OBJECTIVE

This work studied the presence of inflammatory and atherogenic lipoprotein markers that could explain the high incidence of cardiovascular disease (CVD) reported in rheumatoid arthritis (RA) patients.

METHODS

Inflammatory markers were 1) soluble adhesion molecules (intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]), 2) C-reactive protein (CRP), <em>3</em>) fibrinogen (Fb), 4) cytokines (<em>interferon</em>-gamma [IFNgamma], tumor necrosis factor <em>alpha</em> [TNF<em>alpha</em>]), and 5) secretory group IIA phospholipase A2 (sPLA2-IIA). Atherogenic lipoprotein markers were 1) the size distribution of plasma lipoprotein subclasses, and 2) the binding affinity of low-density lipoprotein (LDL) to chondroitin 6-sulfate glycosaminoglycan (GAG).

RESULTS

RA patients (n = <em>3</em>1) and matched controls (n = 28) had similar plasma concentrations of total cholesterol, triglycerides, Apo B, Apo A-I, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein (HDL). RA patients had significantly higher plasma levels of sPLA2-IIA, ICAM, CRP, Fb, TNF<em>alpha</em>, and IFNgamma compared with controls. RA patients also had significantly higher levels of small, dense LDL-1 (P < 0.05) and lower levels of small HDL-2 particles (P < 0.001) compared with controls. In addition, LDL from RA patients had a significantly higher binding affinity (Kd) to GAG (mean +/- SD Kd 204+/-22.4 nM Apo B) than did LDL from control subjects (Kd <em>3</em>12+/-<em>3</em>6 nM Apo B) (P < 0.05). This Kd value showed a significant negative correlation with the plasma levels of LDL-1 (r = -0.566, P < or = 0.004). In RA patients, a significant positive correlation was obtained between sPLA2-IIA and CRP, ICAM, and LDL-1. HDL-2 showed a negative correlation with sPLA2-IIA.

CONCLUSIONS

These atherogenic lipoprotein factors combined with the presence of chronic inflammation may contribute to the high CVD-related mortality in RA patients.

Many homosexual men with the acquired immunodeficiency syndrome (AIDS) have an unusual acid-labile form of human leukocyte, or <em>alpha</em>, <em>interferon</em> in their serum. Male patients with classic hemophilia treated with lyophilized clotting-factor concentrates are also at high risk for the development of AIDS. To determine whether the level of <em>alpha</em> <em>interferon</em> may be a preclinical marker of early subclinical disease, we examined stored plasma and serum from three hemophilic patients with AIDS. Persistently elevated levels of the acid-labile form of <em>alpha</em> <em>interferon</em> were found in all three patients. In two patients the appearance of circulating <em>alpha</em> <em>interferon</em> preceded the onset of clinical disease by <em>3</em> to 10 months. In contrast, <em>alpha</em>-<em>interferon</em> levels were not elevated in 4<em>3</em> of 46 unselected patients with hemophilia; three of these patients had transient elevations. These results suggest that acid-labile <em>alpha</em> <em>interferon</em> may be a marker that can be used to identify affected asymptomatic members of high-risk groups before the onset of clinical disease.

The secreted cytokine <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) binds its receptor to activate the Jak-STAT signal transduction pathway, leading to formation of the heterotrimeric IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>) transcription complex for induction of IFN-stimulated genes (ISGs) and establishment of an antiviral state. Many viruses have evolved countermeasures to inhibit the IFN pathway, thereby subverting the innate antiviral response. Here, we demonstrate that the mildly myocarditic reovirus type 1 Lang (T1L), but not the nonmyocarditic reovirus type <em>3</em> Dearing, represses IFN induction of a subset of ISGs and that this repressor function segregates with the T1L M1 gene. Concordantly, the T1L M1 gene product, mu2, dramatically inhibits IFN-beta-induced reporter gene expression. Surprisingly, T1L infection does not degrade components of the ISGF<em>3</em> complex or interfere with STAT1 or STAT2 nuclear translocation as has been observed for other viruses. Instead, infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of <em>interferon</em> regulatory factor 9 (IRF9) in the nucleus. This effect has not been previously described for any virus and suggests that mu2 modulates IRF9 interactions with STATs for both ISGF<em>3</em> function and nuclear export. The M1 gene is a determinant of virus strain-specific differences in the IFN response, which are linked to virus strain-specific differences in induction of murine myocarditis. We find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response and couple virus strain-specific differences in IFN antagonism to the pathogenesis of viral myocarditis.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by <em>interferon</em> and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 <em>alpha</em> and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the <em>3</em>' untranslated region (<em>3</em>'UTR) of human <em>alpha</em>-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (199<em>3</em>) Cell 75, 1107-1117]. Here we report that purified RNA from the <em>3</em>'UTR of human <em>alpha</em>-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin <em>3</em>'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the <em>3</em>'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin <em>3</em>'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin <em>3</em>'UTR RNA exerts its tumor-suppression activity in vivo.

