loci were: leucine-rich repeat containing 20 (LRRC20); protein phosphatase 1 H (PPM1H); lysophosphatidic acid receptor 1 (LPAR1); ankyrin repeat and sterile alpha motif domain 1A (ANKS1A); protein tyrosine phosphatase, receptor type M (PTPRM); ephrin A5 (EFNA5); and V-set and immunoglobulin domain containing 2 (VSIG2).

RESULTS

SNPs in the LRRC20, PPM1H, LPAR1, ANKS1A, and VSIG2 loci each demonstrated strong association with a particular serologic profile (all odds ratios>> 2.2 and P < 3.5 × 10-4). Each of these serologic profiles was associated with increased serum IFN-α. SNPs in both PTPRM and LRRC20 were associated with increased serum IFN-α independent of serologic profile (P = 2.2 × 10-6 and P = 2.6 × 10-3 respectively). None of the SNPs were strongly associated with SLE in case-control analysis, suggesting that the major impact of these variants will be upon subphenotypes in SLE.

CONCLUSIONS

This study demonstrates the power of using serologic and cytokine subphenotypes to elucidate genetic factors involved in complex autoimmune disease. The distinct associations observed emphasize the heterogeneity of molecular pathogenesis in SLE, and the need for stratification by subphenotypes in genetic studies. We hypothesize that these genetic variants play a role in disease manifestations and severity in SLE.

BACKGROUND

Interest in airways inflammatory disease has increasingly focused on innate immunity. We investigated several components of innate immunity in induced sputum of patients with cystic fibrosis (CF), COPD, and asthma, and healthy control subjects.

METHODS

Twenty eight patients with mild CF lung disease (age>> or = 12 years; FEV1, 74 +/- <em>3</em>% predicted [mean +/- SE]), 74 adults with COPD (FEV1, 55 +/- 2% of predicted), <em>3</em>4 adults with persistent asthma (FEV1, 66 +/- 2% of predicted), and 44 adult control subjects (FEV1, 85 +/- 1% of predicted) were studied while in stable clinical condition. Levels of sputum interleukin (IL)-8, IL-10, <em>interferon</em> (IFN)-gamma, tumor necrosis factor (TNF)-<em>alpha</em>, human cationic antimicrobial protein 18 (CAP18), urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 were determined. Cell sources were investigated by flow cytometry and immunohistochemistry. Spirometry was performed prior to sputum induction.

RESULTS

CF patient sputum showed greatest increase in IL-8 compared to that of patients with COPD and asthma (which were also greater than control subjects), and elevated levels of TNF-alpha and IL-10 compared to other groups. There were no differences in IFN-gamma. CAP18 levels were elevated in CF and COPD patients compared to control subjects, while asthma patients had reduced CAP18 levels. uPA levels were similar but uPAR was elevated in CF and COPD patients more so than in asthma patients, while PAI-1 levels were elevated in all three disease groups. CAP18 localized to neutrophil secondary granules; neutrophils were also sources of IL-8 and PAI-1. CAP18 and PAI-1 negatively correlated with pulmonary function.

CONCLUSIONS

Induced-sputum innate immune factor levels discriminate inflammatory changes in CF, COPD, and asthma, suggesting potential roles in pathophysiology and as well as providing disease-specific biomarker patterns.

Previous studies have shown that T cells from human alcoholics overexpress activation or memory markers such as human leukocyte antigen-DR, CD45RO, CD57, and CD11b and may have reduced levels of CD62L. In those studies, we demonstrated that the increased CD57(+) T cell population rapidly produces <em>interferon</em>-gamma (IFN-gamma) and tumor necrosis factor <em>alpha</em>, independent of a second signal requirement, consistent with an increased effector T cell population. In contrast to the length of alcohol abuse by human alcoholics, most work with mice has involved 2-week ethanol exposures or less, which result in decreased IFN-gamma responses. In the present work, we have evaluated C57Bl/6 or BALB/c mice, which were administered 20% w/v ethanol in water for <em>3</em>-1<em>3</em> weeks. In these mice, rapid cytoplasmic IFN-gamma expression by T cells after stimulation through the T cell receptor was significantly increased versus normals. Studies of surface-activation markers showed that T cells from chronically ethanol-fed mice had reduced CD62L expression and an increased percentage of CD44(hi) T cells. The CD44(hi) subset was largely second signal-independent for secreted IFN-gamma and interleukin (IL)-4 production at early times after stimulation. The enriched T cells of chronic ethanol mice secreted more IFN-gamma and IL-4 than controls and equivalent IL-2 at early times after stimulation (6-24 h). The overall results support the concept that in humans and mice, chronic alcohol exposure of sufficient duration results in T cell activation or sensitization in vivo and an increased percentage of the effector/memory subset.

