Inhalation is an important route of infection with Burkholderia pseudomallei, the causative agent of melioidosis. In resistant C57BL/6 mice, activated neutrophils are rapidly recruited to the lungs after intranasal B. pseudomallei infection. Prevention of this response by use of the anti-Gr-1+ cell-depleting monoclonal antibody RB6-8C5 severely exacerbated disease, resulting in an acute lethal infection associated with a 1000-fold increase in lung bacterial loads within 4 days. C57BL/6 <em>interferon</em> (IFN)-gamma(-/-) mice were also acutely susceptible to pulmonary B. pseudomallei infection, dying within <em>3</em> days of challenge; this suggests that IFN-gamma is essential for control in the lungs and precedes the protective role of neutrophils in resistance. In neutrophil-depleted mice, lung concentrations of tumor necrosis factor (TNF)-<em>alpha</em>, IFN-gamma, and interleukin-6 were decreased by up to 98%. Natural killer cells were the principle source of IFN-gamma, and monocytes were the principle source of TNF-<em>alpha</em>, suggesting that neutrophils play an important indirect role in the generation of the early cytokine environment in the lungs.

OBJECTIVE

In the patients with chronic hepatitis C, the addition of ribavirin to interferon (IFN)-alpha significantly increases the virologic responses. Our aim was to assess the antiviral action of ribavirin on hepatitis C virus (HCV) as a function of ribavirin pharmacokinetics and to evaluate the influence of this antiviral effect on IFN-alpha efficacy.

METHODS

Forty-five patients with chronic hepatitis C (genotype 1b) received various schedules of IFN-alpha and/or ribavirin administration. Frequent blood sampling was performed for HCV RNA kinetics and ribavirin pharmacokinetics assessment.

RESULTS

Ribavirin monotherapy induced a significant, moderate, early, and transient viral load decrease in approximately half of the patients. The occurrence of this effect was associated with longer ribavirin clearance half-lives and higher serum ribavirin concentrations. Ribavirin antiviral effect partly reduced the rebound preceding the second IFN-alpha injection in patients receiving standard IFN-alpha 3 times per week plus ribavirin. The magnitude of the rebound was inversely related to ribavirin concentrations. These patients subsequently experienced a slow, but significant, second slope of viral decrease and cleared HCV RNA. The addition of ribavirin to daily IFN-alpha monotherapy did not have any impact on the second phase of viral decline.

CONCLUSIONS

Ribavirin exerts a significant, moderate, and transient antiviral effect in a significant proportion of patients with chronic hepatitis C. The antiviral effect of ribavirin correlates with ribavirin pharmacokinetics and is partly responsible for the improved efficacy of the combination of standard IFN-alpha and ribavirin compared with IFN-alpha monotherapy by increasing the incidence of the initial response.

OBJECTIVE

The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis.

METHODS

Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst <em>3</em><em>3</em><em>3</em>42-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-<em>3</em>beta, activated caspase-<em>3</em> and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors.

RESULTS

Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-<em>3</em> (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05).

CONCLUSIONS

The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.

Normal human peripheral blood lymphocytes were tested for their susceptibility to infection with retroviruses isolated from patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. Of 10 normal individuals tested, lymphocytes from all subjects became infected and produced virus as detected by assay for Mg+2-dependent reverse transcriptase. Lymphocytes from different individuals were demonstrated to be either high or low producers of reverse transcriptase after infection. The kinetics of virus production were similar in cells from both high- and low-producing individuals. A significant correlation was observed between high and low viral-producing lymphocytes and expression of the Leu-<em>3</em>/T4 (CD4) surface molecule. Mitogen-stimulated peripheral blood lymphocytes exposed to HTLV-III/LAV manifested productive viral infection, as reflected by the appearance of early syncytia, followed by reverse transcriptase. Unstimulated peripheral blood lymphocyte cultures displayed late syncytia but no detectable reverse transcriptase upon exposure to virus. The addition of anti-human <em>interferon</em>-<em>alpha</em> did not appear to have an appreciable effect on viral production in normal peripheral blood lymphocytes exposed to the virus.

