Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>) and <em>interferon</em> gamma (IFNgamma) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a <em>3</em>-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement <em>3</em> (C<em>3</em>) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

We reanalyzed the results of a pilot study of recombinant <em>alpha</em>-<em>interferon</em> therapy for chronic non-A, non-B hepatitis in light of the recent discovery of the hepatitis C virus and the development of diagnostic assays for this agent. Stored serum samples from 10 patients treated between 1984 and 1986 were tested for antibody to hepatitis C virus and hepatitis C virus RNA before, during and after therapy. In addition, the current clinical, serum biochemical and virological statuses of these patients were evaluated to determine the long-term effects of <em>interferon</em> therapy. All patients had evidence of hepatitis C virus infection, with hepatitis C viral RNA, antibody to hepatitis C virus or both markers detectable in serum. Serum hepatitis C virus RNA was found to disappear in seven of eight patients whose aminotransferase levels became normal with <em>interferon</em> therapy but remained present in two patients who did not respond to therapy. Levels of hepatitis C virus RNA decreased and disappeared when serum aminotransferases fell to normal levels but rose with subsequent elevation of aminotransferase levels in two patients who had relapses in disease when <em>interferon</em> was stopped. During a follow-up of <em>3</em> to 6 yr, hepatitis C virus RNA remained undetectable in the six patients whose serum aminotransferase levels remained normal after <em>interferon</em> therapy. However, neither initial titers of hepatitis C virus RNA nor disappearance of viral RNA from serum during treatment predicted a sustained response. Thus long-term beneficial responses to <em>alpha</em>-<em>interferon</em> can occur in patients with chronic hepatitis C and are associated with sustained loss of hepatitis C virus RNA from serum.

BACKGROUND

We postulate that hypercytokinemia plays a role in immunopathogenesis of severe human influenza.

METHODS

We prospectively studied 39 consecutive patients who were hospitalized with severe influenza A virus infection. On laboratory confirmation of the diagnosis, paired acute-phase (obtained at hospital admission) and convalescent-phase (obtained >10 days after hospital admission) plasma samples were collected for assay of 11 cytokines and chemokines (interleukin [IL] 1 beta; IL-6; IL-10; IL-12p70; tumor necrosis factor alpha; IL-8; monokine induced by interferon [IFN]-gamma; IFN-inducible protein 10; monocyte chemoattractant protein 1; regulated upon activation, normal T cell-expressed and secreted; and IFN-gamma) using cytometric bead-array analysis and enzyme-linked immunosorbent assay. Simultaneously, virus concentration in the acute-phase nasopharyngeal aspirate was determined using real-time quantitative reverse-transcriptase polymerase chain reaction. Intracellular signaling molecules regulating lymphocyte activation, phospho-p38 mitogen-activated protein kinase and phospho-extracellular signal-regulated protein kinase in CD4+ and CD8+ T lymphocytes were studied in the acute-phase samples using flow cytometric analysis and were compared with results for samples from healthy control subjects.

RESULTS

Statistically significant increases in plasma IL-6 (3.7-fold increase), IL-8 (2.6-fold increase), IFN-induced protein 10 (4.9-fold increase), and monokine induced by IFN-gamma (2.3-fold increase) concentrations were detected during acute illness (P < .01 for all, by Wilcoxon signed-rank test); the highest concentrations were observed on symptom days 3 and 4. Corresponding plasma cytokine and chemokine concentrations and nasopharyngeal viral loads showed statistically significant correlations (rho = 0.41, 0.49, 0.54, and 0.46, respectively; P < or = .01). Phospho-p38 mitogen-activated protein kinase expression in CD4+ lymphocytes was increased, correlating with cytokine concentrations (e.g., for IFN-induced protein 10, rho = 0.78; P < .01); phospho-extracellular signal-regulated protein kinase was suppressed. Advanced age and comorbidity were associated with aberrant IL-6, IL-8, and monokine induced by IFN-gamma responses (P < .05, by Mann-Whitney U test). An elevated IL-6 concentration was independently associated with prolonged hospitalization (hospitalization for >5 days; P = .02), adjusted for age, comorbidity, and virus load.

