We examined the immunobiological responses to Histoplasma capsulatum in lungs of gamma interferon (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 alpha (TNF-alpha) 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 3 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-alpha 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.

To elucidate whether the fraction of CD28(-) T cells within the CD4(+) T-cell population is a major source of Th1-like and proinflammatory cytokine production driving Wegener's granulomatosis (WG) granuloma formation, we analyzed the phenotype and functional characteristics of peripheral blood CD4(+)CD28(-) T cells and of T cells in granulomatous lesions of 12 patients with active WG. Surface markers and intracytoplasmic cytokine and perforin expression were assessed by flow cytometry. Cytokine secretion was measured by enzyme-linked immunosorbent assay. Immunohistological studies demonstrated interferon-gamma and tumor necrosis factor-alpha cytokine positivity attributable to CD4(+)CD28(-) T cells in granulomatous lesions. Peripheral blood CD4(+)CD28(-) T cells expressed CD57, also found on natural killer cells, and intracytoplasmic perforin. They were generally CD25 (interleukin-2 receptor)-negative. CD18 (adhesion molecule beta(2)-integrin) was strongly up-regulated on CD4(+)CD28(-) T cells, whereas only a minority of CD4(+)CD28(+) T cells expressed CD18. CD4(+)CD28(-) T cells appeared as a major source of interferon-gamma and tumor necrosis factor-alpha. In contrast, CD4(+)CD28(+) T cells were able to produce and secrete a wider variety of cytokines including interleukin-2. One-quarter of CD4(+)CD28(+) T cells expressed the activation marker CD25, but they lacked perforin. Thus, CD4(+)CD28(-) T cells appeared more differentiated than CD4(+)CD28(+) T cells. They displayed Th1-like cytokine production and features suggestive of the capability of CD4(+) T-cell-mediated cytotoxicity. CD4(+)CD28(-) T cells may be recruited into granulomatous lesions from the blood via CD18 interaction, and may subsequently promote monocyte accumulation and granuloma formation through their cytokine secretion in WG.

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 p53 positively regulates the transcription of TLR3, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p53 site in the TLR3 promoter. TLR3 expression was lower in HCT116 p53(-/-) cells than in HCT116 p53(+/+) cells. Activation of p53 by 5-fluorouracil increased the TLR3 mRNA in epithelial cell lines with wild-type p53 but not in cell lines harboring mutant p53. Knockdown of p53 by small interfering RNA decreased the TLR3 expression. TLR3 mRNA was also lower in liver and intestine of p53(-/-) mice than in p53(+/+) mice. Furthermore, the poly(I-C)-induced phosphorylation of IkappaB-alpha, nuclear translocation of NF-kappaB, and phosphorylation of interferon regulatory transcription factor 3, were drastically reduced in HCT116 p53(-/-) cells, indicating a dysregulation of the two signaling pathways governed by TLR3. Consequently, induction of interleukin-8 and beta interferon after poly(I-C) stimulation was impaired in HCT116 p53(-/-) cells. These results suggest that p53 influences TLR3 expression and function and highlight a role of p53 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-alpha 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-3 (stromelysin-1) in astrocytes. In addition, activated microglia release matrix metalloproteinase-9. The 'coactivator' of monocytic phagocytes, interferon-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.

Tricyclic antidepressants (TCAs) are widely used in treating depressive disorders. It has been demonstrated that, for instance, IL-1 beta and IL-6 inhibit the HPA axis, which plays a role in the development of depressions. Therefore. we were interested in investigating how TCAs influence cytokine release by T lymphocytes and monocytes respectively. Cells were incubated with either 5 microM clomipramine, 15 microM imipramine or 20 microM citalopram. IL-2 release was suppressed to 60% of the control values by clomipramine and imipramine (p = 0.001; p = 0.000), but citalopram was found to cause a much weaker inhibition (only 18%) (p = 0.16). INF-gamma release was affected to a lower degree than IL-2 release, and imipramine (34%) (p = 0.054) was more potent than clomipramine (24%) (p = 0.16) and citalopram (12%) (p = 0.059) in this case. Monocytes incubated with TCA for 4 h exhibited only limited inhibition of IL-1 beta and IL-6 release, i.e., 6-25% for all three compounds. The corresponding value for TNF-alpha release was 20-45% inhibition, with citalopram being the weakest inhibitor. After 10 h of monocytes to LPS exposure, all three compounds exerted a strong inhibition of IL-1 beta and TNF-alpha release, i.e., 60-70% with p-values below 0.012 for all of them. However the inhibition of IL-6 release was less than 35%. Citalopram was equality as potent as imipramine and clomipramine in inhibiting IL-6 release after long-term exposure of monocytes to LPS. All three TCAs elevated intracellular cAMP concentrations significantly in T lymphocytes and monocytes (p < 0.001).