We show that <em>interferon</em>-induced transmembrane protein 1 (IFITM-1), IFITM-2, and IFITM-<em>3</em> exhibit a broad spectrum of antiviral activity against several members of the Bunyaviridae family, including Rift Valley fever virus (RVFV), La Crosse virus, Andes virus, and Hantaan virus, all of which can cause severe disease in humans and animals. We found that RVFV was restricted by IFITM-2 and -<em>3</em> but not by IFITM-1, whereas the remaining viruses were equally restricted by all IFITMs. Indeed, at low doses of <em>alpha</em> <em>interferon</em> (IFN-α), IFITM-2 and -<em>3</em> mediated more than half of the antiviral activity of IFN-α against RVFV. IFITM-2 and -<em>3</em> restricted RVFV infection mostly by preventing virus membrane fusion with endosomes, while they had no effect on virion attachment to cells, endocytosis, or viral replication kinetics. We found that large fractions of IFITM-2 and IFITM-<em>3</em> occupy vesicular compartments that are distinct from the vesicles coated by IFITM-1. In addition, although overexpression of all IFITMs expanded vesicular and acidified compartments within cells, there were marked phenotypic differences among the vesicular compartments occupied by IFITMs. Collectively, our data provide new insights into the possible mechanisms by which the IFITM family members restrict distinct viruses.

Chemokines have been shown to chemoattract and activate different leukocyte populations. Here we report the in vitro effect of macrophage inflammatory protein (MIP)-1<em>alpha</em>, MIP-1beta, regulated on activation, normal T-cells, expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), <em>interferon</em> inducible protein-10 (IP-10), neutrophil-activating peptide-2 (NAP-2), growth-related protein (GRO)-<em>alpha</em> and GRO-gamma, on the migration of <em>3</em> human breast carcinoma cell lines, MCF-7, T47D and ZR-75-1, using a microchemotaxis chamber to assess migration across fibronectin-coated polycarbonate membranes. MCF-7 cells responded chemotactically to all chemokines tested in a pattern which was dose and time dependent, although responses to the different chemokines were variable. ZR-75-1 responded to MIP-1beta and GRO-<em>alpha</em>, giving maximum migration indices of <em>3</em>.7 and 5.<em>3</em>, respectively, and exhibited a migratory response to MIP-1<em>alpha</em>, IL-8 and MCP-1 although to a lower degree. T47D was unresponsive to the chemokines tested, but both MCF-7 and T47D cells bound radiolabelled ligands with binding constants (Kd) ranging from 0.6 to 2.2 nM and 0.6 to 2.1 nM, respectively. The specificity of the chemotactic response of MCF-7 to MIP-1<em>alpha</em> and MIP-1beta was confirmed using chemokine-specific neutralising antibodies and heat denaturation, and was demonstrated to involve G protein and cyclic AMP signalling pathways. MIP-1beta and MIP-1<em>alpha</em> were shown to induce changes in the organisation of the actin cytoskeleton and the level of F-actin in MCF-7 cells, as determined using flow cytometric analysis and confocal microscopy. Our results show that breast carcinoma cells can respond to chemokines, and suggests a potential role for these molecules in the process of tumour cell migration, invasion and metastasis.

We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor <em>alpha</em> [TNF-<em>alpha</em>]; Th1, interleukin-12 [IL-12] and gamma <em>interferon</em> [IFN-gamma]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day <em>3</em> [PID <em>3</em>]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-<em>alpha</em> and IFN-gamma at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-<em>alpha</em> and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-gamma and TNF-<em>alpha</em> CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the host's local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

The early systemic production of <em>interferon</em> (IFN)-<em>alpha</em>beta is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-<em>alpha</em>beta response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-<em>alpha</em>beta mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-<em>alpha</em>beta production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-<em>alpha</em>beta response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and <em>interferon</em> regulatory factor (IRF)-<em>3</em>, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-<em>alpha</em>beta and IL-6 in vivo by distinct pathways and confirm that IFN-<em>alpha</em>beta positively regulates the IL-6 response. Finally, by measuring TNF-<em>alpha</em> responses to LPS in Ad-infected wild type and IFN-<em>alpha</em>betaR(-/-) mice, we show that IFN-<em>alpha</em>beta is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-<em>alpha</em>beta response, which is responsible for the bulk of IFN-<em>alpha</em>beta production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-<em>alpha</em>beta induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

