CD8(+) T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8(+) T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8(+) T cells, using a flow-cytometric cytotoxicity assay based on caspase-<em>3</em> activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8(+) T cells secreting <em>interferon</em>-gamma but no tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P =.57), a subset of CD8(+) T cells secreting both inter-feron-gamma and TNF-<em>alpha</em> was substantially more strongly associated with cytotoxicity (R = 0.67, P <.001). This subset of CD8(+) T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8(+) T cells secreting solely <em>interferon</em>-gamma following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8(+) T cells is preferentially mediated by a subset of CD8(+) T cells secreting both <em>interferon</em>-gamma and TNF-<em>alpha</em>.

In this study, the contribution of type I <em>interferons</em> (IFNs) to protection against infection with enterovirus 71 (EV71) was investigated using a murine model where the virus was administrated to neonatal Institute of Cancer Research (ICR) mice by either the intraperitoneal (i.p.) or the oral route. In i.p. inoculated mice, post-infection treatment of dexamethasone (5 mg kg(-1) at 2 or <em>3</em> days after infection) exacerbated clinical symptoms and increased the tissue viral titre. In contrast, polyriboinosinic : polyribocytidylic acid [poly(I : C); 10 or 100 microg per mouse at 12 h before infection], a potent IFN inducer, improved the survival rate and decreased the tissue viral titres after EV71 challenge, which correlated with an increase in serum IFN-<em>alpha</em> concentration, the percentage of dendritic cells, their expression of major histocompatibility complex class II molecule and IFN-<em>alpha</em> in spleen. Treatment with a neutralizing antibody for type I IFNs (10(4) neutralizing units per mouse, 6 h before and 12 h after infection) resulted in frequent deaths and higher tissue viral load in infected mice compared with control mice. In contrast, an early administration of recombinant mouse IFN-<em>alpha</em>A (10(4) U per mouse for <em>3</em> days starting at 0, 1 or <em>3</em> days after infection) protected the mice against EV71 infection. In vitro analysis of virus-induced death in three human cell lines showed that human type I IFNs exerted a direct protective effect on EV71. It was concluded that type I IFNs play an important role in controlling EV71 infection and replication.

Flavonoids may play an important role for adjunct nutritional therapy of chronic intestinal inflammation. In this study, we characterized the molecular mechanisms by which quercetin and its enteric bacterial metabolites, taxifolin, alphitonin, and <em>3</em>, 4-dihydroxy-phenylacetic acid, inhibit tumor necrosis factor <em>alpha</em> (TNF)-induced proinflammatory gene expression in the murine small intestinal epithelial cell (IEC) line Mode-K as well as in heterozygous TNFDeltaARE/WT mice, a murine model of experimental ileitis. Quercetin inhibited TNF-induced <em>interferon</em>-gamma-inducible protein 10 (IP-10) and macrophage inflammatory protein 2 (MIP-2) gene expression in Mode-K cells with effective inhibitory concentration of 40 and 44 micromol/L, respectively. Interestingly, taxifolin, alphitonin, and <em>3</em>,4-dihydroxy-phenylacetic acid did not inhibit TNF responses in IEC, suggesting that microbial transformation of quercetin completely abolished its anti-inflammatory effect. At the molecular level, quercetin inhibited Akt phosphorylation but did not inhibit TNF-induced RelA/I-kappaB phosphorylation and IkappaB degradation or TNF-<em>alpha</em>-induced nuclear factor-kappaB transcriptional activity. Most important for understanding the mechanism involved, chromatin immunoprecipitation analysis revealed inhibitory effects of quercetin on phospho-RelA recruitment to the IP-10 and MIP-2 gene promoters. In addition, and consistent with the lack of cAMP response element binding protein (CBP)/p<em>3</em>00 recruitment and phosphorylation/acetylation of histone <em>3</em> at the promoter binding site, quercetin inhibited histone acetyl transferase activity. The oral application of quercetin to heterozygous TNFDeltaARE/WT mice [10 mg/(d x kg body wt)] significantly inhibited IP-10 and MIP-2 gene expression in primary ileal epithelial cells but did not affect tissue pathology. These studies support an anti-inflammatory effect of quercetin in epithelial cells through mechanisms that inhibit cofactor recruitment at the chromatin of proinflammatory genes.

OBJECTIVE

To explore safety, pharmacokinetics, and pharmacodynamics of oral administration of resiquimod, a Toll-like receptor 7 and 8 agonist that induces endogenous interferon-alpha, in subjects with chronic hepatitis C virus infection.