Iodine is an essential element for thyroid hormone synthesis. The thyroid gland has the capacity and holds the machinery to handle the iodine efficiently when the availability of iodine becomes scarce, as well as when iodine is available in excessive quantities. The latter situation is handled by the thyroid by acutely inhibiting the organification of iodine, the so-called acute Wolff-Chaikoff effect, by a mechanism not well understood 52 years after the original description. It is proposed that iodopeptide(s) are formed that temporarily inhibit thyroid peroxidase (TPO) mRNA and protein synthesis and, therefore, thyroglobulin iodinations. The Wolff-Chaikoff effect is an effective means of rejecting the large quantities of iodide and therefore preventing the thyroid from synthesizing large quantities of thyroid hormones. The acute Wolff-Chaikoff effect lasts for few a days and then, through the so-called "escape" phenomenon, the organification of intrathyroidal iodide resumes and the normal synthesis of thyroxine (T4) and triiodothyronine (T<em>3</em>) returns. This is achieved by decreasing the intrathyroidal inorganic iodine concentration by down regulation of the sodium iodine symporter (NIS) and therefore permits the TPO-H202 system to resume normal activity. However, in a few apparently normal individuals, in newborns and fetuses, in some patients with chronic systemic diseases, euthyroid patients with autoimmune thyroiditis, and Graves' disease patients previously treated with radioimmunoassay (RAI), surgery or antithyroid drugs, the escape from the inhibitory effect of large doses of iodides is not achieved and clinical or subclinical hypothyroidism ensues. Iodide-induced hypothyroidism has also been observed in patients with a history of postpartum thyroiditis, in euthyroid patients after a previous episode of subacute thyroiditis, and in patients treated with recombinant <em>interferon</em>-<em>alpha</em> who developed transient thyroid dysfunction during <em>interferon</em>-a treatment. The hypothyroidism is transient and thyroid function returns to normal in 2 to <em>3</em> weeks after iodide withdrawal, but transient T4 replacement therapy may be required in some patients. The patients who develop transient iodine-induced hypothyroidism must be followed long term thereafter because many will develop permanent primary hypothyroidism.

In human beings, as in mice, two distinct patterns of cytokine secretion have been defined among CD4+ helper T-cell clones. Human type 1 helper (Th1), but not type 2 helper (Th2), cells produce interleukin-2 (IL-2), gamma-<em>interferon</em> (IFN-gamma), and tumor necrosis factor-beta, whereas Th2, but not Th1, cells secrete IL-4 and IL-5, but not IL-2 or IFN-gamma. Other cytokines, such as IL-<em>3</em>, IL-6, GM-CSF, or TNF-<em>alpha</em>, are produced by both Th1 and Th2 cells. Th0 cells, a third Th subset, show combined production of Th1- and Th2-type cytokines. The different cytokine patterns are associated with different functions. In general, Th2 cells provide an excellent helper function for B-cell antibody production, particularly of the IgE class. On the other hand, Th1 cells are responsible for delayed type hypersensitivity reactions and are cytolytic for autologous antigen-presenting cells, including B cells. Most allergen- or helminth-antigen-specific human CD4+ T-cell clones exhibit a Th2 phenotype, whereas most clones specific for bacterial antigens show a Th1 profile. Allergen-specific Th2 cells seem to play a crucial role in atopy. These cells induce IgE production via IL-4 and favor the proliferation, differentiation, and activation of eosinophils via IL-5. In addition, Th2-derived IL-<em>3</em> and IL-4 are mast-cell growth factors that act in synergy, at least in vitro. Recent evidence indicates that allergen-specific Th2 cells are selectively enriched in tissues affected by allergic inflammation, such as the bronchial mucosa of subjects with allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the <em>interferon</em> stimulation response element (ISRE), which mediates <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>)-dependent transcription, and the IFN-gamma activation site (GAS), which is required for IFN-gamma-mediated stimulation. The ISRE binds a factor called ISGF-<em>3</em> that is activated by IFN-<em>alpha</em> but not by IFN-gamma. The GAS binds a protein that is activated by IFN-gamma, which we have termed GAF (IFN-gamma activation factor; T. Decker, D. J. Lew, J. Mirkovitch, and J. E. Darnell, Jr., EMBO J., in press; D. J. Lew, T. Decker, I. Strehlow, and J. E. Darnell, Jr., Mol. Cell. Biol. 11:182-191, 1991). We now find that the GAS is also an IFN-<em>alpha</em>-responsive element in vivo and that IFN-<em>alpha</em> (in addition to activating ISGF-<em>3</em>) rapidly activates a GAS-binding factor, the IFN-<em>alpha</em> activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-<em>3</em> could be distinguished. Therefore, not only do IFN-<em>alpha</em> and IFN-gamma stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-<em>alpha</em> receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.

