Human beta-defensins are antimicrobial peptides produced by epithelial cells. To date, 28 beta-defensins have been described and the expression of a select few has been classified as constitutive or inducible. Most studies have evaluated expression and regulation using a limited number of primary cell cultures or immortalized cell lines. The goal of this study was to quantitatively assess the in vitro expression and inducibility profiles of human beta-defensins, HBD-1, HBD-2, and HBD-<em>3</em> across a number of primary gingival keratinocyte cultures. Cultured cells from 14 human subjects were stimulated with interleukin-1 beta (IL-1beta), IL-2, IL-6, IL-8, IL-12, tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), gamma <em>interferon</em> (IFN-gamma) or Escherichia coli lipopolysaccharide (LPS) and analyzed by reverse transcription (RT)-PCR. A subset of cultures were quantitatively assessed by real-time PCR. HBD-1 presented the highest and most heterogeneous expression at the basal level (non-stimulated) as compared to expression of HBD-2 and HBD-<em>3</em>, which was significantly lower and homogeneous. IFN-gamma was a primary inducer for HBD-1 and HBD-<em>3</em>, while IL-1beta and TNF-<em>alpha</em> were primary inducers for HBD-2. Sporadic induction was seen for IL-2, IL-6 and LPS. Synergistic expression was seen when various cytokines were combined. Interestingly, the induction potential of each beta-defensin was directly correlated to its basal expression. An inhibitor of JAK2 kinase (Janus kinase), down-regulated IFN-gamma-induced HBD-1 and HBD-<em>3</em> expression, suggesting a role for the JAK/signal transducer and activator of transcription (STAT) signaling pathway in their expression. HBD-2 protein expression of supernatants and cell lysates paralleled mRNA expression. The results suggest that beta-defensin expression and induction in gingival keratinocytes is similar to that seen in other tissue. However, the novel finding of considerable variation among induction levels and the correlation of the induction with basal expression suggests that these innate response elements may play a key role in susceptibility or resistance to disease in the oral cavity.

We examined the influence on the <em>interferon</em> (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR<em>3</em>. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to <em>3</em> h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-<em>alpha</em> or IFN-gamma, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U9<em>3</em>7 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.

<em>Interferon</em> <em>alpha</em> and ribavirin (RBV) combination therapy is associated with decreases in haemoglobin (Hb) concentrations and anaemia. The aim of this analysis was to better characterize the magnitude and frequency of Hb changes and risk factors. This retrospective analysis evaluated treatment-related changes in Hb in 677 patients who participated in either of two <em>interferon</em> <em>alpha</em>-2b plus RBV studies for chronic hepatitis C virus (HCV) infection. Study 1 included 192 <em>interferon</em> <em>alpha</em>-naïve patients randomized to receive RBV 1000-1200 mg/day plus <em>interferon</em> <em>alpha</em>-2b <em>3</em> million IU daily or three times weekly for 48 weeks. Study 2 included 485 <em>interferon</em> <em>alpha</em>-experienced patients randomized to receive RBV 1000-1200 mg daily plus <em>interferon</em> <em>alpha</em>-2b <em>3</em> million IU daily or three times weekly for 4 weeks, followed by three times weekly dosing for 44 weeks. More than 50% of all patients experienced a decrease in Hb>> or =<em>3</em>0 g/L. Women were 4.4 times as likely as men to experience a Hb level of <100 g/L; however, men were at a 40% higher risk to experience a Hb decline of>><em>3</em>0 g/L from baseline. Daily use of <em>interferon</em> <em>alpha</em>-2b did not impact the magnitude of Hb decrease. In this pooled analysis, RBV dose reduction resulted in increases in Hb concentration of approximately 10 g/L. Lower baseline creatinine clearance, higher baseline Hb levels and increased age were independently associated with increased risk of Hb decreases of >27.7%. Lower baseline weight was not associated with increased risk of Hb decrease. Substantial Hb decreases occur frequently with <em>interferon</em> <em>alpha</em>/RBV combination therapy. Sex, the magnitude of the Hb decline and renal function are potentially important factors to consider in patients receiving RBV. Further research is needed to determine the impact on virological response and to develop strategies to manage the medical consequences.