METHODS

Two randomized, double-blind phase IIa studies of resiquimod administered two times per week for 4 weeks. Multicenter study (U.S.): 12 subjects received resiquimod 0.01 mg/kg and 4 received placebo. Single center study (France): 6 subjects received 0.01 mg/kg, 11 received 0.02 mg/kg and 6 received placebo.

RESULTS

Resiquimod 0.01 mg/kg was tolerated; two 0.2 mg/kg subjects discontinued treatment. More subjects reported severe grade adverse events at 0.02 mg/kg; events were consistent with systemic cytokine induction, including fever, headache, shivering, and lymphopenia. Mean maximum serum resiquimod concentrations were 3.82+/-1.47 and 7.55+/-4.17 ng/mL for 0.01 mg/kg and 0.02 mg/kg, respectively. At 0.02 mg/kg, two, three and one subjects had maximal reductions in viral levels of at least 1-, 2- and 3-logs, respectively; reductions were generally transient. Interferon-alpha levels appeared correlated with decreases in viral titer and lymphocyte counts, as well as increase in neutrophil counts.

CONCLUSIONS

Oral administration of resiquimod 0.02 mg/kg transiently reduced viral levels but was associated with adverse effects similar to interferon-alpha.

OBJECTIVE

To study the presence of interferogenic autoantibodies in systemic sclerosis (SSc) and their correlation with clinical manifestations, serum levels of interferon alpha (IFNalpha) and chemokines of importance in the disease process.

METHODS

Peripheral blood mononuclear cells (PBMCs) or purified plasmacytoid dendritic cells (pDCs) from healthy donors were stimulated with sera from patients with SSc (n=70) or healthy individuals (n=30), together with necrotic or apoptotic cell material. The IFNalpha produced and serum levels of IFNalpha, IFN-inducible protein-10 (IP-10)/chemokine (C-X-C motif) ligand 10, monocyte chemoattractant protein-1 (MCP-1)/(C-C motif) ligand-2 (CCL-2), macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL-3 and RANTES/CCL-5 were measured and correlated with the presence of autoantibodies and clinical manifestations in the patients with SSc.

RESULTS

Sera from both diffuse SSc and limited SSc contained interferogenic antibodies, which correlated with the presence of anti-ribonucleoprotein and anti-Sjögren syndrome antigen autoantibodies. The pDCs were responsible for the IFNalpha production which required interaction with FcgammaRII and endocytosis. Increased serum levels of IP-10 were associated with vascular manifestations such as cardiac involvement (p=0.027) and pulmonary arterial hypertension (p=0.036). Increased MCP-1 or IFNalpha serum levels were associated with lung fibrosis (p=0.019 and 0.048, respectively). Digital ulcers including digital loss were associated with increased serum levels of IFNalpha (p=0.029).

CONCLUSIONS

An activated type I IFN system previously seen in several other systemic autoimmune diseases is also present in SSc and may contribute to the vascular pathology and affect the profibrotic process.

Administration of <em>interferon</em> (IFN) <em>3</em> times weekly in patients with chronic hepatitis C (CHC) is associated with low sustained responses, which may be, in part, related to this regimen's inability to maintain IFN concentrations sufficient to suppress viral replication. An enhanced IFN molecule produced by the covalent attachment of a branched 40-kd polyethylene glycol moiety to IFN <em>alpha</em>-2a (PEG[40kd] IFN <em>alpha</em>-2a) exhibits sustained absorption, a restricted volume of distribution, and reduced clearance compared with unmodified IFN <em>alpha</em>-2a. One hundred fifty-nine patients with CHC participated in a randomized, ascending-dose (45 or 90, 180, 270 microg) study comparing PEG(40kd) IFN <em>alpha</em>-2a administered once weekly with <em>3</em> MIU IFN <em>alpha</em>-2a administered <em>3</em> times weekly for 48 weeks to determine the most appropriate PEG(40kd) IFN <em>alpha</em>-2a dose for subsequent clinical trials. Efficacy was assessed by measuring hepatitis C virus (HCV) RNA following a 24-week treatment-free period. Sustained virological responses for PEG(40kd) IFN <em>alpha</em>-2a once weekly were 10% (45 microg; not significant), <em>3</em>0% (90 microg; P = .009), <em>3</em>6% (180 microg; P = .0006), and 29% (270 microg; P = .004), compared with <em>3</em>% for the <em>3</em>-times-weekly <em>3</em>-MIU IFN <em>alpha</em>-2a regimen. The types and frequencies of adverse events and laboratory abnormalities were similar among all groups. In conclusion, once-weekly PEG(40kd) IFN <em>alpha</em>-2a was associated with a higher number of sustained virological responses compared with IFN <em>alpha</em>-2a <em>3</em> times weekly in patients with CHC, but had a similar safety profile. The 180-microg PEG(40kd) IFN <em>alpha</em>-2a dose appeared to be the optimal dose based on sustained virological response and its associated side-effect profile.

