The recent observation made in our laboratory that cellular myc (c-myc) mRNA has a very short half-life in a variety of normal and transformed human cells emphasized the potential importance of post-transcriptional regulation of c-myc gene expression. Jonak and Knight [Jonak, G. J. & Knight, E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 1747-1750] have reported a selective reduction of c-myc mRNA accumulation in lymphoblastoid Daudi cells treated with human beta interferon. This provided a suitable situation in which to examine a possible action of negative modulators of c-myc expression at the level of mRNA stability. Our results confirm the observation by Jonak and Knight that c-myc mRNA level is depressed in cells treated with beta interferon and extend it to alpha 2 interferon. Furthermore, we now demonstrate that interferon has no effect on c-myc transcription rate in isolated nuclei but rather reduces the half-life of its mRNA. Conversely, we show that it increases the level of HLA-A2 mRNA by stimulating its transcription.

The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.

OBJECTIVE

We measured the serum concentrations of interleukin-6 (IL-6) in 70 patients with established septic shock caused predominantly by gram-negative bacteria. The aims of the study were to determine whether and for how long IL-6 was detectable in the circulation of these patients, to assess whether IL-6 levels were associated with patients' outcomes, and, finally, to examine the interplay between IL-6, tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma).

RESULTS

IL-6 was detected in 64% of the patients at study entry but in only 18% on Day 1 and 2% on Day 10. Serum levels of IL-6 were higher (median: 3.5 ng/mL, range: less than 0.1 to 305 ng/mL) in patients dying of fulminant septic shock than in those surviving (median: 0.5 ng/mL, range: less than 0.1 to 135 ng/mL; p = 0.003) or in those with a transient reversal of shock but who ultimately died of a relapse of shock (median: less than 0.1 ng/mL, range: less than 0.1 to 12.5 ng/mL; p = 0.005). However, no cutoff values of IL-6 confidently predicted the outcome of an individual patient. The serum concentrations of IL-6 measured at study entry correlated with the duration of survival (r = -0.51, p = 0.004) and with the levels of TNF-alpha (r = 0.53; p less than 0.0001) but not with the levels of either IL-1 beta (r = 0.01, p = 0.90) or IFN-gamma (r = 0.06, p = 0.60).

CONCLUSIONS

These results indicate that circulating levels of IL-6 are detectable in a majority of patients with gram-negative septic shock. Concentrations of IL-6 peaked near the onset of shock and rapidly decreased to undetectable levels within approximately 24 hours in most patients. Levels of IL-6 measured at study entry correlated with levels of TNF and with patients' outcomes. Yet, IL-6 does not appear to be a clinically useful laboratory test for predicting the outcome of an individual patient.

OBJECTIVE

To elucidate extracellular matrix (ECM) changes underlying intestinal fibrosis, a frequent complication of inflammatory bowel disease, we developed a murine model of chronic colitis associated with intestinal fibrosis.

METHODS

Chronic inflammation was established by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS). In 2 variations of the model an antisense oligonucleotide for nuclear factor kappa B (NF-kappa B) p65 was given prophylactically or therapeutically to block chronic inflammation-associated fibrosis. Colonic inflammation and fibrosis were determined by histology. Total collagen level was estimated by hydroxyproline quantification. Colonic expression of collagens (Col1a2, Col3a2), ECM remodeling genes (matrix metalloproteinase [MMP]-1, -3, and tissue inhibitor of matrix metalloproteinase [TIMP]-1), and inflammation-modulating cytokines (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma], transforming growth factor beta 1 [TGF-beta 1], and insulin-like growth factor 1 [IGF-1]) were assessed by semiquantitative reverse-transcription polymerase chain reaction. Control and TNBS-treated colonic mesenchymal cells were characterized by morphology, phenotype, and functional response to TNF-alpha and IFN-gamma.