Among 49 patients with carcinoid tumors given long term therapy (mean, 8 months; range, <em>3</em>-<em>3</em>6) with human leukocyte-derived <em>interferon</em>-<em>alpha</em> (huLe-IFN <em>alpha</em>), hypothyroidism occurred in 5 and thyrotoxicosis in 2. Antibodies against thyroid microsomal antigen and/or thyroglobulin were found in 1<em>3</em> patients. In 7 of these, <em>3</em> of whom developed hypothyroidism, the antibodies appeared after the start of therapy. During treatment, an increase in the proportions of circulating activated surface HLA-DR-positive T-helper and T-suppressor cells occurred after <em>3</em>-4 days, and the proportions remained elevated at <em>3</em> and 6 months. Incubation of T cells of normal individuals in vitro with the huLe-IFN <em>alpha</em> preparation induced a rise in activated T-helper and T-suppressor cells. This effect was mimicked by recombinant IFN gamma (r-IFN gamma), but not by r-IFN <em>alpha</em>. Further, the huLe-IFN <em>alpha</em> preparation employed induced HLA-DR expression on human thyroid cells in tissue culture as did r-IFN gamma, but not r-IFN <em>alpha</em>, suggesting the presence of bioactive IFN gamma in the huLe-IFN <em>alpha</em> preparation. The results demonstrate that thyroid autoimmune disease can occur as a side-effect of treatment with huLe-IFN <em>alpha</em> and suggest that IFNs may play important regulatory roles, at both the effector and target cell levels, in the development of human autoimmune disorders.

The ability of human neutrophils to aid in defense against pulmonary infection with Mycobacterium tuberculosis is controversial. In this study, we have shown that neutrophils respond to and phagocytose M. tuberculosis in human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum of M. tuberculosis within 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor <em>alpha</em> but not by gamma <em>interferon</em>. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellular M. tuberculosis to neutrophil defensins. In vitro, human neutrophil peptides 1 to <em>3</em> were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection with M. tuberculosis.

<em>Interferon</em>-<em>alpha</em> (IFN <em>alpha</em>) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFN <em>alpha</em>-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFN <em>alpha</em>, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFN <em>alpha</em> stimulation failed to induce <em>interferon</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFN <em>alpha</em>-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT<em>3</em> were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFN <em>alpha</em> in CTCL cells may result from lack of STAT1 expression.

Cytokines are suspected of playing an important role in the pathophysiology of septic shock. This study was undertaken to determine whether tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) induces the production of other cytokines and mediates mortality in a neonatal rat model of sepsis caused by group B streptococci (GBS). We have measured TNF-<em>alpha</em>, interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), interleukin-6 (IL-6), and gamma <em>interferon</em> (IFN-gamma) levels in neonatal rats infected with different strains (H7<em>3</em>8, 259, and 90) and doses (1 50% lethal dose [LD50] and 5 90% lethal doses [LD90]) of type III GBS. TNF-<em>alpha</em> and IL-6 were detected by the L929 cytotoxicity and the B9 proliferation assays, respectively, in serial plasma samples. IL-1 <em>alpha</em> and IFN-gamma were measured in spleen homogenates by enzyme-linked immunosorbent assay kits by using antibodies raised against the corresponding mouse cytokines. Plasma TNF-<em>alpha</em> levels significantly rose above baseline values within 12 h after intraperitoneal challenge with 5 LD90 of GBS strain H7<em>3</em>8, corresponding to <em>3</em> x 10(<em>3</em>) CFU. A mean peak TNF-<em>alpha</em> concentration of 2<em>3</em>2 +/- 124 U/ml was reached at 20 h. Peak IL-1 <em>alpha</em> and IL-6 levels of 766 +/- 404 U/g and 1,0<em>3</em><em>3</em> +/- 520 U/ml, respectively, were reached at 24 h after bacterial challenge. Maximal spleen concentrations of IFN-gamma (449 +/- 28<em>3</em> U/g) were measured at <em>3</em>6 h. Concentrations of TNF-<em>alpha</em>, but not other cytokines, remained significantly elevated at 72 h, a time when mortality approached 100%. Significant correlations were found between concentrations of each of the cytokines tested and the logs of CFU concentrations in the blood. In order to ascertain whether TNF-<em>alpha</em> influenced the production of other cytokines, rat pups received two injections of anti-murine TNF-<em>alpha</em> or normal rabbit serum at 2 h before and at 26 h after challenge with live GBS. Plasma TNF-<em>alpha</em> bioactivity was undetectable in anti-TNF-<em>alpha</em>-treated animals, while IL-6 and IFN-gamma, but not IL-1 <em>alpha</em>, levels were significantly reduced, compared with normal serum controls. Rat pups pretreated with anti-TNF-<em>alpha</em> serum and infected with 1 and 5 LD90 of strains H7<em>3</em>8 and 259 showed enhanced early (48 to 72 h) survival. However, by 96 h this protection was no longer apparent.