OBJECTIVE

Up-regulation of whole blood type I interferon (IFN)-driven transcripts and chemokines has been described in a number of autoimmune diseases. An IFN gene expression "signature" is a candidate biomarker in patients with dermatomyositis (DM). This study was performed to evaluate the capacity of IFN-dependent peripheral blood gene and chemokine signatures and levels of proinflammatory cytokines to serve as biomarkers for disease activity in adult and juvenile DM.

METHODS

Peripheral blood samples and clinical data were obtained from 56 patients with adult or juvenile DM. The type I IFN gene signature in the whole blood of patients with DM was defined by determining the expression levels of 3 IFN-regulated genes (IFIT1, G1P2, and IRF7) using quantitative real-time reverse transcription-polymerase chain reaction. Multiplexed immunoassays were used to quantify the serum levels of 4 type I IFN-regulated chemokines (IFN-inducible T cell alpha chemoattractant, IFNgamma-inducible 10-kd protein, monocyte chemotactic protein 1 [MCP-1], and MCP-2) and the serum levels of other proinflammatory cytokines, including interleukin-6 (IL-6).

RESULTS

DM disease activity correlated significantly with the type I IFN gene signature (r = 0.41, P = 0.007) and with the type I IFN chemokine signature (r = 0.61, P < 0.0001). Furthermore, the serum levels of IL-6 were significantly correlated with disease activity (r = 0.45, P = 0.001). In addition, correlations between the serum levels of IL-6 and both the type I IFN gene signature (r = 0.47, P < 0.01) and the type I IFN chemokine signature (r = 0.71, P < 0.0001) were detected in patients with DM.

CONCLUSIONS

These results suggest that serum IL-6 production and the type I IFN gene signature are candidate biomarkers for disease activity in adult and juvenile DM. Coregulation of the expression of IFN-driven chemokines and IL-6 suggests a novel pathogenic linkage in DM.

Infection with Toxoplasma gondii is naturally acquired through the oral route by ingestion of undercooked or raw meat containing cysts of the parasite or through ingestion of contaminated water or food contaminated with cysts or oocysts. Following peroral infection with 100 cysts of the ME49 strain of T. gondii, C57BL/6 mice die within 1<em>3</em> days after infection, whereas BALB/c mice survive. At day 7 of infection, massive necrosis of the villi and mucosal cells in the ilea is observed in C57BL/6 but not BALB/c mice. CD4(+) T cells, <em>interferon</em>-gamma, tumor necrosis factor-<em>alpha</em>, and inducible nitric oxide synthase mediate the development of necrosis. These findings indicate a Th1-type immunopathology, with parasite replication appearing to be involved in the first <em>3</em> days of infection. Murine and human studies on the immunopathogenesis of inflammatory bowel disease (e.g., Crohn's disease) also indicate a Th1-type immunopathology. The shared and distinct features of oral infection of mice with T. gondii and murine models of inflammatory bowel disease are discussed herein.

The <em>interferon</em> (IFN)-inducible chemokines, specifically, IFN-gamma-inducible protein-10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell <em>alpha</em>-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR<em>3</em>). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH(2)-terminal dipeptides by CD26/DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti-human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR<em>3</em> are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were <em>3</em>- and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/DPP IV resulted in reduced CXCR<em>3</em>-binding properties, loss of calcium-signaling capacity through CXCR<em>3</em>, and more than 10-fold reduced chemotactic potency. Moreover, IP-10 and I-TAC cleaved by CD26/DPP IV acted as chemotaxis antagonists and CD26/DPP IV-truncated IP-10 and Mig retained their ability to inhibit the angiogenic activity of interleukin-8 in the rabbit cornea micropocket model. These data demonstrate a negative feedback regulation by CD26/DPP IV in CXCR<em>3</em>-mediated chemotaxis without affecting the angiostatic potential of the CXCR<em>3</em> ligands IP-10 and Mig.