mdr1a-deficient mice lack P-glycoprotein and spontaneously develop colitis with age. Helicobacter spp. are gram-negative organisms that have been associated with colitis in certain mouse strains, but Helicobacter spp. have been excluded as contributing to the spontaneous colitis that develops in mdr1a-/- mice. We wished to determine whether infection with either H. bilis or H. hepaticus would accelerate the development of inflammatory bowel disease (IBD) in mdr1a-/- mice. We found that H. bilis infection induced diarrhea, weight loss, and IBD in mdr1a-/- mice within 6 to 17 weeks post-inoculation and before the expected onset of spontaneous IBD. Histopathology of H. bilis-induced IBD included crypt hyperplasia, inflammatory cell infiltrates, crypt abscesses, and obliteration of normal gut architecture. Reverse transcription-polymerase chain reaction and Taqman analysis from colonic tissue showed increased transcripts for interferon-gamma and interleukin-10 from H. bilis-infected colitic mdr1a-/- mice. Additionally, mesenteric lymph nodes had increased cellularity with expansion of CD4+ and CD8+ T cells and B cells and increased proliferation to soluble H. bilis antigens with elaboration of interferon-gamma, tumor necrosis factor-alpha and interleukin-10. In contrast, H. hepaticus infection of mdr1a-/- mice did not accelerate disease but rather delayed the onset of spontaneous colitis which was milder in severity. mdr1a-/- mice infected with Helicobacter spp. may provide a useful tool to explore the pathogenesis of microbial-induced IBD in a model with a presumed epithelial cell "barrier" defect.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, drug-induced reaction that involves both the skin and the viscera. Evidence for reactivation of herpes family viruses has been seen in some DRESS patients. To understand the immunological components of DRESS and their relationship to viral reactivation, we prospectively assessed 40 patients exhibiting DRESS in response to carbamazepine, allopurinol, or sulfamethoxazole. Peripheral blood T lymphocytes from the patients were evaluated for phenotype, cytokine secretion, and repertoire of CD4+ and CD8+ and for viral reactivation. We found Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), or HHV-7 reactivation in 76% of the patients. In all patients, circulating CD8+ T lymphocytes were activated, exhibited increased cutaneous homing markers, and secreted large amounts of tumor necrosis factor-alpha and interferon-gamma. The production of these cytokines was particularly high in patients with the most severe visceral involvement. In addition, expanded populations of CD8+ T lymphocytes sharing the same T cell receptor repertoire were detected in the blood, skin, liver, and lungs of patients. Nearly half of these expanded blood CD8+ T lymphocytes specifically recognized one of several EBV epitopes. Finally, we found that the culprit drugs triggered the production of EBV in patients' EBV-transformed B lymphocytes. Thus, cutaneous and visceral symptoms of DRESS are mediated by activated CD8+ T lymphocytes, which are largely directed against herpes viruses such as EBV.

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-alpha and IFN-beta genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