BACKGROUND

Pediatric eosinophilic esophagitis (EE) is a recently described disorder associated with atopy. Although studies of esophageal tissue suggest that Th2 cytokines and eotaxin-<em>3</em> may be crucial in disease pathogenesis, little is known about the systemic immunological phenotypes of children with EE.

OBJECTIVE

To define the phenotypes of peripheral blood eosinophils and lymphocytes in EE and to examine for correlations between these parameters and tissue eosinophil numbers and disease severity.

METHODS

Blood was collected from children with EE, atopic control children without EE, and nonatopic control children without EE. Flow cytometry was used to measure eosinophil expression of chemokine receptor <em>3</em> (CCR<em>3</em>) and interleukin-5 receptor-alpha (IL-5Ralpha), and intracellular lymphocyte expression of IL-4, IL-5, IL-1<em>3</em>, interferon-gamma, and tumor necrosis factor-alpha. Eosinophil numbers and eotaxin-<em>3</em> mRNA levels were quantitated in esophageal biopsy specimens.

RESULTS

Compared with nonatopic control children, EE patients with active disease had increased peripheral blood eosinophil percentages, mean channel of fluorescence (MCF) of CCR<em>3</em> on eosinophils, and percentage of CD4+ T cells expressing IL-5. Notably, these parameters positively correlated with esophageal eosinophil numbers. Eotaxin-<em>3</em> tissue expression positively correlated with esophageal eosinophil numbers and peripheral blood eosinophil CCR<em>3</em> MCF. The percentage of peripheral blood eosinophils, eosinophil CCR<em>3</em> MCF, and CD4+ T cell expression of IL-5 were lower in EE patients in disease remission than in patients with active disease.

CONCLUSIONS

Collectively, these studies demonstrate cooperation between systemic CD4+ Th2-cell-mediated immunity and an enhanced eosinophil-CCR<em>3</em>/eotaxin-<em>3</em> pathway in EE pathogenesis. Furthermore, the imbalanced Th2 immunity and increased CCR<em>3</em> expression are reversible with disease remission.

OBJECTIVE

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of antinuclear autoantibodies. Increased apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE, but the underlying mechanisms remain elusive. During apoptosis apoptotic blebs are formed in which autoantigens are clustered. The cellular remnants after blebbing are referred to as apoptotic cell bodies. We undertook this study to compare the effects of apoptotic blebs and apoptotic cell bodies on maturation of dendritic cells (DCs) and their T cell stimulatory capacity in a murine setting.

METHODS

The uptake by DCs of apoptotic blebs and apoptotic cell bodies was analyzed by flow cytometry and confocal microscopy. DC maturation and DC-induced T cell activation were determined by measuring expression of costimulatory molecules using flow cytometry and by measuring production of cytokines using enzyme-linked immunosorbent assay.

RESULTS

DCs internalized apoptotic blebs more efficiently than apoptotic cell bodies. Incubation of DCs with apoptotic blebs resulted in increased CD40 and CD86 expression and increased interleukin-6 (IL-6) and tumor necrosis factor <em>alpha</em> production, while apoptotic cell bodies had no stimulatory effects. Using chloroquine, apoptotic bleb-induced DC maturation was shown to be independent of Toll-like receptors <em>3</em>, 7, and 9. Interestingly, in cocultures with allogeneic T cells, bleb-matured DCs induced production of IL-2, <em>interferon</em>-gamma, and, in particular, IL-17, suggesting a Th1/Th17 response.

CONCLUSIONS

Apoptotic blebs, in contrast to apoptotic cell bodies, induce DC maturation, thereby providing DCs with increased Th17 cell stimulatory capacity. These data imply that apoptotic bleb-induced DC maturation represents an important driving force in the autoimmune response in SLE.

BACKGROUND

Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine.