OBJECTIVE

C1q deficiency is the strongest risk factor known for the development of systemic lupus erythematosus (SLE), since almost all humans with a genetic deficiency of C1q develop this disease. Low C1q serum concentration is also a typical finding in SLE during flares, emphasizing the involvement of C1q in SLE pathogenesis. Recent studies have revealed that C1q has a regulatory effect on Toll-like receptor-induced cytokine production. Therefore, we undertook this study to investigate whether C1q could regulate production of interferon-alpha (IFNalpha).

METHODS

Peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) were stimulated with 3 known interferogenic stimuli and cultured with physiologic concentrations of C1q. IFNalpha production was determined by an immunoassay.

RESULTS

C1q significantly inhibited PBMC IFNalpha production induced by RNA-containing immune complexes (ICs), herpes simplex virus (HSV), and CpG DNA. C1q also inhibited PDC IFNalpha production induced by ICs and CpG DNA but increased PDC IFNalpha production induced by HSV. The regulatory role of C1q was not specific for IFNalpha but was also seen for interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha. We demonstrated binding of C1q to PDCs both by surface plasmon resonance interaction analysis and by flow cytometry, and we also demonstrated intracellular detection of 2 C1q binding proteins.

CONCLUSIONS

Our findings contribute to the understanding of why C1q deficiency is such a strong risk factor for SLE and suggest an explanation for the up-regulation of the type I IFN system seen in SLE patients.

BACKGROUND

Both bacillus Calmette-Guérin (BCG) and interferon-alpha (IFN-alpha) express activity against superficial bladder cancer. The results of a national multicenter phase II trial of a combination of these 2 agents used in a wide range of patients are reported.

METHODS

Patients previously having BCG failure received IFN-alpha (50 million units) plus reduced dose BCG, while patients naïve to BCG received the same IFN-alpha dose with standard dose BCG. All patients who were relapse free received an additional 3 series of 3-week reduced dose BCG plus IFN-alpha treatments at 3, 9, and 15 months after completing induction. Any relapse during the 3-month evaluation was counted as a failure of therapy for Kaplan-Meier analysis. Multivariate analysis was performed to identify factors associated with recurrence.

RESULTS

Of 1,007 valuable patients, 59% and 45% of patients naïve to BCG and those having BCG failure, respectively, remained disease free at 24-month median follow-up. Stage T1, tumor size>5 cm, prior BCG failure more than once, and multifocality were all statistically significant risk factors for recurrence.

CONCLUSIONS

Although BCG plus IFN-alpha can be effectively applied to both patients naïve to BCG and those having BCG failure, certain patient and tumor characteristics influence durable response.

OBJECTIVE

Daclizumab is an interleukin 2 receptor alpha chain specific humanized monoclonal antibody that has shown promising therapeutic effects in multiple sclerosis (MS). Daclizumab treatment in patients with relapsing and remitting MS was administered to determine effects on MRI and clinical outcomes.

METHODS

Patients with MS on interferon (IFN) therapy but with continuing relapses and contrast enhancing lesions (CEL) were selected. Patients were evaluated with monthly MRI scans and clinical rating scales starting 3 months prior to treatment and then at 0.5 to 27.5 months during treatment. Daclizumab (1 mg/kg IV) was administered twice in the first month (initiated and administered again in 2 weeks), followed by treatments every 4 weeks. IFN was continued until 5.5 months after daclizumab was initiated. Patients were then placed on daclizumab monotherapy. Patients with recurrent CEL were restarted on IFN with daclizumab therapy at (1.5 mg/kg IV) every 28 days.

RESULTS

Nine patients qualified for inclusion and completed the trial. Efficacy measured by both total CEL and new CEL (p < 0.001), relapses, timed ambulation, Expanded Disability Status Scale, and Neurologic Rating Scale (p < 0.05 to p < 0.01) was observed.

CONCLUSIONS

Daclizumab was effective in reducing contrast enhancing lesions and improving clinical scores in patients with relapsing and remitting multiple sclerosis with active disease not controlled by interferon therapy. These results provide evidence for long-term efficacy and support further clinical development of daclizumab.