OBJECTIVE

Interferon plus ribavirin is the most effective therapy for chronic hepatitis C. The aim of this study was to evaluate the effect of chronic hepatitis C and therapy on health-related quality of life and work functioning.

METHODS

Nine hundred and twelve patients with hepatitis C infection were randomized in a controlled trial of Interferon alpha 2b 3 MU tiw for 24 or 48 weeks plus ribavirin 1000-1200 mg or placebo. Questionnaire-based assessments of health-related quality of life and work functioning were performed before, during, and after treatment. Outcome measures included the SF-36 Health Survey and additional generic and specific scales. Work functioning was assessed as missed days, shorter hours or less productivity at work.

RESULTS

Pre-treatment, patients had significant impairment in five of eight SF-36 concepts compared to matched population norms. Sustained responders had a return to normal for four of these five concepts. Quality of life did not improve in non-responders. Improvements in histology, viral load or ALT values predicted improvements in quality of life. Sustained responders also had improvements in work functioning and productivity.

CONCLUSIONS

Hepatitis C patients had impaired quality of life. After combination therapy, sustained virologic responders achieved benefits in their quality of life and work functioning.

Transcription factor ISGF-<em>3</em> is a multiprotein, <em>interferon</em> <em>alpha</em>-activated transcription complex consisting of a 48-kDa DNA-binding protein and two proteins termed Stats (for signal transducers and activators of transcription) that become phosphorylated on tyrosine in the cell cytoplasm, a 11<em>3</em>-kDa and either a 91- or 84-kDa polypeptide, the latter two of which arise from differentially spliced mRNAs. Using cell lines lacking the Stat91 or Stat84 proteins, we show that mutations in several different sites in the 91-kDa protein block the <em>interferon</em> <em>alpha</em>-induced phosphorylation of the 91-kDa protein and subsequent ISGF-<em>3</em> formation. Although correct tyrosine phosphorylation on residue 690 of the Stat11<em>3</em> protein occurs independent of the Stat91/84 protein, the Stat11<em>3</em> phosphoprotein by itself moves to the cell nucleus much less efficiently in the absence of phosphorylated Stat91/84 protein.

We investigated the antiproliferative effect of partially purified human leukocyte <em>interferon</em> (HuIFN-<em>alpha</em>) given in a dose of 9-15 X 10(6) U daily by intramuscular injection to 7 patients with chronic myelogenous leukemia (CML). Hematologic remission of the disease was obtained in 5 patients. Among the responding patients, the mean white blood cell count decreased from 97.4 X 10(<em>3</em>)/cu mm (range from <em>3</em>5 X 10(<em>3</em>)/cu mm to 2<em>3</em>9 X 10(<em>3</em>)/cu mm) to 4.2 X 10(2)/cu mm (range from <em>3</em>.0 X 10(<em>3</em>) to 7.9 X 10(<em>3</em>) cu/mm). Parallel reduction occurred in serum B12, from a mean of 1,4<em>3</em>5 pg/ml to a mean of 726 pg/ml, and lactate dehydrogenase levels, from a mean of <em>3</em>25 mU/ml to 112 mU/ml. Enlarged spleens decreased in <em>3</em> of <em>3</em> patients. The 5 responding patients have been maintained on HuIFN-<em>alpha</em>, <em>3</em> X 10(6) U daily or every other day, for 6+-<em>3</em>5+ wk.