CONCLUSIONS

Hypercytokinemia (of proinflammatory and T helper 1 cytokines) is detected in severe influenza, correlating with clinical illness and virus concentration. Hyperactivation of phospho-p38 mitogen-activated protein kinase (in T helper cells) is possibly involved. Early viral suppression may attenuate these potentially deleterious cytokine responses.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin <em>3</em>, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma <em>interferon</em> or tumor necrosis factor <em>alpha</em> to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin <em>3</em>, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

OBJECTIVE

To determine Th1 and Th2 cytokine production in patients with reactive arthritis (ReA) in relation to disease outcome and in comparison with rheumatoid arthritis (RA).

METHODS

Secretion of tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>), <em>interferon</em>-gamma, interleukin-10 (IL-10), and IL-4 by peripheral blood mononuclear cells (PBMC) from 5<em>3</em> patients with early ReA (disease duration <8 weeks, 64% HLA-B27 positive) and <em>3</em>0 patients with early, untreated RA (disease duration <6 months) was determined by enzyme-linked immunosorbent assay (ELISA) after ex vivo stimulation. Intracellular cytokine staining with quantification of positive T cells by fluorescence-activated cell sorting (FACS) was performed in 12 ReA patients and 12 RA patients. In 27 ReA patients, cytokine secretion was measured again after <em>3</em> months. Patients were followed up for 1 year, and cytokine patterns were correlated with disease duration.

RESULTS

TNF<em>alpha</em> secreted by whole PBMC and by T cells was significantly lower, by ELISA and by FACS, in ReA patients than in RA patients, while no significant differences were detected for the other cytokines. ReA patients with a disease duration of>> or =6 months showed significantly lower TNF<em>alpha</em> secretion than patients with a disease duration of <6 months (mean +/- SD <em>3</em>85 +/- 207 pg/ml versus 684 +/- 277 pg/ml; P = 0.00<em>3</em>). Furthermore, low TNF<em>alpha</em> secretion after <em>3</em> months also correlated significantly with a more chronic course of disease. HLA-B27 positive patients secreted less TNF<em>alpha</em> than did those who were B27 negative (<em>3</em><em>3</em>8 +/- 214 pg/ml versus 512 +/- 207 pg/ml; P = 0.05), and patients with a more chronic course had a higher frequency of B27 positivity (47% versus 80%; P = 0.01). Among the 27 HLA-B27 positive patients, TNF<em>alpha</em> secretion in those with a disease duration of>> or = 6 months was lower than that in the 7 with a disease duration of <6 months (<em>3</em>08 +/- 167 pg/ml versus 562 +/- <em>3</em>08 pg/ml; P = 0.04).

CONCLUSIONS

Low TNFalpha secretion and HLA-B27 status correlate with longer disease duration in ReA patients, possibly with an additive effect. The diminished TNFalpha production might reflect a state of relative immunodeficiency contributing to bacterial persistence in ReA.

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via <em>interferon</em> (IFN) regulatory factor <em>3</em> (IRF-<em>3</em>)-dependent pathways. The absence of IRF-<em>3</em> would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-<em>3</em>(-/-) mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 4<em>3</em>4:772-777, 2005). In contrast, the present study demonstrated that IRF-<em>3</em>(-/-) mice are significantly more susceptible to HSV infection via the corneal and intracranial routes. Following corneal infection with 2 x 10(6) PFU of HSV-1 strain McKrae, 50% of wild-type mice survived, compared to 10% of IRF-<em>3</em>-deficient mice. Significantly increased viral replication and inflammatory cytokine production were observed in brain tissues of IRF-<em>3</em>(-/-) mice compared to control mice, with a concomitant deficit in production of both IFN-beta and IFN-<em>alpha</em>. These data demonstrate a critical role for IRF-<em>3</em> in control of central nervous system infection following HSV-1 challenge. Furthermore, this work underscores the necessity to evaluate multiple routes of infection and animal models in order to fully determine the role of host resistance factors in pathogenesis.