<em>Interferon</em> (IFN)-<em>alpha</em>, IFN-beta, and IFN-gamma are currently available for the treatment of malignancies, viral infections (e.g., hepatitis C virus), multiple sclerosis (MS), and skin conditions. In addition to their therapeutic effects, IFNs commonly cause various side effects. Most common among the side effects of IFN are "flu-like" symptoms such as chills, fever, and muscle soreness. However, IFN can also cause significant neuropsychiatric side effects, particularly symptoms of depression. A literature search was conducted in order to summarize current information on (1) the frequency, characteristics, and risk factors of IFN-induced depression, (2) possible biochemical mechanisms associated with IFN-induced depression, and (<em>3</em>) the treatment strategies for IFN-induced depression. Review of the literature suggests that symptoms of depression induced by IFN therapy, in particular IFN-<em>alpha</em> therapy, are common and can limit the treatment utility, often necessitating discontinuation of IFN therapy or the use of psychopharmacologic agents. Depression is also a suspected side effect of therapy with IFN-beta and IFN-gamma; however, the association has not been as convincingly confirmed. Importantly, IFNs affect neurochemical pathways putatively involved in the etiology of depression. While these depressive side effects usually resolve after the completion of IFN therapy, they can persist or reappear with dose escalations. It is recommended that health care providers, patients and their families be informed about the potential risk of the psychiatric disturbances that can occur with IFN-<em>alpha</em> therapy. Screening and monitoring, ideally using symptom rating scales for depression, and early antidepressant treatment intervention appear necessary to optimize IFN therapy for the majority of patients.

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed <em>3</em>-11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-<em>alpha</em> and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp<em>3</em>8 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4+ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88-/- mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive <em>interferon</em>-gamma-dependent CD4+ T cell polarization and antibody switching.

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, <em>3</em>, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I <em>interferon</em> (IFN) and tumor necrosis factor (TNF)-<em>alpha</em> genes. A network-based analysis suggests that the synergy between IFN-beta and TNF-<em>alpha</em> results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.

Sinus histiocytosis with massive lymphadenopathy (SHML) is a rare disorder of unknown etiology, usually associated with lymph node enlargement in various superficial or deep sites. It usually shows a prolonged clinical course with occasional exacerbation and remission phases. We describe the long-term follow-up of a case of SHML that showed typical clinical features and in which various therapeutic strategies were attempted. Chemotherapy and <em>alpha</em>-<em>interferon</em> (IFN) were ineffective; surgery was ultimately required with satisfactory results. From an extensive literature review we found different treatment strategies in SHML in the 80 cases published between 1969 and 2000. Spontaneous resolution of adenopathies is frequently observed: <em>3</em>2 out of 40 cases which did not receive chemotherapy, radiotherapy, or surgery were healthy at the time of publication. Radiotherapy alone showed conflicting results: <em>3</em> complete remissions (CR) were obtained in the 9 patients treated. Surgical debulking when required was effective--8/9 CR--while chemotherapy showed generally negative results. IFN has been previously employed in only one case. In conclusion, clinical observation without treatment is advisable when possible. In the presence of vital organ compression and/or extranodal localization with important clinical signs, surgical debulking may be necessary. Radiotherapy has shown limited efficacy, while chemotherapy is in general ineffective. More experience is needed to evaluate the role of IFN.