RESULTS

Colons of TNBS-treated mice contained acute and chronic inflammatory infiltrates, increased collagen, fibrogenic tissue architecture, and increased expression of TNF-alpha, TGF-beta 1, IGF-1, Col1a2, MMP-1, and TIMP-1. Colonic mesenchymal cells from TNBS-treated mice were also morphologically distinct from those of the control mice, with increased TIMP-1 expression in response to IFN-gamma treatment. Fibrosis persisted for 2-4 weeks after cessation of the TNBS treatment. In mice given NF-kappa B antisense prophylactically, 67% were fibrosis-free, whereas of those treated after establishing chronic inflammation, 43% were free of fibrosis.

CONCLUSIONS

Extended TNBS treatment of mice yielded chronic intestinal inflammation-associated fibrosis with extensive fibrogenic ECM changes that could be counteracted by specific blockade of NF-kappa B.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5' untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-alpha/beta) activity in VEE pathogenesis. In IFN-alpha/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-alpha/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-alpha/beta pathway. In cells derived from IFN-alpha/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-alpha/beta plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-alpha/beta between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-alpha/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-alpha/beta plays a major role in the in vivo attenuation of V3043.

BACKGROUND

An important function of skin is to serve as a barrier and thus provide protection from the external environment. The epidermal keratinocyte establishes this barrier by producing an intact stratum corneum. In the past, keratinocytes were appreciated only for this rather inert, passive structural responsibility and not for their potential dynamic contribution to inflammatory or immune-mediated reactions.

OBJECTIVE

Our purpose was to examine the cascade of molecular and cellular events that occur when the barrier function of human skin is abrogated by repeated tape stripping, which physically removes the stratum corneum without inducing any cytopathic effects on the underlying epidermal keratinocytes.

METHODS

Eight healthy human volunteers underwent repeated tape stripping and sequential punch biopsy specimens of skin obtained between 1 and 24 hours after tape stripping were analyzed for protein antigens by immunostaining of cryostat-cut sections. The presence or absence of various messenger RNAs (mRNAs) were detected by polymerase chain reaction.

RESULTS

After repeated tape stripping, keratinocytes became activated within hours. The responses included up-regulation of keratin-16 expression and keratinocyte proliferation accompanied by production of a specific profile of cytokine and adhesion molecule mRNAs and proteins in both epidermal and dermal compartments. Polymerase chain reaction amplification of RNA species isolated from the epidermal portion of skin revealed increases 6 hours after tape stripping in mRNA coding for tumor necrosis factor-alpha, IL-8, IL-10, interferon gamma, intercellular adhesion molecule-1, transforming growth factor-alpha, and transforming growth factor-beta. There was no increase in tumor necrosis factor-alpha, IL-8, IL-10, or transforming growth factor-alpha mRNAs in the dermal samples. Immunostaining revealed that keratinocyte intercellular adhesion molecule-1 was increased 6 hours after stripping and was accompanied by endothelial cell expression of E-selectin (endothelial cell adhesion molecule-1) and vascular cell adhesion molecule-1. These molecular events, which occurred after 6 hours in tape-stripped skin, preceded any movement of inflammatory cells from the circulation into dermis or epidermis and hence reflect changes that occur in cells indigenous to normal human skin. None of these changes occurred in persons who underwent limited tape strippings without barrier perturbation.

CONCLUSIONS

The results highlight the rapid and distinctive responses of epidermal keratinocytes and demonstrate that these cells can actively participate in a far greater number of homeostatic responses other than the production of the epidermal barrier.

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10(8) CFU and observed for 2 days. No clinical signs of illness developed in animals given AS- EBS- organisms, and all survived. All rabbits given AS- EBS+ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+ EBS- organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+ EBS+ organisms developed signs of illness and died. None of the rabbits receiving AS+ EBS+ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+ EBS+ organisms. In contrast, cell extracts from AS- EBS- organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-alpha) and TNF-beta, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 x 10(9) CFU of test organisms. None of the animals given AS- EBS- organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS- EBS+ or AS+ EBS- organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+ EBS+ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS- EBS- or AS+ EBS+ organisms developed vegetations, but animals given AS+ EBS+ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.

BACKGROUND

We studied the balance between ileal T-effector cells versus T-regulatory cells in active and inactive Crohn's disease (CD).