The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma <em>interferon</em> at concentrations of 8 to 16 U/ml. The <em>interferon</em> was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma <em>interferon</em> for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma <em>interferon</em> were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma <em>interferon</em> had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma <em>interferon</em> had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% <em>3</em> days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant <em>alpha</em> and beta <em>interferons</em> had no effect on the growth of T. gondii.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of a nuclear transcription factor family, has been previously demonstrated to have antiinflammatory activity. The effects of PPARgamma activation in the development of an immune response are less well characterized. Through evaluation of PPARgamma heterozygote mice (PPARgamma(+/-) and specific PPARgamma agonist ligand binding, we evaluated the immunologic effects of PPARgamma activation in a well-described model of colitis. Increased susceptibility to dextran sodium sulfate (DSS)-induced colitis as defined by body weights, histologic injury, and survival was observed in the PPARgamma(+/-) mice in comparison to wild-type mice. Three different PPARgamma ligands (troglitazone, pioglitazone, and rosiglitazone) demonstrated beneficial dose-related treatment effects when administered prior to the onset of colitis. However, no protection was observed when PPARgamma ligand activation occurred after the onset of colitis. The reduction in DSS-induced inflammation noted with PPARgamma ligand treatment was associated with decreased <em>interferon</em>-gamma and tumor necrosis factor-<em>alpha</em> and increased interleukin (IL)-4 and IL- 10 levels as assessed by quantitative reverse transcriptase-polymerase chain reaction. Consistent with this shift towards a T helper (Th2) cytokine dominance, PPARgamma ligand treatment stimulated increased GATA-<em>3</em> expression. These results indicate that the protective effects exhibited by PPARgamma ligands in intestinal inflammation may be due to immune deviation away from Th1 and towards Th2 cytokine production.

Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of <em>interferon</em>-alfa-1b (IFN-<em>alpha</em>-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-<em>alpha</em> at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day for treated groups, and <em>3</em> x 10(7) U/kg/day; tumor volume was 8,475 mm(<em>3</em>) +/- 2,6<em>3</em>6 mm(<em>3</em>), 7,96<em>3</em> mm(<em>3</em>) +/- <em>3</em>,214 mm(<em>3</em>), 769 mm(<em>3</em>) +/- 287 mm(<em>3</em>), and 1<em>3</em> mm(<em>3</em>) +/- 9 mm(<em>3</em>); incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 +/- 4 days, 5<em>3</em> +/- 8 days, 81 +/- 6 days, and 105 +/- 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 x 10(5) U/kg/day, 1 x 10(6) U/kg/day, 4 x 10(6) U/kg/day, 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day, and <em>3</em> x 10(7) U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-<em>alpha</em> inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. In conclusion, high-dose and long-term therapy with IFN-<em>alpha</em> dose-dependently inhibits tumor growth and recurrence after resection of HCC. The effect of IFN-<em>alpha</em> may be attributed to antiangiogenesis in this experiment. These results provide potential clinical implication, particularly for the prevention of recurrence after curative resection of HCC.

Shigella infection is accompanied by an intestinal activation of epithelial cells, T cells, and macrophages within the inflamed colonic mucosa. A prospective study was carried out to elucidate the cytokine pattern in Shigella infection linked to development of immunity and eradication of bacteria from the local site and also to correlate the cytokine profile with histological severity. An indirect immunohistochemical technique was used to determine the production and localization of various cytokines at the single-cell level in cryopreserved rectal biopsies from 24 patients with either Shigella dysenteriae type 1 (n = 18) or Shigella flexneri (n = 6) infection. The histopathological profile included presence of chronic inflammatory cells with or without neutrophils and microulcers in the lamina propria, crypt distortion, branching, and less frequently crypt abscesses. Patients had significantly higher (P < 0.005) numbers of cytokine producing cells for all of the cytokines studied, interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), IL-1 beta, IL-1ra, tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), IL-6, IL-8, IL-4, IL-10, gamma <em>interferon</em>, TNF-beta, and transforming growth factor beta 1-<em>3</em>, in the biopsies than the healthy controls (n = 1<em>3</em>). The cytokine production profile during the study period was dominated by IL-1 beta, transforming growth factor beta 1-<em>3</em>, IL-4, and IL-10. Significantly increased frequencies of cytokine-producing cells (P < 0.05) were observed for IL-1, IL-6, gamma <em>interferon</em>, and TNF-<em>alpha</em> in biopsies with severe inflammation in comparison with those with mild inflammation. During the acute stage of the disease, 20 of 24 patients exhibited acute inflammation in the rectal biopsies and the cellular infiltration was still extensive <em>3</em>0 days after the onset of diarrhea, although the disease was clinically resolved. In accordance with the histological findings, cytokine production was also upregulated during the convalescent phase; there was no significant difference (P>> 0.05) in the incidence of cytokine-producing cells between acute (2 to 8 days after the onset of diarrhea) and convalescent (<em>3</em>0 days after onset) stages.