In vitro infection of freshly isolated human peripheral blood mononuclear cells (HPBMC) with Chlamydia pneumoniae was found to induce a production of tumour necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>). The secretion was dependent on the amount of infecting chlamydiae and most of it occurred during the first 12 to 24 h. Lipopolysaccharide (LPS) of Salmonella minnesota Rechemotype, used as a positive control for HPBMC activation, induced a release of TNF-<em>alpha</em>, IL-1 beta and IL-6, but not of IFN-<em>alpha</em>, similar to the effect of C. pneumoniae. Viable chlamydiae could not be recovered from HPBMCs infected immediately after their isolation, whereas HPBMCs which were cultured in vitro for <em>3</em> to 9 days before infection were able to maintain the growth of C. pneumoniae. Growth inside HPBMCs as well as induction of cytokine response may have a role in the pathogenesis of C. pneumoniae infection.

Natural <em>interferon</em>-producing cells (NIPC), also referred to as immature plasmacytoid dendritic cells (PDC), constitute a small population of leucocytes secreting high levels of type I <em>interferons</em> in response to certain danger signals. Amongst these signals are those from DNA containing unmethylated CpG motifs. The present work demonstrated that the CpG oligonucleotides (CpG-ODN) 2216, D<em>3</em>2 and D19 induce high amounts of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), tumour-necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and interleukin (IL)-12 in porcine peripheral blood mononuclear cells (PBMCs). Swine workshop cluster <em>3</em> (SWC<em>3</em>)1ow CD4high cells, with high IL-<em>3</em>-binding activity, representing NIPC, were the exclusive cytokine-producing cells responding to the CpG-ODN. These cells did not express CD6, CD8 or CD45RA. Importantly, monocyte-derived DC did not respond to CpG-ODN by secretion of IFN-<em>alpha</em> or TNF-<em>alpha</em> or by the up-regulation of costimulatory molecule expression. CpG-ODN up-regulated MHC class II and CD80\86 expression on the NIPC, but were unable to promote NIPC survival. Interestingly, certain CpG-ODN, incapable of inducing NIPC to secrete IFN-<em>alpha</em> or up-regulate MHC class II and CD80\86, did promote NIPC viability. Taken together, the influence of CpG-ODN on porcine NIPC, monocytes and myeloid DCs relates to that observed with their human equivalents. These results represent an important basis for the application of CpG-ODN as adjuvants for the formulation of novel vaccines and demonstrate the importance of the pig as an alternative animal model for this approach.

OKT<em>3</em> monoclonal antibody, a human T cell mitogen, induced <em>interferon</em> production by cultured mononuclear cells at 10(-11) M concentrations. <em>Interferon</em> was secreted only under conditions wherein OKT<em>3</em> was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, <em>interferon</em> secretion reached a peak <em>3</em> days after OKT<em>3</em> stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT<em>3</em> for induction. The <em>interferon</em> was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-<em>alpha</em> or -beta <em>interferon</em> antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT<em>3</em> stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of <em>interferon</em>. Studies with selective pretreatments with mitomycin C suggested that gamma-<em>interferon</em> was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.