Cytokine effects on permanent cell lines of transformed mouse pancreatic <em>alpha</em>- and beta-cells were compared. The beta-tumor cell 1 (beta TC1) line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat insulin II promoter) produced insulin predominantly, although small quantities of intracellular glucagon (100:1 insulin to glucagon) were detectable by radioimmunoassay. The <em>alpha</em> TC1 line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat preproglucagon promoter) produced not only glucagon but also considerable quantities of insulin (4:1 glucagon to insulin) and preproinsulin mRNA. We therefore cloned <em>alpha</em> TC1 cells and obtained 12 glucagon-producing clonal cell lines that did not produce levels of insulin detectable by radioimmunoassay. Analysis by Northern blotting of total RNA from two lines, <em>alpha</em> TC1 clones 6 and 9, confirmed the absence of preproinsulin mRNA. No somatostatin or pancreatic polypeptide was detected by immunohistochemical staining in <em>alpha</em> TC1 clones 6 or 9 or beta TC1 cells. Rat recombinant gamma-<em>interferon</em> (IFN-gamma; 5-250 U/ml) or mouse recombinant interleukin 1 (IL-1; 1-25 U/ml) individually inhibited DNA synthesis in beta TC1 cells after <em>3</em> days of treatment. The two cytokines in combination acted synergistically to further depress DNA synthesis and increase cytotoxicity. In contrast, <em>alpha</em> TC1 clone 9 cells were not sensitive to inhibition of DNA synthesis by each cytokine individually, although glucagon synthesis was inhibited. The combination of these cytokines caused marked inhibition of DNA and glucagon syntheses in <em>alpha</em> TC1 clone 9 cells. <em>alpha</em> TC1 clone 9 cells were somewhat more resistant to the cytotoxic action of the combined cytokines than were beta TC1 cells. Incubation with 50 U/ml IFN-gamma induced class II MHC molecules (I-Ab, I-Ad, and I-Ed) and enhanced the constitutive expression of class I molecules (H-2Kb and H-2Kd) on the cell surfaces of beta TC1, uncloned <em>alpha</em> TC1, and <em>alpha</em> TC1 clones 6 and 9. Thus, these cell lines are heterozygous for MHC alleles derived from both parental strains used in the construction of the transgenic mice [C57BL/6J (H-2b) and DBA/2J (H-2d)]. Class II gene transcription induced by IFN-gamma was confirmed in beta TC1 and <em>alpha</em> TC1 clone 9 cells by Northern blot analysis with A <em>alpha</em>-, A beta-, E <em>alpha</em>, and E beta-DNA probes.(ABSTRACT TRUNCATED AT 400 WORDS)

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was <em>interferon</em> (IFN)-gamma and that to inhibit viral growth was IFN-<em>alpha</em>/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS-insensitive C<em>3</em>H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including <em>3</em> of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G-C motif(s) are required for induction of IFNs and activation of NK cells with synthetic <em>3</em>0-mer oligonucleotides.

OBJECTIVE

Human herpesvirus 8/Kaposi's sarcoma herpesvirus (HHV-8/KSHV) contains, in addition to genes required for viral replication, an unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that show homology to the transcription factors of the interferon regulatory factor (IRF) family. In this study we demonstrate that one of these ORFs (vIRF-2) encodes a protein with mobility of 18 kd which has distinct pattern of expression and properties from the cellular IRFs and the previously characterized vIRF-1.

METHODS

We cloned vIRF-2 by polymerase chain reaction (PCR) and studied its expression by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). Biologic activities were tested by chloramphenicol acetyltransferase (CAT) assay in transiently transfected mammalian cells. We characterized its DNA binding specificity by electrophoretic mobility shift analysis (EMSA) and its protein-protein interactions by in vitro pull-down assay.