Treatment with recombinant interleukin 2 and lymphokine-activated killer cells (rIL-2/LAK) has produced a clinical antitumor effect in preliminary human trials. The cytokines gamma-<em>interferon</em> (IFN-gamma), tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), and tumor necrosis factor beta (TNF-beta, lymphotoxin) have potent in vitro antitumor activity and some clinical toxicities similar to interleukin 2 (IL-2)/LAK. This study sought to determine whether these cytokines were detectable in sera of IL-2/LAK-treated patients. Ten patients were treated with a protocol of 5-day i.v. rIL-2 bolus priming (10(5) units/kg, every 8 h), followed by 5 daily phereses with harvested lymphocytes cultured in vitro to generate LAK, and 5 days of rIL-2 bolus with infusion of LAK cells. Five patients were treated with a protocol modified to a <em>3</em>-day rIL-2 prime and 6-day continuous infusion rIL-2 (<em>3</em> x 10(6) units/m2/day) with infusion of LAK cells. Serum specimens were obtained prior to and 0.5, 2, <em>3</em>, and 5 h after IL-2 or LAK cell administrations. IFN-gamma was detected by enzyme-linked immunosorbent assay, TNF-<em>alpha</em> by WEHI 164 bioassay or enzyme-linked immunosorbent assay, and TNF-beta by WEHI 164 cell bioassay. During the prime, few patients manifested in vivo detectable serum cytokines: IFN-gamma, three of ten, 5-day prime (1.0<em>3</em> +/- 0.46 ng/ml), and zero of five, <em>3</em>-day prime; TNF-<em>alpha</em>, one of ten, 5-day prime, and one of three, <em>3</em>-day prime; TNF-beta, one of ten, 5-day prime. The supernatants of in vitro LAK generation cultures had detectable levels of cytokines at 24 h which increased progressively until culture harvest at Day 4 (IFN-gamma, 2.56 +/- 0.<em>3</em>4 ng/ml; TNF-<em>alpha</em>, <em>3</em>56 +/- 110 pg/ml; TNF-beta, 8.2 +/- 4.4 units/ml). The highest levels of in vivo serum cytokines occurred following LAK cell infusion and were more often elevated in patients receiving rIL-2 by bolus than by continuous infusion: IFN-gamma, four of six bolus, zero of three continuous infusion; TNF-<em>alpha</em>, six of six bolus (maximum 679 pg/ml) versus two of three continuous infusion (maximum, 106 pg/ml). LAK cells in vitro responded with cytokine release on stimulation by tumor cell lines (IFN-gamma, 0.88 +/- 0.06 ng/ml; TNF-<em>alpha</em>, 426 +/- 16 pg/ml; TNF-beta, 0.64 +/- 0.06 units/ml). In summary, this preliminary study has detected circulating cytokines in sera of patients receiving IL-2/LAK therapy. The greatest cytokine elevations followed LAK cell infusion.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

Inflammatory and immune reactions raised in response to periodontopathogens are thought to trigger periodontal tissue destruction. We therefore investigated the expression of matrix metalloproteinases (MMPs) and the osteoclastogenic factor RANKL (receptor activator of nuclear factor-kappaB ligand), their respective inhibitors TIMPs (tissue inhibitors of metalloproteinases) and OPG (osteoprotegerin) and their possible correlation with the expression of inflammatory and regulatory cytokines in the course of experimental periodontal disease in mice.

METHODS

We characterized the time course of leukocyte migration and alveolar bone loss in C57BL/6 mice infected with Actinobacillus actinomycetemcomitans. Quantitative polymerase chain reaction (RealTime PCR) and ELISA were performed to determine the expression of MMPs, TIMPs, RANKL, OPG and cathepsin K, interleukin-1beta, tumor necrosis factor-alpha, interferon-gamma, interleukin-12, interleukin-4 and interleukin-10 in periodontal tissue samples harvested throughout the course of experimental disease.