We measured de novo lipogenesis in human immunodeficiency virus (HIV) infected men using a newly developed stable isotope method. HIV-infected subjects with a history of weight loss (n = 17, mean weight loss 14.9 +/- <em>3</em>.2 kg), asymptomatic HIV-seropositive subjects with normal CD4 T-cell counts (n = 7) and healthy HIV seronegative controls (n = 11) were studied. Hepatic lipogenesis was determined by infusion of [2-1<em>3</em>C]-acetate, using the recently described xenobiotic probe technique with mass isotopomer analysis. Hepatic acetyl-coenzyme A enrichment was measured by high performance liquid chromatography/mass spectrometry of secreted sulfamethoxazole-acetate, with measurement of incorporation into very low density lipoprotein-fatty acids by gas chromatography-mass spectrometry. Circulating tumor necrosis factor (TNF), interleukin-1 (IL-1), <em>interferon</em> <em>alpha</em> (IFN <em>alpha</em>), insulin, and triglycerides were measured concurrently, and 7-day weighed food records were performed. De novo hepatic lipogenesis was increased <em>3</em>- to 4-fold in HIV-infected subjects with weight loss compared to normal controls (P < 0.05 for palmitate and stearate in both overnight-fasted and fed states), and was also significantly increased in asymptomatic HIV seropositive subjects. Circulating TNF and IL-1 were not measurable in any subject (detection limit 2 pg/ml for IL-1 and 20 pg/ml for TNF). Serum IFN <em>alpha</em> was measurable in 11 out of 17 subjects with wasting and correlated significantly with de novo lipogenesis in overnight-fasted but not fed states. Serum IFN <em>alpha</em> was unmeasurable in asymptomatic HIV-infected subjects despite elevated lipogenic rates. Serum triglyceride concentrations were elevated in subjects with weight loss (2.09 +/- 0.28 mmol/L) and asymptomatic HIV-positives (1.<em>3</em>4 +/- 0.<em>3</em>4 mmol/L) in comparison to controls (0.67 +/- 0.08 mmol/L), and correlated with lipogenesis. Food intake correlated inversely with lipogenesis in the overnight-fasted state. We conclude that HIV infection is characterized by abnormal fat anabolism. This applies to subjects with reduced lean body mass and to asymptomatic HIV-positive subjects with normal T-cell counts. The former observation may have implications for the pathophysiology and treatment of the wasting syndrome. The latter observation is consistent with activation of the immune response and a state of viral nonlatency in early HIV disease.

The mechanism by which respiratory syncytial virus (RSV) suppresses T-cell proliferation to itself and other antigens is poorly understood. We used monocyte-derived dendritic cells (MDDC) and CD4 T cells and measured [(<em>3</em>)H]thymidine incorporation to determine the factors responsible for RSV-induced T-cell suppression. These two cell types were sufficient for RSV-induced suppression of T-cell proliferation in response to cytomegalovirus or Staphylococcus enterotoxin B. Suppressive activity was transferable with supernatants from RSV-infected MDDC and was not due to transfer of live virus or RSV F (fusion) protein. Supernatants from RSV-infected MDDC, but not MDDC exposed to UV-killed RSV or mock conditions, contained <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>; median, 4<em>3</em> pg/ml) and IFN-lambda (approximately 1 to 20 ng/ml). Neutralization of IFN-<em>alpha</em> with monoclonal antibody (MAb) against one of its receptor chains, IFNAR2, or of IFN-lambda with MAb against either of its receptor chains, IFN-lambdaR1 (interleukin 28R [IL-28R]) or IL-10R2, had a modest effect. In contrast, blocking the two receptors together markedly reduced or completely blocked the RSV-induced suppression of CD4 T-cell proliferation. Defining the mechanism of RSV-induced suppression may guide vaccine design and provide insight into previously uncharacterized human T-cell responses and activities of <em>interferons</em>.

OBJECTIVE

A relationship between chronic hepatitis C virus (HCV) infection and lipid metabolism has recently been suggested. The aim of this study was to determine the correlation between lipid profile and virology, histologic lesions, and response to alpha interferon therapy in noncirrhotic, nondiabetic patients with hepatitis C.

METHODS

A total of 109 consecutive untreated chronic hepatitis C patients were studied to assess the following: 1) the effects of HCV genotype, viral load, steatosis, hepatic fibrosis, and body mass index (BMI) on lipid profile; and 2) whether lipid parameters could predict response to antiviral therapy.