METHODS

We compared effector and regulatory T-cell-related markers such as interleukin (IL)-17, interferon (IFN)-gamma, IL-4, and Foxp3 transforming growth factor (TGF)-beta CTLA-4 and markers for innate immune activation such as IL-6, IL-10, IL-18, IL-23, tumor necrosis factor (TNF)-alpha, and IL-12p70, studied with immunohistochemistry and RT-PCR in ileal biopsies from patients with active or inactive CD and from control subjects. IL-17 in fecal samples was detected by ELISA. The effect of IL-17 on IL-8 and TNF-alpha mRNA expression in epithelial cell line Caco-2 was studied.

RESULTS

The numbers of IL-4-, IL-17-, and IL-23(p19)-positive cells in the lamina propria were higher in patients with CD, both active and inactive, than in the controls. mRNA expression of IL-17A, IL-6, and Foxp3 was increased in the biopsies both from patients with active disease and those in remission, whereas mRNA expression of IL-23 was increased only in active disease. Fecal IL-17 concentration was increased in patients with active disease. IL-17 enhanced the IL-8 and TNF-alpha response of the epithelial cell line to lipopolysaccharide (LPS) in vitro.

CONCLUSIONS

Our findings suggest that activation of the IL-23/IL-17 axis is fundamentally connected to the etiology of CD and may represent the basis for the relapsing nature of the disease by increasing the sensitivity of epithelium to microbial LPS.

Rabies virus (RV) phosphoprotein P is an interferon (IFN) antagonist counteracting transcriptional activation of type I IFN (K. Brzózka, S. Finke, and K. K. Conzelmann, J. Virol 79:7673-7681, 2005). We here show that RV P in addition is responsible for preventing IFN-alpha/beta- and IFN-gamma-stimulated JAK-STAT signaling in RV-infected cells by the retention of activated STATs in the cytoplasm. Expression of IFN-stimulated response element- and gamma-activated sequence-controlled genes was severely impaired in cells infected with RV SAD L16 or in cells expressing RV P protein from transfected plasmids. In contrast, a recombinant RV expressing small amounts of P had lost the ability to interfere with JAK-STAT signaling. IFN-mediated tyrosine phosphorylation of STAT1 and STAT2 was not impaired in RV P-expressing cells; rather, a defect in STAT recycling was suggested by distinct accumulation of tyrosine-phosphorylated STATs in cell extracts. In the presence of P, activated STAT1 and STAT2 were unable to accumulate in the nucleus. Notably, STAT1 and STAT2 were coprecipitated with RV P only from extracts of cells previously stimulated with IFN-alpha or IFN-gamma, whereas in nonstimulated cells no association of P with STATs was observed. This conditional, IFN activation-dependent binding of tyrosine-phosphorylated STATs by RV P is unique for a viral IFN antagonist. The 10 C-terminal residues of P are required for counteracting JAK-STAT signaling but not for inhibition of transcriptional activation of IFN-beta, thus demonstrating two independent functions of RV P in counteracting the host's IFN response.

The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-alpha/beta); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in approximately 15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-beta promoter resulted in approximately 6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing individual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-beta promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-beta promoter-driven transcription, showed approximately 7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-beta promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.

The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.

Leukaemic cells from most cases of B-chronic lymphocytic leukaemia die rapidly by apoptosis in vitro unless they are cultured in the presence of interleukin-4 or interferon alpha or gamma. We now report prolonged survival of purified CLL cells cultured on bone marrow (BM) derived stromal cells in the absence of exogenous growth factors. In 10 cases of CLL examined 0-61% (mean 14.7%) of the cells were viable after 10 d culture in medium alone, whereas in the presence of BM stromal cells 10-102% (mean 47.0%) of cells were recovered alive (P < 0.005) in 7/10 cases of CLL, cells remained viable after 30 d of culture in BM stromal cells with cell recovery of 12-65%. These long-term cultured CLL cells were Epstein Barr virus negative, shown by the failure to detect the ENBA-2 and BZLF1 genes of EBV by PCR analysis. Identity between day 0 and day 30 CLL cells was demonstrated by sequence analysis of their clonal IgH CDR3 region. Adherence of CLL cells to BM stromal cell layers was critical for their protection from apoptosis. Separation of CLL cells from stroma by 0.45 micron culture filters resulted in loss of the protective effect of the stromal cells. Stromal cells were also able to protect CLL cells from hydrocortisone-induced apoptotic cell death. Our findings provide an in vitro system that can be used to analyse the growth requirements of CLL cells and their chemosensitivity in an in vitro environment that mimics the in vivo milieu.