The co-localization of activated macrophages and damaged neurons observed in brain injury and degenerative brain diseases may hint to macrophage-induced neuronal cytotoxicity. Recently, macrophages have been found to secrete neurotoxic molecules such as radical oxygen intermediates and glutamate, the latter interacting with N-methyl-D-aspartate (NMDA) receptors. As shown in the present study, brain macrophages termed microglial cells co-cultured with differentiated cerebellar neurons excert potent neurotoxic effects. Neurotoxicity is unlikely to be due to cytokines since tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6 and interferon (IFN)-alpha/IFN-beta/IFN-gamma had no such effects. In contrast, when treating neurons with H2O2 or oxygen radical-generating systems cytotoxicity was induced. Furthermore, microglia were found to produce O2- and H2O2 when triggered with phorbol 12-myristate 13-acetate. However, in co-cultures of neurons and microglia, oxygen-radical scavengers catalase and superoxide dismutase, failed to protect neurons from microglia-induced killing. Moreover, when using undifferentiated neurons which are susceptible to H2O2 but not to NMDA receptor-dependent killing, microglia did not destroy the neurons. Thus, the amount of reactive oxygen intermediates produced by microglia in co-culture do not reach the critical concentrations required for neurotoxicity. As dibenzocyclohepteneimide, an antagonist to NMDA receptors neutralized neurotoxicity in microglia-neuronal co-cultures, excitatory amino acids released by microglia are suggested to compose the major determinant of neurotoxicity.

Measles virus, a member of the Morbillivirus family, infects millions of people each year despite the availability of effective vaccines. The V protein of measles virus is an important virulence factor that can interfere with host innate immunity by inactivating alpha/beta interferon (IFN-alpha/beta) and IFN-gamma signaling through protein interactions with signal transducer and activator of transcription proteins STAT1 and STAT2. Here we demonstrate that although STAT1 interference results from protein interactions within a V protein N-terminal region encompassed by amino acids 110 to 130, detection of STAT1 interaction and IFN-gamma signaling inhibition requires the presence of cellular STAT2. Cell-specific variability in STAT1 interference was observed to correlate with V protein expression level. A more direct target for measles virus V protein-mediated IFN-alpha/beta evasion is STAT2. Results indicate that the widely conserved C-terminal zinc finger domain of measles virus V protein is both necessary and sufficient to bind STAT2 and disrupt IFN-alpha/beta signal transduction. Mutagenesis and molecular modeling define a contact surface for STAT2 association that includes aspartic acid residue 248 as critical for STAT2 interference and IFN antiviral immune suppression. These findings clearly define the molecular determinants for measles virus IFN evasion and validate specific targets as candidates for therapeutic intervention.

To understand the processes regulating inflammation and fibrosis in the human lung, we characterized the effects of recombinant interleukin-1, tumor necrosis factor, and gamma interferon on fibroblast proliferation, collagen production, interleukin-1-alpha production, interleukin-1-beta production, and interleukin-6-production. These studies demonstrated the existence of complex cytokine networks by which inflammatory cells regulate fibroblast function and fibroblasts, in turn, feed back to regulate inflammatory cell function. They also demonstrated that, in this complex network, the effect of an individual cytokine varies with the state of activation of the target cell, the presence of other cytokines in the local microenvironment, and the ability of the target cell to produce bioactive autocoids such as prostaglandins. Aspects of this cytokine network are discussed and a testable hypothesis for granuloma and abscess formation is detailed.

Dengue virus (DENV) infects human immune cells in vitro and likely infects dendritic cells (DCs) in vivo. DENV-2 productive infection induces activation and release of high levels of chemokines and proinflammatory cytokines in monocyte-derived DCs (moDCs), with the notable exception of alpha/beta interferon (IFN-alpha/beta). Interestingly, DENV-2-infected moDCs fail to prime T cells, most likely due to the lack of IFN-alpha/beta released by moDCs, since this effect was reversed by addition of exogenous IFN-beta. Together, our data show that inhibition of IFN-alpha/beta production by DENV in primary human moDCs is a novel mechanism of immune evasion.

BACKGROUND

The herpes simplex virus type 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase, which is encoded within the HSV-1 latency-associated locus. When ICP0 is not synthesized, the HSV-1 genome is acutely susceptible to cellular repression. Reciprocally, when ICP0 is synthesized, viral replication is efficiently initiated from virions or latent HSV-1 genomes. The current study was initiated to determine if ICP0's putative role as a viral interferon (IFN) antagonist may be relevant to the process by which ICP0 influences the balance between productive replication versus cellular repression of HSV-1.