Mice with a fat-specific insulin receptor knock-out (FIRKO) have reduced adipose tissue mass, are protected against obesity, and have an extended life span. White adipose tissue of FIRKO mice is also characterized by a polarization into two major populations of adipocytes, one small (<50 microm) and one large (>100 microm), which differ with regard to basal triglyceride synthesis and lipolysis, as well as in the expression of fatty acid synthase, sterol regulatory element-binding protein 1c, and CCAAT/enhancer-binding protein <em>alpha</em> (C/EBP-<em>alpha</em>). Gene expression analysis using RNA isolated from large and small adipocytes of FIRKO and control (IR lox/lox) mice was performed on oligonucleotide microarrays. Of the 12,488 genes/expressed sequence tags represented, 111 genes were expressed differentially in the four populations of adipocytes at the p < 0.001 level. These alterations exhibited 10 defined patterns and occurred in response to two distinct regulatory effects. 6<em>3</em> genes were identified as changed in expression depending primarily upon adipocyte size, including C/EBP-<em>alpha</em>, C/EBP-delta, superoxide dismutase <em>3</em>, and the platelet-derived growth factor receptor. 48 genes were regulated primarily by impairment of insulin signaling, including transforming growth factor beta, <em>interferon</em> gamma, insulin-like growth factor I receptor, activating transcription factor <em>3</em>, aldehyde dehydrogenase 2, and protein kinase Cdelta. These data suggest an intrinsic heterogeneity of adipocytes with differences in gene expression related to adipocyte size and insulin signaling.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the <em>interferon</em> <em>alpha</em>/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor <em>interferon</em> <em>alpha</em> signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-<em>3</em>) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in <em>interferon</em> <em>alpha</em>/beta receptor signaling.

BACKGROUND

Hepatitis C virus (HCV) infection has been linked to an increased risk of insulin resistance and carotid atherosclerosis.

OBJECTIVE

To investigate the association between HCV infection and stroke, and the effect of interferon-based therapy (IBT) on stroke risk in chronic hepatitis C (CHC) patients.

METHODS

We conducted a retrospective cohort study that followed up 3113 subjects with a newly detected HCV infection and 12 452 age- and gender-matched subjects without HCV infection selected from a random sample of 10(6) beneficiaries from the Taiwan National Health Insurance Program up to 5 years. Use of IBT was defined as treatment with interferon alpha, pegylated interferon alpha-2a or pegylated interferon alpha-2b for at least 3 months. The hazard ratio (HR) for newly detected stroke was calculated for subjects with HCV compared to those without HCV, and for IBT-treated HCV patients compared to non-IBT-treated HCV patients while adjusting for possible confounding factors.

RESULTS

The overall person-years of follow-up were 8624.11 in patients with HCV, 54,533.69 in patients without HCV, 666.65 in IBT-treated patients, and 7886.49 in nontreated patients. The multivariable-adjusted hazard ratio (HR) for newly detected stroke was 1.23 for subjects with HCV compared to the age- and sex-matched subjects without HCV (adjusted HR = 1.23, 95% CI = 1.06-1.42, P = 0.008). Moreover, use of IBT significantly reduced the risk of stroke in HCV patients (adjusted HR = 0.39, 95% CI = 0.16-0.95, P = 0.039) after adjusting for known prognostic factors.

CONCLUSIONS

Interferon-based therapy may reduce the long-term risk of stroke in patients with chronic HCV infection.

Respiratory syncytial virus (RSV) belongs to Paramyxoviridae family of enveloped negative-strand RNA viruses and causes severe bronchiolitis and pneumonia in children younger than 2 years of age. As members of Paramyxoviridae family, RSV and parainfluenza type <em>3</em> (PIV<em>3</em>) have similar modes of infection and replication. A variety of negative-strand RNA virus infections, including that of PIV<em>3</em>, are inhibited by human MxA protein, a type I <em>interferon</em> (IFN)-inducible GTPase. We tested whether the MxA protein, induced either by type I human IFNs or by stable transfection of human MxA gene in human (U-87) or simian (Vero) cells, confers resistance to these cells against infection by RSV strain A2. RSV infection was resistant to antiviral effects induced by 0-10,000 U/ml type I IFNs (IFN-<em>alpha</em> or -beta) in both human lung epithelial, A549, and fibroblast, MRC-5 cells. RSV virus yield was reduced only by 10- to 20-fold, and viral protein synthesis was not significantly affected under conditions of IFN treatment where PIV<em>3</em> yield was reduced by 1000- to 10,000-fold. Human or simian cell lines constitutively expressing MxA were protected against infection by PIV<em>3</em> but not by RSV. Our results indicate that RSV A2 is resistant to the antiviral effects of MxA, even though RSV and PIV<em>3</em> have similar replication strategies. In IFN-treated coinfected cultures, IFN-resistant RSV A2 did not prevent the IFN-mediated inhibition of PIV<em>3</em> multiplication. Hence the resistance of RSV A2 to type I IFNs does not appear to be due to soluble factors released into the medium or a disruption in the cellular antiviral machinery brought about by RSV A2 infection.