RESULTS

Oral inoculation of A. actinomycetemcomitans results in an intense and widespread migration of leukocytes to the gingival tissues, besides marked alveolar bone resorption. Our data also demonstrate two distinct patterns of MMP/TIMP and RANKL/OPG expression in the course of experimental periodontal disease. The expression of MMPs (MMP-1, 2 and 9) and RANKL was correlated with the expression of interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma, in a time period characterized by the intense increase of inflammatory reaction and alveolar bone loss. On the other hand, interleukin-4 and interleukin-10 were associated with higher expression of TIMPs (TIMP 1, 2 and 3) and OPG, with a lower expression of MMPs and RANKL, and with reduced rates of increase of cellular infiltration in periodontal tissues and alveolar bone loss.

CONCLUSIONS

It is possible that the pattern of cytokines produced in periodontal tissues determines the progression and the severity of experimental periodontal disease, controlling the breakdown of soft and bone tissues through the balance between MMPs/TIMP and RANKL/OPG expression in gingival tissues.

BACKGROUND

Hypoxia is a microenvironmental feature in the inflamed joint, which promotes survival advantage for cells. The aim of this study was to examine the relationship of partial oxygen pressure in the synovial tissue (tPO(2)) in patients with inflammatory arthritis with macroscopic/microscopic inflammation and local levels of proinflammatory mediators.

METHODS

Patients with inflammatory arthritis underwent full clinical assessment and video arthroscopy to quantify macroscopic synovitis and measure synovial tPO(2) under direct visualisation. Cell specific markers (CD3 (T cells), CD68 (macrophages), Ki67 (cell proliferation) and terminal deoxynucleotidyl transferase dUTP nick end labelling (cell apoptosis)) were quantified by immunohistology. In vitro migration was assessed in primary and normal synoviocytes (synovial fibroblast cells (SFCs)) using a wound repair scratch assay. Levels of tumour necrosis factor alpha (TNFalpha), interleukin 1beta (IL1beta), interferon gamma (IFNgamma), IL6, macrophage inflammatory protein 3alpha (MIP3alpha) and IL8 were quantified, in matched serum and synovial fluid, by multiplex cytokine assay and ELISA.

RESULTS

The tPO(2) was 22.5 (range 3.2-54.1) mm Hg and correlated inversely with macroscopic synovitis (r=-0.421, p=0.02), sublining CD3 cells (-0.611, p<0.01) and sublining CD68 cells (r=-0.615, p<0.001). No relationship with cell proliferation or apoptosis was found. Primary and normal SFCs exposed to 1% and 3% oxygen (reflecting the median tPO(2) in vivo) induced cell migration. This was coupled with significantly higher levels of synovial fluid tumour necrosis factor alpha (TNFalpha), IL1beta, IFNgamma and MIP3alpha in patients with tPO(2) <20 mm Hg (all p values <0.05).

CONCLUSIONS

This is the first study to show a direct in vivo correlation between synovial tPO(2), inflammation and cell migration, thus it is proposed that hypoxia is a possible primary driver of inflammatory processes in the arthritic joint.

There has been a recent paradigm shift in the indications and endpoints of treatment for chronic hepatitis B (CHB). Hepatitis B e antigen (HBeAg)-negative disease is being increasingly recognized. Antiviral treatment for both HBeAg-positive and HBeAg-negative patients should aim at long-term suppression of HBV DNA, with the ultimate ideal endpoint of hepatitis B surface antigen (HBsAg) seroconversion. Conventional <em>interferon</em> <em>alpha</em> (IFN-α), the only agent licensed in 1991, has been superseded by pegylated IFN-α. HBeAg seroconversion using pegylated IFN-α is <em>3</em><em>3</em>%, with only 25% of HBeAg-positive patients achieving undetectable HBV DNA by polymerase chain reaction (PCR) assay. Five nucleoside/nucleotide analogues have been licensed since 1998. Lamivudine, an L-nucleoside, is limited by the development of resistance in 76% of patients after 5 years of therapy. Telbivudine, another L-nucleoside, is more potent than lamivudine but resistance still develops in 25% of HBeAg-positive and 11% HBeAg-negative patients after 2 years. Adefovir, an acyclic phosphonate, is relatively weak, but is effective against lamivudine- and telbivudine- resistant mutations, for which it should be used in combination (add-on therapy) rather than substituted. Resistance to adefovir develops slowly, rising to 29% for HBeAg-negative patients by year 5, but more rapidly when used alone for lamivudine-resistant HBV. Currently the two first line nucleoside/nucleotides are entecavir and tenofovir. Entecavir, a cyclopentane (D-nucleoside), is very potent, with 94% of patients having undetectable HBV DNA after 5 years. Resistance develops in only 1.2% of treatment-naïve patients. Tenofovir, another acyclic nucleotide, is more potent with less renal toxicity compared to adefovir. It is effective against lamivudine-resistant mutations when used alone. No resistance to tenofovir has been described after its use for <em>3</em> years or longer, often for patients with human immunodeficiency virus/HBV co-infection. With these current, potent antiviral agents associated with very low rates of resistance, long-term HBV DNA suppression and possibly even reversal of cirrhosis can now be achieved in a proportion of patients. In addition, long-term treatment with these antiviral agents is associated with a reduced risk of development of hepatocellular carcinoma.