RESULTS

The control group showed a significantly higher apolipoprotein B (apoB) concentration compared with patients with chronic hepatitis C. Hypobetalipoproteinemia (apo B <0.7 g/L) was found in 27 (24.7%) chronic HCV patients and in five (5.3%) control subjects (p = 0.0002). Levels of apo B were negatively correlated with steatosis and HCV viral load (r = -0.22; p = 0.03). This last correlation was strong for non-1 genotype and genotype 3 (r = -0.48; p = 0.0005, and r = -0.47; p = 0.007, respectively) but was not found in genotype 1. In multivariate analysis, low apo B concentration was significantly associated with fibrosis grade 2 or 3 versus grade 0 or 1 (p < 0.001), steatosis >5% (p < 0.001), low body mass index (p < 0.001), and high HCV viral load (p < 0.014). No correlation was found in the 76 treated patients between apo B and response to interferon therapy.

CONCLUSIONS

In chronic HCV patients, hypobetalipoproteinemia occurs already in the early stages of HCV infection before the development of liver cirrhosis. The correlation between apo B levels and HCV viral load seems to confirm the interaction between hepatitis C infection and beta-lipoprotein metabolism.

<em>Alpha</em>/beta <em>interferons</em> (IFN-<em>alpha</em>/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-<em>alpha</em>/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-<em>alpha</em>/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-<em>alpha</em>/beta in mDCs while fusion-defective virus did not induce IFN-<em>alpha</em>/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor <em>3</em>. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-<em>alpha</em>/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-<em>alpha</em>/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

There have been several descriptions of mouse models that manifest select immunological and clinical features of autoimmune cholangitis with similarities to primary biliary cirrhosis in humans. Some of these models require immunization with complete Freund's adjuvant, whereas others suggest that a decreased frequency of T regulatory cells (Tregs) facilitates spontaneous disease. We hypothesized that antimitochondrial antibodies (AMAs) and development of autoimmune cholangitis would be found in mice genetically deficient in components essential for the development and homeostasis of forkhead box <em>3</em> (Foxp<em>3</em>)(+) Tregs. Therefore, we examined Scurfy (Sf) mice, animals that have a mutation in the gene encoding the Foxp<em>3</em> transcription factor that results in a complete abolition of Foxp<em>3</em>(+) Tregs. At <em>3</em> to 4 weeks of age, 100% of animals exhibit high-titer serum AMA of all isotypes. Furthermore, mice have moderate to severe lymphocytic infiltrates surrounding portal areas with evidence of biliary duct damage, and dramatic elevation of cytokines in serum and messenger RNAs encoding cytokines in liver tissue, including tumor necrosis factor <em>alpha</em>, <em>interferon</em>-gamma, interleukin (IL)-6, IL-12, and IL-2<em>3</em>.

CONCLUSIONS

The lack of functional Foxp<em>3</em> is a major predisposing feature for loss of tolerance that leads to autoimmune cholangitis. These findings reflect on the importance of regulatory T cells in other murine models as well as in patients with primary biliary cirrhosis.

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX(<em>3</em>)C subclasses. Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-<em>3</em>-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 micro g/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to approximately 1<em>3</em>% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting approximately 49% of the approximately 6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1 <em>alpha</em> and -1 beta, which are important in RSV-induced pulmonary pathology, and <em>interferon</em> (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-beta expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1 <em>alpha</em>, STAT-1 beta, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.

OBJECTIVE

Human tears contain hundreds of proteins that may exert a significant influence on tear film stability, ocular surface integrity, and visual function. The authors hypothesise that many of these proteins originate from the meibomian gland. This study's aim was to begin to develop the proteomic methodology to permit the testing of their hypothesis.

METHODS

Meibomian gland secretions were collected from the lower eyelids of adult volunteers and placed in a chloroform-methanol mixture. Samples were partitioned in a biphasic system and non-lipid phase materials were reduced, alkylated, and trypsin digested to obtain peptides for protein identification. This peptide mixture was separated by micro-capillary reverse phase chromatography and the effluent examined by nano-electrospray MS and data dependent MS/MS. SEQUEST software was used to identify proteins from the MS/MS spectra.