METHODS

Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-<em>3</em> to replicon cells. The antiviral effect of chloroquine and/or <em>interferon</em>-<em>alpha</em> (IFN<em>alpha</em>) was evaluated.

RESULTS

The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNalpha enhanced the antiviral effect of IFNalpha and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNalpha but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine.

CONCLUSIONS

The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.

The CC chemokine RANTES is synthesized, stored, and upregulated in response to <em>interferon</em>-gamma (IFN-gamma) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-gamma to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 +/- 15 pg/mL per 2 x 10(6) cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-gamma stimulation. This effect was not observed in eosinophils treated with IFN-<em>alpha</em>, interleukin-<em>3</em> (IL-<em>3</em>), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-gamma. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) <em>3</em> and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL<em>3</em> in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of <em>3</em>T<em>3</em>-L1 adipocytes with LPS or cytokines (TNF <em>alpha</em>, IL-1 beta, and <em>interferon</em> gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.

Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral 'adaptation'. It was previously demonstrated that animals infected intranasally with 10(7) TCID50 of a recent H<em>3</em>N2 influenza, A/Wuhan/<em>3</em>59/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day <em>3</em>. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly <em>3</em> weeks. Interleukin 1 <em>alpha</em> (IL1<em>alpha</em>), <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>), IL6, tumour necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), GRO<em>alpha</em> and MIP-1beta mRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-gamma, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.

OBJECTIVE

Kupffer cells (KC) are important innate immune cells of the liver, functioning as scavenging sinusoidal phagocytes and transducers of pattern recognition signals, including those of toll-like receptors (TLRs). The hepatitis C virus core protein (HCVc) engages TLR2 on peripheral blood monocytes and induces production of multiple inflammatory cytokines. We examined the effects of HCVc on human primary KC functions.

METHODS

KC were isolated from living donor allografts and stimulated with HCVc and/or ligands for TLRs. KC were examined for production of cytokines, expression of programmed death-ligand 1 (PD-L1), secretion of type 1 interferons (IFNs), and expression of the apoptosis-inducing protein tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).

RESULTS

HCVc acts as a ligand for TLR2 on human KC, inducing them to secrete interleukin (IL)-1beta, TNF-alpha, and IL-10 and up-regulate cell surface PD-L1. HCVc blocked TLR3-mediated secretion of IFN-alpha, IFN-beta, and cell surface expression of the cytotoxic molecule TRAIL. Inhibition of phosphoinositide 3 kinase with LY294002 blocked the up-regulation of PD-L1 by TLR ligands and the TLR3-specific induction of TRAIL and type 1 IFNs.

CONCLUSIONS

KC are intravascular macrophages that are continuously exposed to, and tolerant of, bacterial TLR ligands, which are delivered via the portal circulation. By mimicking a bacterial TLR2 ligand and effectively blocking the TLR3-mediated, double-stranded RNA-induced antiviral response, HCVc might appear to exploit this unique aspect of immunity in the liver.

<em>Interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) has been implicated in the pathogenesis of Sjögren's syndrome and systemic lupus erythematosus. Ro52, which was recently identified as an E<em>3</em> ligase with anti-proliferative and pro-apoptotic properties, is a major autoantigen targeted in both these conditions. Microarray analyses have indicated up-regulation of Ro52 by IFN-<em>alpha</em>, and the objective of the present study was to address the potential link between IFN-<em>alpha</em> and Ro52. To investigate the influence of IFN-<em>alpha</em> on Ro52 protein levels and cellular localization, we generated a panel of monoclonal antibodies to different domains of Ro52. These novel monoclonal antibodies were characterized by immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay using cell lysates, recombinant Ro52 protein, and synthetic peptides. Ro52 was up-regulated in HeLa cells and human B cells at the messenger RNA and protein levels in response to IFN-<em>alpha</em> stimulation as detected by reverse transcriptase polymerase chain reaction and Western blot. After up-regulation, Ro52 translocated from the cytoplasm to the nucleus. The nuclear translocation of Ro52 was observed after staining with generated monoclonal antibodies specific for both the RING, coiled-coil, and B<em>3</em>0.2 domains of Ro52 and the nuclear translocation of Ro52 preceded IFN-<em>alpha</em>-induced apoptotic cell death detected by caspase-<em>3</em> and TUNEL staining in the treated cultures. In conclusion, our data show that IFN-<em>alpha</em> first induces up-regulation of Ro52 protein and then prompts translocation of the up-regulated Ro52 protein in to the nucleus. The translocation precedes apoptosis of the IFN-<em>alpha</em> exposed cells, suggesting a role for Ro52 in mediating the anti-proliferative or pro-apoptotic effects of the autoimmune-related cytokine IFN-<em>alpha</em>.