RESULTS

Wild-type (ICP0+) strains of HSV-1 produced lethal infections in scid or rag2-/- mice. The replication of ICP0- null viruses was rapidly repressed by the innate host response of scid or rag2-/- mice, and the infected animals remained healthy for months. In contrast, rag2-/- mice that lacked the IFN-alpha/beta receptor (rag2-/- ifnar-/-) or Stat 1 (rag2-/- stat1-/-) failed to repress ICP0- viral replication, resulting in uncontrolled viral spread and death. Thus, the replication of ICP0- viruses is potently repressed in vivo by an innate immune response that is dependent on the IFN-alpha/beta receptor and the downstream transcription factor, Stat 1.

CONCLUSIONS

ICP0's function as a viral IFN antagonist is necessary in vivo to prevent an innate, Stat 1-dependent host response from rapidly repressing productive HSV-1 replication. This antagonistic relationship between ICP0 and the host IFN response may be relevant in regulating whether the HSV-1 genome is expressed, or silenced, in virus-infected cells in vivo. These results may also be clinically relevant. IFN-sensitive ICP0- viruses are avirulent, establish long-term latent infections, and induce an adaptive immune response that is highly protective against lethal challenge with HSV-1. Therefore, ICP0- viruses appear to possess the desired safety and efficacy profile of a live vaccine against herpetic disease.

OBJECTIVE

Synovitis is a common feature of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the pattern of joint involvement differs in each disease. This study was undertaken to investigate the global gene expression profiles in synovial biopsy tissue from the swollen knees of untreated SLE patients (n = 6), RA patients (n = 7), and osteoarthritis (OA) patients (n = 6).

METHODS

Synovial biopsy samples were obtained from the affected knees of patients in the 3 groups by needle arthroscopy. Half of the material was used for extraction of total RNA, amplification of complementary RNA, and high-density oligonucleotide spotted hybridization arrays. On the remaining tissue samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical experiments were performed to confirm the microarray data.

RESULTS

SLE synovial biopsy tissue displayed a significant down-regulation of genes involved in extracellular matrix (ECM) homeostasis and a significant up-regulation of interferon-inducible (IFI) genes. Real-time RT-PCR experiments confirmed the up-regulation of selected IFI genes (IFI27, IFI44, and IFI44L) in the SLE synovial tissue. Immunohistochemical analyses showed that 3 molecules involved in ECM regulation, chondroitin sulfate proteoglycan 2, latent transforming growth factor beta binding protein 2, and fibroblast activation protein alpha, were significantly down-regulated in SLE synovium. In contrast, immunostaining for IFI27, Toll-like receptor 4, and STAT-1 resulted in higher quantitative scores in SLE synovial tissue, which could be attributed to the fact that the RA samples had a large population of inflammatory cell infiltrates that were negative for these markers.

CONCLUSIONS

Arthritis in SLE has a very distinct molecular signature as compared with that in OA and RA, characterized by up-regulation of IFI genes and down-regulation of genes involved in ECM homeostasis.

Type I interferon (IFN-alpha/beta) is expressed rapidly after infection and plays a key role in innate defense against pathogens. Recent studies have shown that a connection exists between IFN-alpha/beta and antigen-presenting dendritic cells (DCs) at two levels. Firstly, a specific DC precursor, the plasmacytoid pre-DC (p-preDC), was identified as a cell type able to secrete very high amounts of IFN-alpha/beta following stimulation with infectious agents. Secondly, IFN-alpha/beta has been shown to act as a differentiation/maturation factor for DCs. These findings will be discussed in association with evidence indicating that IFN-alpha/beta can enhance and modulate immune responses in vivo. Taken together, the available data suggest that IFN-alpha/beta serves as a link between the innate response to infection and the adaptive immune response.