RESULTS

The methodological approach to date has permitted the identification of more than 90 proteins in human meibomian gland secretions. Proteins include the <em>alpha</em>2-macroglobulin receptor, IgA <em>alpha</em> chain, farnesoid X activated receptor, <em>interferon</em> regulatory factor <em>3</em>, lacritin precursor, lactotransferrin, lipocalin 1, lysozyme C precursor, potential phospholipid transporting ATPase IK, seven transmembrane helix receptor (also termed somatostatin receptor type 4), testes development related NYD-SP21 (also termed high affinity IgE receptor beta subunit), and TrkC tyrosine kinase.

CONCLUSIONS

These findings indicate that the meibomian gland secretes a number of proteins into the tear film. It is quite possible that these proteins contribute to the dynamics of the tear film in both health and disease.

Macrophages have a central role in innate-immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram-positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL<em>3</em>/macrophage-inflammatory protein-1<em>alpha</em> (MIP-1<em>alpha</em>), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP-<em>3</em>, CCL19/MIP-<em>3</em>beta, and CCL20/MIP-<em>3</em><em>alpha</em> and CXC chemokine ligands CXCL8/interleukin (IL)-8, CXCL9/monokine induced by <em>interferon</em>-gamma (IFN-gamma), and CXCL10/IFN-inducible protein 10. Bacteria-induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria-induced IFN-<em>alpha</em>/beta production. CCL19 and CCL20 mRNA expression was up-regulated by IL-1beta or tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), and in addition, IFN-<em>alpha</em> together with TNF-<em>alpha</em> further enhanced CCL19 gene expression. Synergy between IFN-<em>alpha</em> and TNF-<em>alpha</em> was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria-stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus-induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C<em>3</em>H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and interleukin-12 compared to C<em>3</em>H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C<em>3</em>H/HeJ, C<em>3</em>H/HePas, TLR2 knockout, and wild-type mice at 1, <em>3</em>, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks <em>3</em> and 6 after infection in animals that lacked functional TLR4 (C<em>3</em>H/HeJ) compared to C<em>3</em>H/HePas that paralleled the reduced gamma <em>interferon</em> production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Transcription factors of the nuclear factor of activated T cells (NFAT) family are thought to regulate the expression of a variety of inducible genes such as interleukin-2 (IL-2), IL-4, and tumor necrosis factor-<em>alpha</em>. However, it remains unresolved whether NFAT proteins play a role in regulating transcription of the <em>interferon</em>- gamma (IFN-gamma) gene. Here it is shown that the transcription factor NFAT1 (NFATc2) is a major regulator of IFN-gamma production in vivo. Compared with T cells expressing NFAT1, T cells lacking NFAT1 display a substantial IL-4-independent defect in expression of IFN-gamma mRNA and protein. Reduced IFN-gamma production by NFAT1(-/-)x IL-4(-/-) T cells is observed after primary in vitro stimulation of naive CD4+ T cells, is conserved through at least 2 rounds of T-helper cell differentiation, and occurs by a cell-intrinsic mechanism that does not depend on overexpression of the Th2-specific factors GATA-<em>3</em> and c-Maf. Concomitantly, NFAT1(-/-)x IL-4(-/-) mice show increased susceptibility to infection with the intracellular parasite Leishmania major. Moreover, IFN-gamma production in a murine T-cell clone is sensitive to the selective peptide inhibitor of NFAT, VIVIT. These results suggest that IFN-gamma production by T cells is regulated by NFAT1, most likely at the level of gene transcription.