This study was designed to define the lipopolysaccharide (LPS) sensitivity of aged mice in terms of lethality and cytokine production and to determine down-regulating responses of corticosterone and interleukin 10 (IL-10). The 50% lethal doses of LPS in young (6- to 7-week-old) and aged (98- to 102-week-old) mice were 601 and 9<em>3</em> microg per mouse (25.6 and 1.6 mg per kg of body weight), respectively. Aged mice were approximately 6.5-fold more sensitive to the lethal toxicity of LPS in micrograms per mouse (16-fold more sensitive in milligrams per kilogram) than young mice. Levels in sera of tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) IL-1<em>alpha</em>, and IL-6 after intraperitoneal injection of 100 microg of LPS peaked at 1.5, <em>3</em>, and <em>3</em> h, respectively, and declined thereafter in both groups of mice. However, the peak values of these cytokines were significantly higher in aged than in young mice (P < 0.05). Gamma <em>interferon</em> (IFN-gamma) was detectable at <em>3</em> h, and sustained high levels were still detected after 12 h in both age groups. Although there were no significant differences in levels of IFN-gamma in sera from both groups, aged mice showed higher IFN-gamma levels throughout the <em>3</em>- to 12-h study period. Administration of increasing doses of LPS revealed that aged mice had a lower threshold to IL-1<em>alpha</em> production than young mice. In addition, aged mice were approximately 4-fold more sensitive to the lethal toxicity of exogenous TNF in units per mouse (10-fold more sensitive in units per kilogram) than young mice. With regard to down-regulating factors, corticosterone amounts were similar at basal levels and no differences in kinetics after the LPS challenge were observed, whereas IL-10 levels in sera were significantly higher in aged mice at 1.5 and <em>3</em> h than in young mice (P < 0.01). These results indicate that aged mice are more sensitive to the lethal toxicities of LPS and TNF than young mice. We conclude that a relatively activated, or primed, state for LPS-induced cytokine production, in spite of full down-regulating responses by corticosterone and IL- 10, may explain at least in part LPS sensitivity in aged mice.

<em>Interferon</em> (IFN) consensus sequence binding protein (ICSBP) is a transcription factor expressed mostly in the cells of the immune system. ICSBP belongs to the IFN regulatory factor (IRF) family, which also includes IRF-1, IRF-2, and the IFN-<em>alpha</em>-stimulated gene factor <em>3</em> gamma (ISGF<em>3</em> gamma). We show here that ICSBP forms a complex with IRF-1 or IRF-2 both in vivo and in vitro and, in the presence or absence of the target DNA, with the IFN-stimulated response element (ISRE). Further, electrophoretic mobility shift assays show that this interaction greatly enhances the otherwise very low binding affinity of ICSBP to the ISRE. We show, on the other hand, that ICSBP inhibits binding of the IFN-<em>alpha</em>-stimulated gene factor <em>3</em> gamma to the ISRE. Through these interactions ICSBP is likely to exert complex modulatory functions in the regulation of IFN-stimulated genes.

Despite considerable progress, peritonitis and sepsis remain life-threatening conditions. To improve the understanding of the pathophysiology encountered in sepsis, a new standardized and highly reproducible murine model of abdominal sepsis termed colon ascendens stent peritonitis (CASP) was developed. In CASP, a stent is inserted into the ascending colon, which generates a septic focus. CASP employing a stent of 14-gauge diameter (14G stent) results in a mortality of 100% within 18 to 48 h after surgery. By inserting stents of small diameters, mortality can be exactly controlled. Thus, CASP surgery with insertion of a 22G or 18G stent (22G or 18G CASP surgery) results in <em>3</em>8 or 68% mortality, respectively. 14G CASP surgery leads to a rapid invasion of bacteria into the peritoneum and the blood. As a consequence, endotoxemia occurs, inflammatory cells are recruited, and a systemic inflammatory response syndrome develops. Interestingly, the most pronounced upregulation of inflammatory cytokines (gamma <em>interferon</em> [IFN-gamma], tumor necrosis factor <em>alpha</em> [TNF-<em>alpha</em>] and interleukin-12) is observed in spleen and lungs. CASP surgery followed by stent removal at specific time intervals revealed that all animals survived if intervention was performed after <em>3</em> h, whereas removal of the septic focus after 9 h did not prevent death, suggesting induction of autonomous mechanisms of a lethal inflammatory response syndrome. 18G CASP surgery in IFN-gamma receptor-deficient (IFNgammaR-/-) mice revealed an essential role of IFN-gamma in survival of sepsis, whereas TNF receptor p55-deficient (TNFRp55-/-) mice did not show altered survival rates. In summary, this study describes a novel animal model that closely mimics human sepsis and appears to be highly suitable for the study of the pathophysiology of abdominal sepsis. Importantly, this model demonstrates a protective role of IFN-gamma in survival of bacterial sepsis.