<em>Interferons</em> (IFNs) play critical roles in host defense by modulating gene expression via activation of signal transducer and activator of transcription (STAT) factors. IFN-<em>alpha</em>/beta also activates another transcription factor, nuclear factor kappaB (NF-kappaB), which protects cells against apoptotic stimuli. NF-kappaB activation requires the IFN-dependent association of STAT<em>3</em> with the IFNAR1 chain of the IFN receptor. IFN-dependent NF-kappaB activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol <em>3</em>-kinase (PI-<em>3</em>K) and Akt. Whereas constitutively active PI-<em>3</em>K and Akt induce NF-kappaB activation, Ly294002 (a PI-<em>3</em>K inhibitor), dominant-negative PI-<em>3</em>K, and kinase-dead Akt block IFN-dependent NF-kappaB activation. Moreover, dominant-negative PI-<em>3</em>K blocks IFN-promoted degradation of kappaBox <em>alpha</em>. Ly294002, a dominant-negative PI-<em>3</em>K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT<em>3</em>, PI-<em>3</em>K, and Akt are components of an IFN signaling pathway that promotes cell survival through NF-kappaB activation.

Neural trauma, such as traumatic brain injury or stroke, results in a vigorous inflammatory response at and near the site of injury, with cytokine production by endogenous glial cells and invading immune cells. Little is known of the effect that these cytokines have on neural stem cell function. Here we examine the effects of two inflammatory cytokines, <em>interferon</em>-gamma (IFN gamma) and tumour necrosis factor-<em>alpha</em> (TNF<em>alpha</em>), on adult neural stem cells. Neural stem cells grown in the presence of either cytokine failed to generate neurospheres. Cytotoxicity assays showed that TNF <em>alpha</em> but not IFN gamma was toxic to the neural stem cells under proliferative conditions. Under differentiating conditions, neither cytokine was toxic; however, IFN gamma enhanced neuronal differentiation, rapidly increasing beta III-tubulin positive cell numbers <em>3</em>-4 fold and inhibiting astrocyte generation. Furthermore, neurite outgrowth and the number of neurites per neuron was enhanced in cells differentiated in the presence of IFN gamma. Therefore, both inflammatory cytokines examined have substantial, but different effects on neural stem cell function and suggests that regulation of the inflammatory environment following brain injury may influence the ability of neural stem cells to repair the damage.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through imperfect base pairing with the <em>3</em>' untranslated region (<em>3</em>'UTR) of target mRNA. We studied the regulation of <em>alpha</em> 1 (I) collagen (Col1A1) expression by miRNAs in human stellate cells, which are involved in liver fibrogenesis. Among miR-29b, -14<em>3</em>, and -218, whose expressions were altered in response to transforming growth factor-beta1 or <em>interferon</em>-<em>alpha</em> stimulation, miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 <em>3</em>'UTR. miR-29b also had an effect on SP1 expression. These results suggested that miR-29b is involved in the regulation of type I collagen expression by <em>interferon</em>-<em>alpha</em> in hepatic stellate cells. It is anticipated that miR-29b will be used for the regulation of stellate cell activation and lead to antifibrotic therapy.

To determine if the clearance of hepatitis C genotype 1 virus (HCV) is dependent on the dose of <em>interferon</em> alfa-2b (IFN-<em>alpha</em>2b), the acute clearance of HCV after a single dose of either <em>3</em>, 5, or 10 mIU of IFN-<em>alpha</em> was compared in patients with chronic hepatitis C. HCV-RNA levels following IFN-<em>alpha</em> administration were measured. At 24 hours, mean percentage serum viral reduction was 41.4%, 6<em>3</em>.7%, and 85.5% for <em>3</em>, 5, and 10 mIU, respectively (P < .001). At 48 hours, the mean viral reduction was consistently less than the reduction at 24 hours, averaging 22.9%, 61.9%, and 74.<em>3</em>%, respectively (P < .001), indicating that the drug effect diminishes before 48 hours. Regression analysis showed a positive correlation between dose and percent reduction of HCV-RNA levels (r = .6; P < .001). A mathematical model showed that such dose dependence is expected if IFN-<em>alpha</em> partially blocks viral production. Minimum clearance and production rates of HCV were estimated from measurements of HCV-RNA levels after the 10-mIU dose. HCV decay followed an exponential decline with a minimum estimate of the viral clearance rate constant of 2.8 per day, corresponding to a virion half-life of 0.<em>3</em> days or less. A minimal estimate of the daily HCV production and clearance is <em>3</em>.7 x 10(11) virions per day, indicating a high rate of replication and turnover. These results indicate that there is a dose-dependent effect of IFN-<em>alpha</em> in clearance of HCV genotype 1. Because the virion production rate is very rapid and because the current recommended dose of IFN-<em>alpha</em> (<em>3</em> mIU) is often ineffective, larger doses should be considered to treat genotype 1-infected patients.