To understand the effects of cytokines on epithelial cells in asthma, we have investigated the effects of interleukin (IL)-4, IL-1<em>3</em>, and <em>interferon</em> (IFN)-gamma on barrier function and wound healing in Calu-<em>3</em> human lung epithelial cells. IL-4 and IL-1<em>3</em> treatment of Calu-<em>3</em> cells grown on Transwell filters resulted in a 70-75% decrease in barrier function as assessed by electrophysiological and [(14)C]mannitol flux measurements. In contrast, IFN-gamma enhanced barrier function threefold using these same parameters. Cells treated concurrently with IFN-gamma and IL-4 or IL-1<em>3</em> showed an initial decline in barrier function that was reversed within 2 days, resulting in barrier levels comparable to control cells. Analysis of the tight junction-associated proteins ZO-1 and occludin showed that IL-4 and IL-1<em>3</em> significantly reduced ZO-1 expression and modestly decreased occludin expression compared with controls. IFN-gamma, quite unexpectedly given its enhancing effect on barrier function, reduced expression of ZO-1 and occludin to almost undetectable levels compared with controls. In wound-healing assays of cells grown on collagen I, IL-4 and IL-1<em>3</em> decreased migration, whereas IFN-gamma treatment enhanced migration, compared with control cells. Addition of IFN-gamma, in combination with IL-4 or IL-1<em>3</em>, restored migration of cells to control levels. Migration differences observed between the various cytokine treatments was correlated with expression of the collagen I-binding <em>alpha</em>(2)beta(1)-integrin at the leading edge of cells at the wound front; <em>alpha</em>(2)beta(1)-integrin expression was decreased in IFN-gamma-treated cells compared with controls, whereas it was highest in IL-4- and IL-1<em>3</em>-treated cells. These results demonstrate that IL-4 and IL-1<em>3</em> diminish the capacity of Calu-<em>3</em> cells to maintain barrier function and repair wounds, whereas IFN-gamma promotes epithelial restitution by enhancing barrier function and wound healing.

Cyclophosphamide (CYP)-induced cystitis alters micturition function and produces reorganization of the micturition reflex. This reorganization may involve cytokine expression in the urinary bladder. These studies have determined candidate cytokines in the bladder that may contribute to the reorganization process. An RNase protection assay was used to measure changes in rat bladder cytokine mRNA [<em>interferon</em>-gamma (IFN)-gamma, interleukin-1<em>alpha</em>/beta (IL-1<em>alpha</em>/beta), IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6, IL-10, and tumor necrosis factor-<em>alpha</em>/beta (TNF-<em>alpha</em>/beta)] after acute (4 h), intermediate (48 h), or chronic (10 day) cystitis. The correlation between bladder cytokine mRNA and protein expression was also determined by immunoassay. Although at each time point after cystitis significant changes in bladder cytokine mRNA were observed, the magnitude differed (acute>> intermediate>> chronic). Acute cystitis demonstrated the most robust changes (P </= 0.005; IL-1beta, <em>3</em><em>3</em>0-fold increase; IL-2, 20-fold increase; IL-4, 8-fold increase; IL-6, 80-fold increase) in cytokine mRNA expression and TNF-<em>alpha</em> or TNF-beta mRNA were only increased (2-10-fold) after acute cystitis. More modest increases in cytokine mRNA expression were observed after 48-h or 10-day cystitis. Cytokine protein expression generally paralleled that of mRNA. Increased cytokine expression after CYP-induced cystitis, alone or in combination with other inflammatory mediators or growth factors, may contribute to altered lower urinary tract function after cystitis.

OBJECTIVE

To determine whether <em>interferon</em>-<em>alpha</em> (IFN<em>alpha</em>) reduces the severity of skin involvement in early ((<em>3</em> years) diffuse scleroderma.

METHODS

In a randomized, placebo-controlled, double-blind trial, <em>3</em>5 patients with early scleroderma received subcutaneous injections of either IFN<em>alpha</em> (1<em>3</em>.5 x 10(6) units per week in divided doses) or indistinguishable placebo. Outcomes assessed were the modified Rodnan skin score, as determined by a single observer at baseline, 6 months, and 12 months, as well as data on renal, cardiac, and lung function. Pre- and posttreatment skin biopsy samples were analyzed and blood was obtained for assessment of procollagen peptide levels.

RESULTS

There were 11 withdrawals from the IFN<em>alpha</em> group and <em>3</em> from the placebo group due to either toxicity, lack of efficacy, or death. In the intent-to-treat analysis, there was a greater improvement in the skin score in the placebo group between 0 and 12 months (mean change IFN<em>alpha</em> -4.7 versus placebo -7.5; P = 0.<em>3</em>6). There was also a greater deterioration in lung function in patients receiving active therapy, as assessed by either the forced vital capacity (mean change IFN<em>alpha</em> -8.2 versus placebo +1.<em>3</em>; P = 0.01) or the diffusing capacity for carbon monoxide (mean change IFN<em>alpha</em> -9.<em>3</em> versus placebo +4.7; P = 0.002). Skin biopsy showed no significant decrease in collagen synthesis in the IFN<em>alpha</em> group, and no significant differences in the levels of procollagen peptides were seen between the 2 groups.