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta <em>interferon</em> (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-<em>3</em> and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-<em>3</em> phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor <em>alpha</em>-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.

OBJECTIVE

To investigate whether histone-specific T helper (Th) cells that are able to induce anti-double-stranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones.

METHODS

Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA:

RESULTS

Numerous histone-specific T cell receptor (TCR) <em>alpha</em>/beta+ Th clones were established from 2 of <em>3</em> patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, <em>interferon</em>-gamma (IFNgamma), and IL-4, whereas some produced predominantly IL-2 and IFNgamma. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual.

CONCLUSIONS

Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.

Duchenne muscular dystrophy (DMD), a lethal disorder characterized by dystrophin absence, courses with chronic inflammation, sarcolemmal damage, and skeletal muscle degeneration. Among the multiple pathogenic mechanisms proposed for DMD, oxidative stress and inflammation are directly involved in the dystrophic process. Unfortunately, there is no current treatment for DMD, and the inflammatory process is an important target for therapies. Based on the antioxidant and anti-inflammatory properties of melatonin, we investigated whether melatonin treatment may reduce the dystrophic process. Ten DMD patients aged 12.8 +/- 0.98 yr, were treated with melatonin (60 mg at 21:00 hr plus 10 mg at 09:00 hr), and plasma levels of lipid peroxidation (LPO), nitrites (NO(x)), interleukin (IL)-1beta, IL-2, IL-6, tumor necrosis factor-<em>alpha</em>, <em>interferon</em>-gamma, and plasma markers of muscle injury, were determined at <em>3</em>, 6 and 9 months of treatment. Healthy age- and sex-matched subjects were used as controls. The results show a significant increase in LPO, NO(x), and cytokine levels in plasma of DMD patients compared with controls. Melatonin administration reduced these values to control levels at <em>3</em> months of treatment, decreasing further 9 months later. In parallel, melatonin also reduced plasma levels of creatine kinase (CK; 50%), lactate dehydrogenase (28%), aspartate aminotransferase (28%), alanine aminotransferase (20%), and myoglobin (1<em>3</em>%). These findings strongly support the conclusion that melatonin administration significantly reduced the hyperoxidative and inflammatory process in DMD patients, reducing the muscle degenerative process.

Major impediments to a Chlamydia vaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model against Chlamydia infection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for a Chlamydia vaccine, we evaluated 10 new Chlamydia T cell antigens discovered via immunoproteomics in addition to the <em>3</em> antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6'-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720-CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4(+) T cells coexpressing gamma <em>interferon</em> (IFN-γ) and tumor necrosis factor <em>alpha</em> (TNF-α), and reduced infection by more than <em>3</em> logs. Using DDA-MPL as an adjuvant, we found that 7 of 1<em>3</em> Chlamydia T cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly defined Chlamydia vaccine.

Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas <em>interferon</em> (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-<em>alpha</em> stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-<em>alpha</em> to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-<em>alpha</em> to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-<em>3</em> and MCP-4 but not eotaxin-2. However, no ECA like MCP-<em>3</em> or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.

Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), <em>interferon</em>-gamma (IFNgamma) and tumour necrosis factor <em>alpha</em> (TNF<em>alpha</em>) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNF<em>alpha</em> neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -<em>3</em>. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.