BACKGROUND

Daclizumab high-yield process (HYP) is a humanized monoclonal antibody that binds to CD25 (alpha subunit of the interleukin-2 receptor) and modulates interleukin-2 signaling. Abnormalities in interleukin-2 signaling have been implicated in the pathogenesis of multiple sclerosis and other autoimmune disorders.

METHODS

We conducted a randomized, double-blind, active-controlled, phase 3 study involving 1841 patients with relapsing-remitting multiple sclerosis to compare daclizumab HYP, administered subcutaneously at a dose of 150 mg every 4 weeks, with interferon beta-1a, administered intramuscularly at a dose of 30 μg once weekly, for up to 144 weeks. The primary end point was the annualized relapse rate.

RESULTS

The annualized relapse rate was lower with daclizumab HYP than with interferon beta-1a (0.22 vs. 0.39; 45% lower rate with daclizumab HYP; P<0.001). The number of new or newly enlarged hyperintense lesions on T2-weighted magnetic resonance imaging (MRI) over a period of 96 weeks was lower with daclizumab HYP than with interferon beta-1a (4.3 vs. 9.4; 54% lower number of lesions with daclizumab HYP; P<0.001). At week 144, the estimated incidence of disability progression confirmed at 12 weeks was 16% with daclizumab HYP and 20% with interferon beta-1a (P=0.16). Serious adverse events, excluding relapse of multiple sclerosis, were reported in 15% of the patients in the daclizumab HYP group and in 10% of those in the interferon beta-1a group. Infections were more common in the daclizumab HYP group than in the interferon beta-1a group (in 65% vs. 57% of the patients, including serious infection in 4% vs. 2%), as were cutaneous events such as rash or eczema (in 37% vs. 19%, including serious events in 2% vs. <1%) and elevations in liver aminotransferase levels that were more than 5 times the upper limit of the normal range (in 6% vs. 3%).

CONCLUSIONS

Among patients with relapsing-remitting multiple sclerosis, daclizumab HYP showed efficacy superior to that of interferon beta-1a with regard to the annualized relapse rate and lesions, as assessed by means of MRI, but was not associated with a significantly lower risk of disability progression confirmed at 12 weeks. The rates of infection, rash, and abnormalities on liver-function testing were higher with daclizumab HYP than with interferon beta-1a. (Funded by Biogen and AbbVie Biotherapeutics; DECIDE ClinicalTrials.gov number, NCT01064401.).

Foreign DNA has been shown to impinge on immune cell function by an as yet unidentified mechanism. We and others have demonstrated that single-stranded (ss) DNA containing the motif CpG flanked by two 5' purines and two <em>3</em>' pyrimidines are mitogenic for B cells and activate macrophages to release tumor necrosis factor-<em>alpha</em>, <em>interferon</em>-gamma, interleukin (IL)-6 or IL-12. Because of these pro-inflammatory responses we investigated if ssDNA would serve as a potential vaccine adjuvant. Here we show that CpG-containing oligonucleotides represent a powerful adjuvant for both humoral and cellular immune responses. When ssDNA was incorporated into inocula, specific antibody titers of the IgG2 isotype were enhanced by greater than 100-fold. Primary cytotoxic T lymphocyte responses generated to either unprocessed protein antigen or major histocompatibility complex class I-restricted peptide were exceedingly strong. Evidence is also provided that oligomers directly influenced T cell receptor-triggered T cell proliferation. Thus ssDNA oligomers may serve as inexpensive and safe vaccine adjuvants and, in addition, differential effects due to sequence may allow for directed responses.