CONCLUSIONS

This study suggests that IFNalpha is of no value in the treatment of scleroderma, and that it may in fact be deleterious.

OBJECTIVE

Cholestatic disorders often are associated with portal inflammation, but whether or how inflammation contributes to cholestasis is unknown. Thus we studied the effects of proinflammatory cytokines on bile duct epithelia secretory mechanisms.

METHODS

Isolated bile duct units (IBDUs) were cultured with interleukin (IL)-6, <em>interferon</em> gamma, tumor necrosis factor (TNF)-<em>alpha</em>, and IL-1 alone or in combination. Ductular secretion was measured using video-optical planimetry. Bicarbonate and Cl(-) transport were assessed microfluorimetric measuring pH(i) (BCECF) and [Cl(-)](i) transients (MEQ). Expression of Cl(-)/HCO(<em>3</em>)(-) exchanger (AE-2), cystic fibrosis transmembrane conductance regulator (CFTR), and the secretin receptor (SR) were assessed by ribonuclease protection assay. Cellular cyclic adenosine monophosphate (cAMP) levels were studied by enzymatic immunoassay. Paracellular permeability was assessed using fluorescein-labeled dextrans (FD) in cholangiocyte monolayers (NRC-1).

RESULTS

Although not effective when given alone, each combination of IL-6, interferon gamma, IL-1, and TNF-alpha inhibited secretion in IBDU. Cytokines inhibited cAMP formation, AE-2 activity, and cyclic AMP-dependent Cl(-) efflux, but not that induced by purinergic agonists. AE-2 gene expression was unaffected by proinflammatory cytokines, whereas CFTR and SR expression was increased. In addition, paracellular transit of FD across NRC-1 monolayers was increased.

CONCLUSIONS

Inflammatory cytokines inhibit cAMP-dependent fluid secretion in cholangiocytes and impair the barrier functions of biliary epithelia. These changes may represent the molecular mechanisms by which inflammation leads to ductular cholestasis in vivo.

OBJECTIVE

Severe uveitis is potentially associated with visual impairment or blindness in young patients. Therapeutic strategies remain controversial. The efficacy of <em>interferon</em> <em>alpha</em>-2a (IFN-<em>alpha</em>2a) in severe uveitis, refractory to steroids and conventional immunosuppressive agents, was evaluated.

METHODS

Patients were included after a major relapse of uveitis following corticosteroids and immunosuppressants. IFN-<em>alpha</em>2a (3 million units three times a week) was administered subcutaneously. Efficacy was assessed by improvement in visual acuity, decrease in vitreous haze, resolution of retinal vasculitis and macular oedema, assessed by fundus examination and fluorescein angiography, and decrease in oral prednisone threshold.

RESULTS

45 patients were included. Median age was 32.3 years (range 8-58) and sex ratio (F/M) was 0.66. Uveitis was associated with Behçet's disease in 23 cases (51.1%) and with other entities in 22 cases (48.9%). Median duration of uveitis before interferon therapy was 34.9 months (range 3.4-168.7) and an average of 3.26 relapses following corticosteroids and immunosuppressants was noted. Uveitis was controlled in 82.6% of patients with Behçet's disease and 59% of patients with other types of uveitis (p = 0.07). During a mean follow-up of 29.6 months (range 14-55), median oral prednisone threshold decreased significantly from 23.6 mg/day (range 16-45) to 10 mg/d (range 4-14) (p<0.001). Interferon was discontinued in 10 patients (22.2%) with Behçet's disease and in four patients without Behçet's disease. Relapses occurred in four and one cases, respectively.

CONCLUSIONS

Interferon therapy appears to be an efficient strategy in severe and relapsing forms of Behçet's disease but also in other uveitic entities. However, it seems to act more to suspend rather than cure the disease. Therefore, IFN-<em>alpha</em>2a may be proposed as a secondline strategy after failure of conventional immunosuppressants.