Evidence shows that the CD<em>3</em>8 molecule, recently involved in the two main features of asthma, bronchial hyper-responsiveness and airway inflammation, could represent a new potential therapeutic target for asthma. In this study, we investigated whether glucocorticoid (GC), the most effective treatment for lung diseases, can affect CD<em>3</em>8 expression in human airway smooth muscle (ASM) cells treated with different pro-inflammatory cytokines, such as tumor necrosis factor-<em>alpha</em> (TNF<em>alpha</em>) and <em>interferons</em> (IFNs). We found that CD<em>3</em>8 expression induced by TNF<em>alpha</em> alone was completely abrogated by fluticasone (100 nM), dexamethasone (1 microM), or budesonide (100 nM). In contrast, the synergistic induction of CD<em>3</em>8 by the combination of TNF<em>alpha</em> with IFNgamma or IFNbeta, but not with IL-1beta or IL-1<em>3</em>, was completely insensitive to the GC inhibitory effects. We also found that TNF<em>alpha</em> and IFNgamma impaired GC responsiveness by inhibiting steroid induced both 1) GR<em>alpha</em>-DNA binding activity and 2) GC-responsive element-(GRE)-dependent gene transcription. Although levels of the GC receptor (GR) <em>alpha</em> isoform remained unchanged, expression of GRbeta, the dominant-negative GR isoform, was synergistically increased by TNF<em>alpha</em> and IFNgamma with a GR<em>alpha</em>/GRbeta ratio of 1 to <em>3</em>. More importantly, fluticasone failed to induce GRE-dependent gene transcription and to suppress TNF<em>alpha</em>-induced CD<em>3</em>8 expression in ASM cells transfected with constitutively active GRbeta. We conclude that, upon pro-inflammatory cytokine stimulation, CD<em>3</em>8 expression becomes insensitive to GC action by a mechanism involving the up-regulation of GRbeta isoform, thus providing a novel in vitro cellular model to dissect GC resistance in primary cells.

mRNA expression of two recently described human beta-defensins (hBD-<em>3</em> and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-<em>3</em> and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-<em>3</em> and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-<em>3</em> and hBD-4 mRNA, with higher expression levels of hBD-<em>3</em> mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. <em>Interferon</em> (IFN)-gamma, but not tumour necrosis factor (TNF)-<em>alpha</em> or IL-1beta, augmented hBD-<em>3</em> mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-<em>3</em> and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-<em>3</em> mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-<em>3</em> and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.

Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of <em>interferon</em> (IFN)-<em>alpha</em> and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (<em>3</em>) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection.

The initiation of antituberculosis treatment in patients with severe tuberculosis may be accompanied by clinical deterioration and even death before any improvement occurs. To investigate this phenomenon, newly diagnosed human immunodeficiency virus-negative adults with severe tuberculosis were followed for the first 42 days of standard short-course therapy. Clinical status, serum lactate, plasma cytokine, and plasma cytokine receptor levels were monitored on days 0, <em>3</em>, and 7 and then weekly for up to 42 days. Following 7 days of antituberculosis therapy, a significant transient decrease in mean Karnofsky score (P < .001), a concomitant increase in serum lactate (P = .06), a decrease in patient weight (P = .02), and an increase in plasma tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) concentrations (P = .04) were observed. Plasma levels of soluble interleukin-2 receptor, <em>interferon</em>-gamma, interleukin-6, and TNF-<em>alpha</em> receptor decreased over the 42-day study period. These observations suggest that increases in plasma TNF-<em>alpha</em> levels may be associated with clinical deterioration observed early in the treatment of severe tuberculosis.