We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma <em>interferon</em> (IFN-gamma) knockout mice (GKO mice). Naive GKO mice succumbed by day 9 to intranasal challenge with 2.5 x 10(6) yeasts, whereas all wild-type (WT) mice survived for 45 days. Compared to lungs of WT mice, the lungs of acutely infected GKO mice exhibited dramatically elevated numbers of CFU in lungs and significantly higher levels of tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin-12 (IL-12) or IL-4. To determine if IFN-gamma is necessary in reexposure histoplasmosis, GKO and WT mice were inoculated with 10(4) yeasts intranasally and given amphotericin B for <em>3</em> weeks. Six weeks later, mice were rechallenged with 2.5 x 10(6) yeasts. All GKO mice died by day 6, whereas all WT mice survived for 45 days. Lungs of GKO mice contained substantially elevated numbers of CFU and higher TNF-<em>alpha</em> and GM-CSF levels but not IL-12 or IL-4. Thus, IFN-gamma is requisite for control of pulmonary histoplasmosis in naive and reexposed mice.

<em>Interferon</em> (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p5<em>3</em> mutants, to tumor necrosis factor-<em>alpha</em>-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p5<em>3</em> function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP<em>3</em>2 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-<em>3</em> (ICE-LAP<em>3</em>, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p5<em>3</em>. The IFN-responsive transcriptional activator <em>interferon</em> regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p5<em>3</em>-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Toll-like receptors (TLRs) are important sensors of microbial pathogens and mediators of innate immune responses. Although the signal transduction of TLRs is well elucidated, their basal regulation is largely unexplored. Here we show that the tumor suppressor p5<em>3</em> positively regulates the transcription of TLR<em>3</em>, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p5<em>3</em> site in the TLR<em>3</em> promoter. TLR<em>3</em> expression was lower in HCT116 p5<em>3</em>(-/-) cells than in HCT116 p5<em>3</em>(+/+) cells. Activation of p5<em>3</em> by 5-fluorouracil increased the TLR<em>3</em> mRNA in epithelial cell lines with wild-type p5<em>3</em> but not in cell lines harboring mutant p5<em>3</em>. Knockdown of p5<em>3</em> by small interfering RNA decreased the TLR<em>3</em> expression. TLR<em>3</em> mRNA was also lower in liver and intestine of p5<em>3</em>(-/-) mice than in p5<em>3</em>(+/+) mice. Furthermore, the poly(I-C)-induced phosphorylation of IkappaB-<em>alpha</em>, nuclear translocation of NF-kappaB, and phosphorylation of <em>interferon</em> regulatory transcription factor <em>3</em>, were drastically reduced in HCT116 p5<em>3</em>(-/-) cells, indicating a dysregulation of the two signaling pathways governed by TLR<em>3</em>. Consequently, induction of interleukin-8 and beta <em>interferon</em> after poly(I-C) stimulation was impaired in HCT116 p5<em>3</em>(-/-) cells. These results suggest that p5<em>3</em> influences TLR<em>3</em> expression and function and highlight a role of p5<em>3</em> in innate immune response in epithelial cells.

In neurodegenerative disease or after brain injury, parenchymal cells in the central nervous system are activated to produce inflammatory mediators, mainly consisting of cytokine-induced factors, in a manner similar to, but clearly different from a peripheral inflammatory response. The upregulated expression of several extracellular matrix proteins in astrocytes located surrounding a neuritic plaque in Alzheimer's disease is a good example of such a response. A family of mediators which is cytokine-induced during an inflammatory response in the periphery are the matrix metalloproteinases. Matrix metalloproteinases are calcium-requiring, zinc-containing endopeptidases that constitute a major component of the enzyme cascade responsible for degradation of extracellular matrix proteins such as collagen, proteoglycan and laminin. Little is known about the cellular source or the function of matrix metalloproteinases in the central nervous system or how their expression is regulated in brain. Thus, it was of interest to determine which factors of the so-called 'brain inflammatory response' regulate the expression of these proteases in the nervous system. To this end, we measured the expression of matrix metalloproteinases in cultured rat astrocytes and microglia after treatment with various cytokines. Interleukin-1 beta, tumor necrosis factor-<em>alpha</em> and lipopolysaccharide were potent stimulators of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-9 (gelatinase B) in cultured rat astrocytes; the effect of each secretagogue was inhibited in the presence of glucocorticoid. Interleukin-1 beta and lipopolysaccharide also stimulated the production of matrix metalloproteinase-<em>3</em> (stromelysin-1) in astrocytes. In addition, activated microglia release matrix metalloproteinase-9. The 'coactivator' of monocytic phagocytes, <em>interferon</em>-gamma, rather than augmenting the response to lipopolysaccharide, inhibited it. Thus, cytokines appear to be potent regulators of matrix metalloproteinase production in astrocytes and microglia. The presence of these enzymes in 'inflamed' central nervous system may suggest their involvement in the pathogenesis or progression of neurodegenerative diseases which are associated with an inflammatory component. Much remains to be learned about the potential substrates for these enzymes and the mechanism of their activation in the central nervous system.