The purpose of this study was to determine the relationships among delayed-onset muscle soreness (DOMS), muscle damage and inflammatory responses to eccentric exercise and investigate the underlying mechanisms. Nine healthy males performed one-leg calf-raise exercise with their right leg on a force plate. They performed 10 sets of 40 repetitions of exercise at 0.5 Hz by the load corresponding to the half of their body weight, with a rest for <em>3</em> min between sets. DOMS was evaluated by a visual analogue scale (VAS). Blood and urine samples were collected before and 2, 4, 24, 48, 72 and 96 h post-exercise. Blood samples were analyzed for leucocyte differential counts and neutrophil functions (migratory activity and oxidative burst activity). We also determined a serum marker of muscle damage, myoglobin (Mb), and plasma and urinary prostaglandin E2 as an algesic substance. As for the inflammatory mediators, plasma and urine were analyzed for cytokines (interleukin (IL)-1beta, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, tumour necrosis factor-<em>alpha</em>, <em>interferon</em>-gamma, monocyte chemotactic protein-1, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and granulocyte macrophage colony-stimulating factor), leucocyte activation markers (calprotectin and myeloperoxidase), and neutrophil chemotactic factor complement 5a. All subjects reported muscle soreness on subsequent days and VAS peaked at 72 h after exercise. Serum Mb concentration significantly increased (p < 0.05) at 72 h after exercise as compared with the pre-exercise values which was correlated with the increases in VAS at 72 h (r = 0.7<em>3</em>, p < 0.05). Circulating neutrophil count and migratory activity increased significantly (p < 0.01, and p < 0.05, respectively) at 4 h after exercise, whereas there were no significant changes in the other plasma and urinary inflammatory mediators. These results suggest that neutrophils can be mobilized into the circulation and migrate to the muscle tissue several hours after the eccentric exercise. There were also positive correlations between the exercise-induced increases in neutrophil migratory activity at 4 h and the increases in Mb at 48 h (r = 0.67, p < 0.05). These findings suggest that neutrophil mobilization and migration after exercise may be involved in the muscle damage and inflammatory processes.

BACKGROUND

Interferon beta is 1 of 2 first-line treatments for relapsing-remitting multiple sclerosis (MS). However, not all patients respond to interferon beta therapy, and to date there is a lack of surrogate markers that reliably correlate with responsiveness to interferon beta therapy in MS.

OBJECTIVE

To identify allelic variants that influence response to interferon beta therapy in patients with MS.

METHODS

Genome-wide scan.

METHODS

Academic research. Patients Two hundred patients having relapsing-remitting MS treated with interferon beta and having a follow-up period of at least 2 years were classified as responders or nonresponders to treatment based on stringent clinical criteria.

METHODS

In the first phase of the study, a pooling-based genome-wide association study of 428 867 single-nucleotide polymorphisms (SNPs) was performed in 53 responders and 53 nonresponders to interferon beta therapy. After applying several selection criteria, 383 SNPs were individually genotyped in an independent validation cohort of 49 responders and 45 nonresponders to interferon beta therapy using a different genotyping platform.

RESULTS

Eighteen SNPs had uncorrected P < .05 associated with interferon beta responder status in the validation cohort. Of these, 7 SNPs were located in genes that code for alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-type glutamate receptor GRIA3, type 1 interferon-related proteins ADAR and IFNAR2, cell cycle-dependent protein CIT, zinc finger proteins ZFAT and ZFHX4, and guanosine triphosphatase-activating protein STARD13.

CONCLUSIONS

This study supports an underlying polygenic response to interferon beta treatment in MS and highlights the importance of the glutamatergic system in patient response to interferon beta therapy.

Tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-<em>alpha</em> is dependent on the mitogen-activated protein kinase p<em>3</em>8. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(<em>3</em>-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-<em>3</em>-butyn-1-ol) inhibited the release of TNF-<em>alpha</em> by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of <em>3</em> nM, as well as the release of TNF-<em>alpha</em> from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 1<em>3</em> nM. This compound was approximately 10-fold more potent than the literature standard p<em>3</em>8 kinase inhibitor SB 20<em>3</em>580 in all p<em>3</em>8 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p<em>3</em>8<em>alpha</em> and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 20<em>3</em>580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or <em>interferon</em>-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-<em>alpha</em> production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.