Seventy-nine subjects (19 women and 60 men) with chronic viral hepatitis were studied to determine the role of hepatic iron and its biochemical correlates in determining response to <em>interferon</em> <em>alpha</em> therapy. Each subject was treated for 6 months with <em>interferon</em> <em>alpha</em>. A total of 45 (57%) subjects achieved either a full or partial response. No differences between responders and non-responders were evident for the type of hepatitis, age, initial alanine aminotransferase, serum iron, total iron binding capacity, %sat, or ferritin. In contrast, the hepatic iron content of non-responders was almost twice that of responders (1156 +/- 28<em>3</em> micrograms/g dry weight vs. 6<em>3</em>8 +/- 118; p < 0.05). Hepatic iron correlated with total iron binding capacity (r = 0.4<em>3</em>5) and ferritin (r = 0.585). This study showed that: 1) the hepatic iron content of responders is less than that of non-responders, 2) the relationships of hepatic iron with %sat and ferritin in patients with viral hepatitis are weak, and <em>3</em>) hepatic iron content predicts a response to <em>interferon</em> therapy.

We have previously reported a link between a deficient Th1 response to Leishmania amazonensis (La) parasites and profound impairments in the cytokine/chemokine network at early stages of the infection. To define the molecular basis of these deficiencies, we focused on early and intracellular events in La-infected dendritic cells (DCs) in this study. La amastigote-infected DCs were less mature and less potent antigen-presenting cells (APC) than their promastigote-infected counterparts, as judged by the lower expression of CD40 and CD8<em>3</em>, suppressed cytokine expression (IL-12p40 and IL-10), reduced effectiveness for priming CD4+ T cells from naïve or infected mice. Infection with La promastigotes, but not amastigotes, triggered transient expression of IL-12p40 by DC. Both forms of parasites markedly suppressed IL-12p40, IL-12p70, and IL-6 production and increased IL-10 production when DCs were treated with LPS, IFN-gamma/LPS or IFN-<em>alpha</em>/LPS as positive stimuli. Of note, pre-infection of DCs with live amastigotes resulted in multiple alterations in innate signaling pathways, including degradation of STAT2, decreased phosphorylation of STAT1, 2, <em>3</em> and ERK1/2, and markedly reduced expression of <em>interferon</em> regulatory factor-1 (IRF-1) and IRF-8, some of which were partially reversed by pretreatment of parasites with proteasome or protease inhibitors. The impaired IL-12 production in infected DCs was not attributed to increased IL-10 production. Together, our data suggest that La parasites, especially in their intracellular forms, have evolved unique strategies to actively down-regulate early innate signaling events, resulting in impaired DC function and Th1 activation.

Death-associated protein kinase (DAPK) was identified as a mediator of <em>interferon</em> (IFN)-induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB-Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin <em>3</em> (Cul<em>3</em>) via its Kelch-repeat domain and BTB domain, respectively. The KLHL20-Cul<em>3</em>-ROC1 E<em>3</em> ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20-mediated DAPK ubiquitination is suppressed in cells receiving IFN-<em>alpha</em> or IFN-gamma, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN-induced autophagy. This study identifies KLHL20-Cul<em>3</em>-ROC1 as an E<em>3</em> ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E<em>3</em> ligase, in IFN-induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.

<em>Interferons</em> regulate the expression of a large number of mammalian genes, including the major histocompatibility antigen genes. To investigate the mechanisms involved in <em>interferon</em> action, we have analyzed the ability of murine H-2Ld and H-2Dd DNA sequences to control the responses to <em>interferon</em>. The results indicate that <em>interferon</em> regulation of class I gene expression is complex and involves at least two mechanisms that are dependent on class I sequences located upstream and downstream to the transcription initiation site. In transfected mouse L cells, both of these regions are required for full enhancement of class I gene expression, with the major portion of the response controlled by the sequences located <em>3</em>' to the transcription initiation site. The fine-mapping analysis of the 5' region-encoded response also suggests that recombinant <em>alpha</em> and gamma <em>interferons</em> may exert their effects on class I gene expression by using different cis-acting regulatory sequences.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U9<em>3</em>7). Oligonucleotides HIV<em>3</em>1 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-<em>3</em>') and HIV<em>3</em>8 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-<em>3</em>') were designed to interact with the transcription initiation site (-16 to + 1<em>3</em>) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a <em>3</em>' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+<em>3</em>-<em>3</em>') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The <em>3</em>' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV<em>3</em>1 or HIV<em>3</em>8 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U9<em>3</em>7, was treated with 10 microM HIV<em>3</em>8. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to <em>alpha</em>-<em>interferon</em> induction resulting from oligonucleotide treatment. Both HIV<em>3</em>1 and HIV<em>3</em>8 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.