RESULTS

Although low levels of vIRF-2 mRNAs can be detected in the HHV-8-positive BCBL-1 tumor cell line, 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment does not stimulate expression of vIRF-2 gene together with primary lytic cycle genes. Recombinant vIRF-2, which can form homodimers, does not bind specifically to the oligodeoxynucleotide repeats corresponding to the interferon-stimulated response element (ISRE), but it does bind to the NF-kappa B binding site. The fusion protein generated from vIRF-2 and the RelA (p65) activation domain stimulates transcriptional activity of HIV LTR, which contains two NF-kappa B sites, but does not stimulate the interferon-beta (IFNB) promoter, which contains only one NF-kappa B site. Interaction between recombinant vIRF-2 and cellular IRFs such as IRF-1, IRF-2, and ICSBP was detected by in vitro binding assay, but no interaction between IRF-3 and vIRF-2 was found. Interaction of vIRF-2 with RelA (p65) and the carboxy-terminal part of p300 was also observed. In a transient transfection assay, vIRF-2 inhibits the IRF-1- or IRF-3-mediated transcriptional activation of interferon-alpha (IFNA) gene promoter in infected cells and downmodulates RelA (p65)-stimulated activity of HIV LTR.

CONCLUSIONS

These results suggest that, by interacting with cellular transcription factors and cofactors, vIRF-2 may modulate the expression of the early inflammatory genes and potentially deregulate the immune system.

OBJECTIVE

To study the expression of blood dendritic cell antigen 2 (BDCA-2) and BDCA-4 molecules by plasmacytoid dendritic cells (PDCs) in the blood of patients with systemic lupus erythematosus (SLE), and to study PDC production of interferon-alpha (IFN alpha) and its inhibition by anti-BDCA-2 and anti-BDCA-4 antibodies.

METHODS

Peripheral blood mononuclear cells (PBMCs) from SLE patients (SLE PBMCs) and from healthy controls were induced to produce IFN alpha in vitro by SLE serum containing an endogenous IFN alpha-inducing factor (SLE-IIF) or by herpes simplex virus type 1 (HSV-1). The frequencies and numbers of BDCA-2-, BDCA-3-, and BDCA-4-expressing cells were analyzed by flow cytometry, and the effects of anti-BDCA-2 and anti-BDCA-4 monoclonal antibodies (mAb) on IFN alpha production were investigated.

RESULTS

IFN alpha production by SLE PBMCs induced by SLE-IIF or HSV-1 was decreased compared with that of healthy control PBMCs (P = 0.002 and P = 0.0007, respectively). The proportions of BDCA-2- and BDCA-3-expressing cells in SLE PBMCs were reduced compared with those in PBMCs from healthy controls (P = 0.01 and P = 0.004, respectively). IFN alpha producers in culture, especially among SLE PBMCs, displayed reduced BDCA-2 expression and constituted only a minority of the BDCA-2-positive cells, at least in healthy control PBMCs (median 18%). IFN alpha production by both SLE and healthy control PBMCs stimulated by SLE-IIF or HSV-1 was markedly reduced by anti-BDCA-2 mAb (median 81-98% inhibition). Anti-BDCA-4 mAb only partially inhibited SLE-IIF-induced IFN alpha production.

CONCLUSIONS

SLE patients had a reduced number of BDCA-2-expressing PDCs, also termed natural IFN alpha-producing cells, and their IFN alpha production could be inhibited by anti-BDCA-2/4 mAb. Such mAb may be a therapeutic option for inhibiting the ongoing IFN alpha production in SLE patients.

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD<em>3</em>-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase <em>3</em> expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor <em>alpha</em>, and gamma <em>interferon</em> but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.

Upon viral stimulation, the natural <em>interferon</em> (IFN)-<em>alpha</em>/beta-producing cells (IPCs; also known as pre-dendritic cells (DCs 2) in human blood and peripheral lymphoid tissues rapidly produce huge amounts of IFN-<em>alpha</em>/beta. After performing this innate antiviral immune response, IPCs can differentiate into DCs and strongly stimulate T cell-mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, we have mapped the developmental pathway of pre-DC2/IPCs from CD<em>3</em>4(+) hematopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages were identified, including CD<em>3</em>4(++)CD45RA(-) early progenitor cells, CD<em>3</em>4(++)CD45RA(+) late progenitor cells, CD<em>3</em>4(+)CD45RA(++)CD4(+)interleukin (IL)-<em>3</em>R<em>alpha</em>(++) pro-DC2, and CD<em>3</em>4(-)CD45RA(++) CD4(+)IL-<em>3</em>R<em>alpha</em>(++) pre-DC2/IPCs. Pro-DC2s have already acquired the capacity to produce large amounts of IFN-<em>alpha</em>/beta upon viral stimulation and to differentiate into DCs in culture with IL-<em>3</em> and CD40 ligand. CD<em>3</em>4(++)CD45RA(-) early progenitor cells did not have the capacity to produce large amounts of IFN-<em>alpha</em>/beta in response to viral stimulation; however, they can be induced to undergo proliferation and differentiation into IPCs/pre-DC2 in culture with FLT<em>3</em> ligand.