Carcinoma in situ is a high-grade and aggressive manifestation of transitional-cell carcinoma of the bladder that has a highly variable course. The treatment of CIS has undergone dramatic changes since this malignancy was first recognized. While cystectomy was once recommended as the initial treatment of choice, recognition of the highly variable prognosis and the uniformly high response rate to intravesical BCG has prompted a more conservative approach to management. While it is recommended that patients be offered the option of radical cystectomy, data do not currently exist to confirm that cystectomy provides a superior survival or quality of life compared with an initial trial of BCG immunotherapy followed by salvage cystectomy if needed. With current optimal BCG immunotherapy regimens, which consist of a 6-week course of BCG followed by three weekly instillations at <em>3</em> months, 6 months, and every 6 months for <em>3</em> years, the complete response rate is 82%; and it is estimated that more than 75% of patients having a complete response will remain continuously disease free for 5 or more years. Patients who fail BCG immunotherapy without evidence of progression may yet be candidates for intravesical chemotherapy, photodynamic therapy, or alternative immunotherapies such as <em>alpha</em>-2b <em>interferon</em>, bropirimine, or keyhole limpet hemocyanin.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV) strain. Analyses of T cell repertoires in health care workers who survived SARS-CoV infection during the 200<em>3</em> outbreak revealed that their effector memory V gamma 9V delta 2 T cell populations were selectively expanded ~<em>3</em> months after the onset of disease. No such expansion of their <em>alpha</em> beta T cell pools was detected. The expansion of the V gamma 9V delta 2 T cell population was associated with higher anti-SARS-CoV immunoglobulin G titers. In addition, in vitro experiments demonstrated that stimulated V gamma 9V delta 2 T cells display an <em>interferon</em>- gamma -dependent anti-SARS-CoV activity and are able to directly kill SARS-CoV-infected target cells. These findings are compatible with the possibility that V gamma 9V delta 2 T cells play a protective role during SARS.

BACKGROUND

Epigallocatechin-<em>3</em>-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.

METHODS

Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG1<em>3</em>2) and MAPKs (p<em>3</em>8-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).

RESULTS

Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-<em>3</em>, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-<em>3</em>alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.

CONCLUSIONS

Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.

Strong reactivity for <em>interferon</em>-inducible protein 10 (IP-10), monokine induced by <em>interferon</em> gamma (Mig), and <em>interferon</em>-inducible T-cell <em>alpha</em> chemoattractant (I-TAC) was found in epithelial cells mainly localized to the medulla of postnatal human thymus. The CXC chemokine receptor common to the <em>3</em> chemokines (CXCR<em>3</em>) was also preferentially expressed in medullary areas of the same thymuses and appeared to be a property of 4 distinct populations: CD<em>3</em>+ T-cell receptor (TCR) <em>alpha</em>beta+ CD8+ single-positive (SP) T cells, TCRgammadelta+ T cells, natural killer (NK)-type cells, and a small subset of CD<em>3</em>+(low) CD4+ CD8+ TCR<em>alpha</em>beta+ double-positive (DP) T cells. IP-10, Mig, and I-TAC showed chemoattractant activity for TCR<em>alpha</em>beta+ CD8+ SP T cells, TCRgammadelta+ T cells, and NK-type cells, suggesting their role in the migration of different subsets of mature thymocytes during human thymus lymphopoiesis.

Lactobacillus pentosus strain S-PT84 isolated from Kyoto pickles enhances splenic natural killer (NK) cell activity and exhibit anti-allergic effects by modulating the Th1/Th2 (T-helper1/T-helper2) balance. In the present study, we investigated whether the immune response could be activated by intranasal administration of S-PT84 in the respiratory immune system and protected against influenza virus infection in mice. When BALB/c mice received intranasal administration of S-PT84 once daily for <em>3</em> consecutive days, S-PT84 strongly induced interleukin-12 (IL-12) and gamma <em>interferon</em> (IFN-gamma) production in mediastinal lymph node (MLN) cells. At intranasal infection with influenza virus PR8 (a mouse-adapted H1N1 strain) after S-PT84 treatment, the survival rates of mice improved in a dose-dependent manner, and the titer of influenza virus in bronchoalveolar lavage fluids (BALF) was significantly decreased by S-PT84 administration. Production of IL-12 and <em>alpha</em>-<em>interferon</em> (IFN-<em>alpha</em>) in BALF were significantly higher in mice treated with S-PT84 compared to the control mice. Lung NK activity was also significantly augmented in S-PT84-treated mice. These results suggested that the L. pentosus strain S-PT84 showed inhibitory activity against influenza virus infection.