Nitazoxanide (NTZ), a thiazolide anti-infective, is active against anaerobic bacteria, protozoa, and a range of viruses in cell culture models, and is currently in phase II clinical development for treating chronic hepatitis C. In this report, we characterize the activities of NTZ and its active metabolite, tizoxanide (TIZ), along with other thiazolides against hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in standard antiviral assays. NTZ and TIZ exhibited potent inhibition of both HBV and HCV replication. NTZ was equally effective at inhibiting replication of lamivudine (LMV) and adefovir dipovoxil (ADV)-resistant HBV mutants and against 2'-C-methyl cytidine (2'CmeC) and telaprevir (VX-950)-resistant HCV mutants. NTZ displayed synergistic interactions with LMV or ADV against HBV, and with recombinant interferon alpha-2b (IFN) or 2'CmeC against HCV. Pre-treatment of HCV replicon-containing cells with NTZ potentiated the effect of subsequent treatment with NTZ plus IFN, but not NTZ plus 2'CmeC. NTZ induced reductions in several HBV proteins (HBsAg, HBeAg, HBcAg) produced by 2.2.15 cells, but did not affect HBV RNA transcription. NTZ, TIZ, and other thiazolides are promising new antiviral agents that may enhance current or future anti-hepatitis therapies.

Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-alpha has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-alpha in a dose-dependent manner. Expression of TNF-alpha was induced only by replicating virus. TNF-alpha showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-alpha was greater than that of gamma or alpha interferon. These findings suggest that TNF-alpha serves as the first line of defense against influenza virus infection in the natural host.

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-gamma-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-alpha + IL-1beta. CXCL11/interferon-inducible T cell alpha chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Ikappa-B, while pretreatment with an Ikappa-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-kappaB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.

OBJECTIVE

The aim of this study was to evaluate the long-term sustainability of response in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B treated with pegylated interferon (PEG-IFN) alpha-2b alone or in combination with lamivudine.

METHODS

All 266 patients enrolled in the HBV99-01 study were offered participation in a long-term follow-up (LTFU) study. Patients were treated with PEG-IFN alpha-2b (100 mug/wk) alone or in combination with lamivudine (100 mg/day) for 52 weeks. Initial response was defined as HBeAg negativity at 26 weeks posttreatment. For the LTFU study, patients had one additional visit after the initial study (mean interval, 3.0 +/- 0.8 years).

RESULTS

Of 266 patients enrolled in the initial study, 172 (65%) participated in the LTFU study. At LTFU, HBeAg and hepatitis B surface antigen (HBsAg) negativity were observed in 37% and 11% of 172 patients, respectively. Sixty-four patients were classified as initial responders and 108 as nonresponders. Among the initial responders, sustained HBeAg negativity and HBsAg loss were observed in 81% and 30%, respectively. Significantly higher rates of HBeAg negativity were observed in genotype A-infected initial responders compared with those with genotype non-A (96% vs 76%; P = .06) as well as HBsAg loss (58% vs 11%; P < .001).

CONCLUSIONS

HBeAg loss after treatment with PEG-IFN alpha-2b alone or in combination with lamivudine is sustained in the majority of patients and is associated with a high likelihood of HBsAg loss, particularly in genotype A-infected patients. Therefore, PEG-IFN alpha-2b remains an important treatment option in this era of nucleos(t)ide analogue therapy.

Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles.We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons alpha, beta, omega and gamma, IL12 and TNFalpha; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings.