OBJECTIVE

Atherosclerosis is a chronic inflammatory response of the arterial wall to injury. High-mobility group box 1 (HMGB1) is a DNA-binding protein, which on release from cells exhibits potent inflammatory actions. We examined its expression in atherosclerotic lesions and regulation by cytokines.

RESULTS

In atherosclerotic lesions, HMGB1 protein is expressed by endothelial cells, some intimal smooth muscle cells, and macrophages. As atherosclerosis develops and progresses from fatty streaks to fibrofatty lesion, the number of HMGB1-producing macrophages increases markedly. Studies using the THP-1 cell line indicated that HMGB1 mRNA expression could be markedly upregulated by inflammatory cytokines, <em>interferon</em> (IFN)-gamma, tumor necrosis factor (TNF)-<em>alpha</em> and also transforming growth factor (TGF)-beta. IFN-gamma, TNF-<em>alpha</em>, TWEAK, and TGF-beta induced an intracellular redistribution of HMGB1 and stimulated secretion by THP-1 cells and human blood monocytes. Inhibitors of MEK1/MEK2, protein kinase C, and PI-<em>3</em>/Akt, which inhibit lysosomal degranulation and mRNA translation, attenuated cytokine-induced HMGB1 secretion.

CONCLUSIONS

Macrophage is the major cell type responsible for HMGB1 production in human atherosclerotic lesions. Inflammatory cytokines and TGF-beta increase HMGB1 expression and secretion by monocyte/macrophages. HMGB1 appears to be a common mediator of inflammation induced by inflammatory cytokines and is likely to contribute to lesion progression and chronic inflammation.

A survival benefit for imatinib mesylate versus <em>interferon</em>-<em>alpha</em> therapy could not be demonstrated in the randomized study in newly diagnosed Philadelphia chromosome (Ph)-positive chronic-phase chronic myelogenous leukemia (CML) due to the high rate of crossover (90%) from <em>interferon</em>-<em>alpha</em> to imatinib mesylate within a year of study entry. We compared survival in 279 patients with newly diagnosed CML treated with imatinib mesylate at our institution (2000-2004) to 650 patients treated with <em>interferon</em>-<em>alpha</em> (1982-1997). The complete cytogenetic response rates were 87% with imatinib mesylate and 28% with <em>interferon</em>-<em>alpha</em> (P < .001). The estimated <em>3</em>-year survival rates were 96% with imatinib mesylate and 81% with <em>interferon</em>-<em>alpha</em> (P < .01). Survival rates with imatinib mesylate were significantly better than with <em>interferon</em>-<em>alpha</em> within each of the CML prognostic risks groups. By multivariate analysis, imatinib mesylate therapy was identified as an independent favorable prognostic factor, after accounting for the impact of pretreatment factors (hazard ratio, 0.44; P < .01). By landmark analysis at 12 months, survival within each cytogenetic response category was similar with imatinib mesylate or <em>interferon</em>-<em>alpha</em>, suggesting that the survival benefit of imatinib mesylate (versus <em>interferon</em>-<em>alpha</em> in newly diagnosed CML) is through improving cytogenetic response.

During viral infection, <em>interferon</em>-<em>alpha</em>/beta (IFN-<em>alpha</em>/beta) and many IFN-inducible genes are induced to elicit antiviral responses of the host. Using cells with a gene disruption(s) for the IRF family of transcription factors, we provide evidence that these genes, containing similar IRF-binding cis-elements, are classified into distinct groups, based on the gene induction pathway(s). The IFN-beta gene induction is dependent on either IRF-<em>3</em> or IRF-7, whereas induction of the IFN-<em>alpha</em> gene family is IRF-7-dependent. On the other hand, ISG15, ISG54 and IP-10 are induced by either IRF-<em>3</em> or IFN stimulated gene factor <em>3</em> (ISGF<em>3</em>). We also show that another group of genes is totally dependent on ISGF<em>3</em>. Thus, during viral infection, a given gene responds either directly to a virus or virus-induced IFN-<em>alpha</em>/beta or both through distinct pathways. The differential utilization of these induction pathways for these genes during viral infection may reflect their distinct functional roles in the efficient antiviral response.