The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, <em>interferon</em>-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as <em>3</em> h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -11<em>3</em> of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaB<em>alpha</em> resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, <em>interferon</em> gamma and tumor necrosis factor <em>alpha</em>. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB.

Oligodendrocytes in multiple sclerosis brain may be under a direct attack by proinflammatory cytokines, particularly tumor necrosis factor-<em>alpha</em> (TNF<em>alpha</em>) and <em>interferon</em>-gamma (IFNgamma). In this study, we have examined the in vitro cytotoxic effects of the two cytokines, individually and in combination, on oligodendrocyte lineage cells using morphological criteria, <em>3</em>-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay (MTT), terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL), and agarose-gel electrophoretic analysis of fragmented DNA. IFNgamma exerted a dose-dependent cytotoxic effect on cultured CG4 cells, an oligodendrocyte progenitor cell line, and in primary cultures of purified oligodendrocyte progenitors. TNF<em>alpha</em>, while by itself being only mildly toxic, greatly potentiated the cytotoxicity of IFNgamma. The cytokine effects were developmentally modified in that their cytotoxic and cooperative effects became less evident in more differentiated cells. A cell-permeable peptide inhibitor (i.e., z-VAD.fmk) of caspases partially suppressed apoptotic changes elicited by the cytokine combination in CG4 cells but not in primary oligodendrocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA prepared from cytokine-treated cultures revealed an increased expression of the death receptor, Fas. The results suggest particular vulnerability of oligodendrocyte progenitors to a combination of TNF<em>alpha</em> and IFNgamma involving an activation of the cell death program.

Fractalkine (also known as CX<em>3</em>CL1), a CX<em>3</em>C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX<em>3</em>C receptor 1 (CX<em>3</em>CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and <em>interferon</em>-gamma (IFN-gamma)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX<em>3</em>CR1. TNF-<em>alpha</em> activated nuclear factor kappaB (NF-kappaB) and induced fractalkine and CX<em>3</em>CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory kappaB (IkappaB)-<em>alpha</em>, dnIkappaB-beta, dnIkappaB kinase (IKK)-gamma, kinase-dead (kd) NF-kappaB-inducing kinase (NIK) and kdIKK-beta, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt ('PDTC') or MG-1<em>3</em>2, significantly attenuated TNF-<em>alpha</em>-induced fractalkine and CX<em>3</em>CR1 expression. Furthermore, expression of dn TNF-<em>alpha</em>-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-<em>alpha</em> signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX<em>3</em>CR1 antibodies attenuated TNF-<em>alpha</em>-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-<em>alpha</em>-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide <em>3</em>-kinase (PI <em>3</em>-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-kappaB activation, and induced SMC cell-cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-<em>alpha</em> induces the expression of fractalkine and CX<em>3</em>CR1 in rat aortic SMCs and that this induction is mediated by NF-kappaB activation. We also show that fractalkine induces its own expression, which is mediated by the PI <em>3</em>-kinase/PDK1/Akt/NIK/IKK/NF-kappaB signalling pathway. More importantly, fractalkine increased cell-cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.

<em>Interferon</em>-<em>alpha</em> (IFN <em>alpha</em>) induces rapid tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking protein with multiple tyrosine phosphorylation sites that bind to the Src homology 2 (SH2) domains of various signaling proteins. During IFN <em>alpha</em> stimulation, the p85 regulatory subunit of the phosphatidylinositol <em>3</em>'-kinase binds via its SH2 domains to tyrosine-phosphorylated IRS-1, and phosphatidylinositol <em>3</em>'-kinase activity is detected in association with IRS-1. Thus, IFN <em>alpha</em> responses occur by activation of the IRS signaling system, which it shares with insulin, insulin-like growth factor-1, and interleukin-4.