The treatment of chronic hepatitis C is currently based on a combination of pegylated <em>interferon</em> (IFN)-<em>alpha</em> and ribavirin. When successful, this treatment leads to sustained HCV clearance which, in virtually all cases, signifies viral eradication. However, approximately 20% of patients with hepatitis C virus (HCV) genotype 2 or <em>3</em> infection, and 50% of patients with genotype 1 infection, fail to eradicate the virus. The risk of treatment failure is related to multiple factors, including the treatment schedule, adherence of therapy, host factors, and the severity of HCV-associated disease. Viral factors can also lead to true "HCV resistance". The mechanisms underlying this resistance are unknown, but indirect evidence suggests that chronic infection is associated with phenomena that protect HCV from the antiviral action of IFN-<em>alpha</em> and hinder the clearance of infected cells. This article discusses current knowledge of the mechanisms of action of IFN-<em>alpha</em> and ribavirin, the virological characteristics of chronic hepatitis C treatment success and failure, and possible underlying mechanisms.

OBJECTIVE

To determine whether combined therapy with interferon-alpha and ribavirin was more effective and cost-effective than no treatment for patients with mild chronic hepatitis C.

METHODS

A multicentre, randomised, controlled, non-blinded trial assessed the efficacy of combination therapy. A Markov model used these efficacy data combined with data on transition probabilities, costs and health-related quality of life (HRQoL) to assess the lifetime cost-effectiveness of the intervention.

METHODS

A multicentre NHS setting.

METHODS

Treatment-naive, adult patients with histologically mild chronic hepatitis C (Ishak necroinflammatory scores <4 and fibrosis scores <3 on liver biopsy).

METHODS

Patients were randomised to receive interferon-alpha and ribavirin for 48 weeks or no treatment (control).

METHODS

The primary outcome measure was the proportion of patients having a sustained virological response (SVR), measured at 6 months after cessation of therapy. Secondary outcome measures were: the ability of early phase kinetics to predict the eventual outcome of treating mild disease; HRQoL measured using the Short Form 36 and EuroQol (5 Dimensions) questionnaires, and the cost per quality-adjusted life-year (QALY) of interferon-alpha and ribavirin for mild disease compared with no treatment.

RESULTS

In the treatment group, 32 out of 98 patients (33%) achieved an SVR. Patients infected with genotype 1 had a lower SVR than those infected with genotype non-1 (18% versus 49%, p = 0.02). No patients who failed to achieve a 2-log drop in viral load at 12 weeks achieved an SVR. HRQoL fell during treatment and rose with treatment cessation. For patients having an SVR there were modest improvements in HRQoL at 6 months post-treatment. The mean cost per QALY gained was 4535 pounds sterling for 40-year-old patients with genotype non-1 and 25,188 pounds sterling for patients with genotype 1. For patients with genotype 1 aged 65, providing interferon-alpha and ribavirin for mild disease led to fewer QALYs gained, and a mean cost per QALY of 53,017 pounds sterling. The model using efficacy estimates from the literature, showed that the cost per QALY gained from providing pegylated interferon alpha-2b and ribavirin at a mild stage rather than a moderate stage was 7821 pounds sterling for patients with genotype non-1 and 28,409 pounds sterling for patients with genotype 1.

CONCLUSIONS

Based on the evidence collected in this study, interferon-alpha and ribavirin treatment for mild chronic hepatitis C patients is in general cost-effective at the 30,000 pounds sterling per QALY threshold previously used by policy-makers in the NHS. For patients with chronic hepatitis C aged 65 or over with genotype 1, antiviral treatment at a mild stage does not appear cost-effective. Further research is required on the cost-effectiveness of pegylated interferon and ribavirin, in particular the intervention's long-term impact on HRQoL and health service costs requires further evaluation. Further research is also needed to develop predictive tests, based on pharmacogenomics, that can identify those cases most likely to respond to antiviral therapy. Liver biopsy before treatment no longer appears justified apart from for older patients (aged 65 or over) with genotype 1. However, further research should monitor the impact this strategy would have on costs and outcomes.