The B cell activation antigen BBB-1 is the natural ligand for the T cell antigen CD28 and these two molecules are capable of mediating T-B cell adhesion. Engagement of the CD28 pathway provides a costimulatory signal to T cells leading to enhanced lymphokine production. We report that interferon-gamma (INF-gamma) induces the expression of BBB-1 on monocytes. This induction was very specific since other cytokines and stimuli which activate monocytes including M-CSF, GM-CSF, IL3, TNF-alpha, and LPS were unable to induce BBB-1. Following culture of monocytes with INF-gamma, maximal mRNA and cell surface BBB-1 expression was detected at 12 and 24 hr, respectively. In addition to antigen presentation, optimal T cell activation and lymphokine synthesis require an additional cell to cell contact signal provided by the antigen presenting cell. The induction of BBB-1 on monocytes and subsequent heterophilic interaction of BBB-1 with CD28 may provide a mechanism for the amplification of T cell proliferation and lymphokine production by INF-gamma activated monocytes.

OBJECTIVE

The therapeutic benefit of ribavirin, a nucleoside analog, in the treatment of chronic HCV infection is seen even in the absence of any apparent direct antiviral effect. We surmised that ribavirin may act by eliciting altered virus-specific immune responses. Because antiviral immunity is predominantly mediated by cytotoxic T cells and antiviral cytokines, we sought to determine whether ribavirin could promote antiviral (Type 1) cytokine expression in human T cells.

METHODS

Isolated human T cells were activated in vitro with enterotoxin B or with phorbol ester plus ionomycin. Cytokine ELISAs were performed on culture supernatants, cytokine mRNA was detected following RT-polymerase chain reaction of T cell RNA, and T cell proliferation measured using MTT assay.

RESULTS

Ribavirin enhanced a Type 1 (IL-2, IFNgamma, TNFalpha) while suppressing a Type 2 cytokine response (IL-4, IL-5 and IL-10), at the level of both protein and mRNA expression. Ribavirin mediated comparable effects on cytokine expression both following activation of specific T cell subpopulations with superantigen and following activation of a larger percentage of T cells via pharmacologic means. The in vitro effect on cytokine expression following ribavirin treatment was comparable in both CD4+ or CD8+ T cell subsets and was observed in a dose range that promoted T cell proliferation.

CONCLUSIONS

These data support the view that ribavirin promotes a Type 1 cytokine-mediated immune response, a property which may account in part for its ability to enhance the antiviral activity of interferon-alpha in the treatment of chronic HCV infection.

Alpha/beta interferons (IFN-alpha/beta) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-alpha/beta. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-alpha/beta in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-alpha/beta system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-alpha/beta gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-gamma-producing CD4(+) T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-alpha/beta.

Stimulation of Toll-like receptors (TLRs) on macrophages and dendritic cells (DCs) by pathogen-derived products induces the production of cytokines, which play an important role in immune responses. Here, we investigated the role of the TPL-2 signaling pathway in TLR induction of interferon-beta (IFN-beta) and interleukin-10 (IL-10) in these cell types. It has previously been suggested that IFN-beta and IL-10 are coordinately regulated after TLR stimulation. However, in the absence of TPL-2 signaling, lipopolysaccharide (TLR4) and CpG (TLR9) stimulation resulted in increased production of IFN-beta while decreasing IL-10 production by both macrophages and myeloid DCs. In contrast, CpG induction of both IFN-alpha and IFN-beta by plasmacytoid DCs was decreased in the absence of TPL-2, although extracellular signal-regulated kinase (ERK) activation was blocked. Extracellular signal-related kinase-dependent negative regulation of IFN-beta in macrophages was IL-10-independent, required protein synthesis, and was recapitulated in TPL-2-deficient myeloid DCs by retroviral transduction of the ERK-dependent transcription factor c-fos.

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (alpha/beta), but it had a much more dramatic effect on type II IFNs (gamma) in vitro and in vivo. A comparative analysis of M27(+) and M27(-) MCMV revealed that the antiviral efficiency of IFN-gamma was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-gamma. This effect required IFN receptor expression and was independent of type I IFNs. IFN-gamma induced increasing levels of tyrosine-phosphorylated STAT2 in M27(-) MCMV-infected cells that were essential for the antiviral potency of IFN-gamma. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-gamma responses.