We provide phenotypic and functional evidence of premonocytoid dendritic cells (DCs) and preplasmacytoid DCs in blood and of corresponding DC subsets in secondary lymphoid tissue of rhesus monkeys. Subsets were identified and sorted by 4-color flow cytometry using antihuman monoclonal antibodies cross-reactive with rhesus monkey. To mobilize pre-DC subsets, fms-like tyrosine <em>3</em> kinase ligand (Flt<em>3</em>L; 100 microg/kg subcutaneously) was administered for 10 days. Presumptive pre-DC subsets were identified within the lineage- (Lin-) major histocompatibility complex (MHC) class II+ fraction of blood mononuclear cells. Premonocytoid DCs were CD11c+CD12<em>3</em>- (interleukin-<em>3</em>R<em>alpha</em>- [IL-<em>3</em>R<em>alpha</em>-]). Preplasmacytoid DCs were characterized as CD11c-CD12<em>3</em>++ Flt<em>3</em>L increased the CD11c+ pre-DC (7-fold) and CD12<em>3</em>++ pre-DC subsets (<em>3</em>-fold) in blood. The freshly isolated CD11c+ pre-DC subset induced modest proliferation of naive allogeneic T cells. After overnight culture with granulocyte macro-phage-colony-stimulating factor (GMCSF) and CD40L, both subsets up-regulated surface costimulatory molecules, and CD11c+ pre-DCs became potent allostimulators. Freshly isolated CD12<em>3</em>++ pre-DCs showed typical plasmacytoid morphology and, when cultured with IL-<em>3</em> and CD40L for 72 hours, developed mature DC morphology. Following stimulation with CD40L, CD11c+ pre-DCs secreted increased levels of IL-12p40. Importantly, herpes simplex virus-stimulated CD12<em>3</em>++ pre-DCs, but not CD11c+ pre-DCs, secreted <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>). Corresponding DC subsets were identified by flow analysis and immunohistochemistry in lymph nodes wherein both populations were increased 2- to <em>3</em>-fold by Flt<em>3</em>L administration. CD12<em>3</em>+ pre-DCs produced IFN-<em>alpha</em> in response to in vivo viral infection. Thus, rhesus monkeys exhibit 2 distinct DC precursor populations that closely resemble those of humans. Both are mobilized into blood and lymphoid tissue by Flt<em>3</em>L, offering potential for their further characterization and possible therapeutic application.

BACKGROUND

Myeloid differentiation factor (MyD)-88 is a key adaptor protein that plays a major role in the innate immune pathway. How MyD88 may regulate host response in inflammatory heart disease is unknown.

RESULTS

We found that the cardiac protein level of MyD88 was significantly increased in the hearts of wild-type mice after exposure to Coxsackievirus B<em>3</em> (CVB<em>3</em>). MyD88(-/-) mice showed a dramatic higher survival rate (86%) in contrast to the low survival (<em>3</em>5%) in the MyD88(+/+) mice after CVB<em>3</em> infection (P<0.0001). Pathological examination showed a significant decrease of cardiac and pancreatic inflammation in the MyD88(-/-) mice. Viral concentrations in the hearts were significantly decreased in the MyD88(-/-) mice. Cardiac mRNA levels for interleukin (IL)-1beta, tumor necrosis factor (TNF)-<em>alpha</em>, <em>interferon</em> (IFN)-gamma, and IL-18 were significantly decreased in the MyD88(-/-) mice. Similarly, serum levels of T-helper 1 cytokines were significantly decreased in the MyD88(-/-) mice. In contrast, cardiac protein levels of the activated <em>interferon</em> regulatory factor (IRF)-<em>3</em> and IFN-beta were significantly increased in the MyD88(-/-) mice but not other usual upstream signals to IRF-<em>3</em>. The cardiac expression of coxsackie-adenoviral receptor and p56(lck) were also significantly decreased.

CONCLUSIONS

MyD88 appears to be a key contributor to cardiac inflammation, mediating cytokine production and T-helper-1/2 cytokine balance, increasing coxsackie-adenoviral receptor and p56(lck) expression and viral titers after CVB<em>3</em> exposure. Absence of MyD88 confers host protection possibly through novel direct activation of IRF-<em>3</em> and IFN-beta.