The effect of human interferons (IFNs) (alpha, beta, and gamma) on the in vitro replication of AIDS viruses (LAV, HTLV-III, and ARV-2) in human peripheral blood lymphocytes was investigated. At the time of peak virus production, IFN-alpha preparations (leukocyte, Namalwa, alpha 1, and alpha 2) at 100 U/ml, suppressed LAV, HTLV-III, and ARV-2 replication as measured by reverse transcriptase (RT) activity by greater than 50%. This suppression was dose dependent and high dosages (500 U/ml) of IFN-alpha resulted in almost complete suppression of RT activities (77-99%). A low dose (100 U/ml) of IFN-beta suppressed all three AIDS viruses by 75%. In contrast, human IFN-gamma at a dose range from 100 U/ml to 500 U/ml had no significant effect on the production of infectious viruses. These results indicate that only IFN-alpha and -beta are effective against LAV, HTLV-III, and ARV-2 replication. A continuous supply of IFN appeared to be essential for the constant suppression of RT activity. In fact, upon termination of single IFN treatment, enhanced virus production resulted.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

BACKGROUND

In children with cerebral malaria (CM), serum chemokine levels and associated morbidity and mortality have not been characterized.

METHODS

Serum levels of the cytokines interleukin (IL)-1 beta , IL-6, IL-10, interferon (IFN)-gamma, and tumor necrosis factor-alpha and the chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) were measured in Ugandan children with CM, in children with uncomplicated malaria (UM), and in healthy children from the community, as control subjects (CCs).

RESULTS

Children with CM had lower levels of RANTES and higher levels of all other cytokines and chemokines than CCs (all P<.0001), and they had lower levels of RANTES (P=.004) and higher levels of IL-10 (P=.003), IFN-gamma (P=.007), and IL-1 beta (P=.05) than children with UM. Children with CM who died had lower levels of RANTES (P=.006) and higher of levels of IL-6 (P=.006), IL-10 (P=.01), IFN-gamma (P=.03), and MIP-1 beta (P=.008) than children who survived. After adjustment for other cytokine and chemokine levels, only low levels of RANTES were independently associated with mortality (P=.016). Levels of RANTES correlated with platelet count but were associated with mortality independently of platelet count.

CONCLUSIONS

The serum cytokine and chemokine profile of children who die of CM is similar to that of individuals who die of sepsis. Levels of RANTES are significantly lower in children with CM, and very low levels of RANTES are associated with mortality, independently of other cytokine and chemokine levels.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-alpha/beta, IFN-gamma, and TNF-alpha at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-alpha/beta or IFN-gamma, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-gamma, but not IFN-alpha/beta, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-alpha. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.

Chronic hepatitis B (CHB) infection is the major cause of hepatocellular carcinoma (HCC). Primary prevention of hepatitis B infection by vaccination is effective in reducing the incidence of HCC. In persons with CHB infection, the two accepted treatment modalities are interferon alpha (IFN-α) given subcutaneously for a limited period and nucleoside/nucleotide analogs given orally on a long-term basis. These treatments are effective in suppressing viral activity and improving disease markers in short-term studies. The long-term effect on the development of liver cancers with these two forms of treatment appears to be different. However, there are no studies directly comparing IFN-α and nucleoside/nucleotide analogs. Comparisons across studies are inevitably limited by differences in the baseline characteristics of the study cohorts. Long-term follow-up studies of IFN-α therapy show inconsistent results. The beneficial effect in reducing the development of liver cancer is observed mainly in treatment responders who have preexisting cirrhosis of the liver. The long-term studies of lamivudine (and adefovir) show a consistent reduction in the development of liver cancers in patients with, and without, cirrhosis. This beneficial effect is blunted by the development of resistance. The effects of the newer nucleoside/nucleotide analogs, with higher potency and minimal risk of resistance development, are, as yet, unknown.

OBJECTIVE

To evaluate whether remissions of chronic hepatitis B induced by alpha-interferon therapy are of long duration.

METHODS

Cohort study.

METHODS

Clinical Center of the National Institutes of Health, a tertiary referral center.

METHODS

Sixty-four patients with chronic hepatitis B were treated with alpha-interferon between 1984 and 1986.

METHODS

Patients were followed with frequent examinations and determinations of serum liver biochemical tests and hepatitis B virus (HBV) markers including hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA using blot hybridization and polymerase chain reaction.

RESULTS

Among 64 patients with chronic hepatitis B who were treated with alpha-interferon, 23 (36%) responded to treatment with loss of HBeAg and improvement in serum aminotransferases. All 23 have been followed for 3 to 7 years (mean, 4.3 years). During follow-up, 3 of 23 patients relapsed, with reappearance of HBeAg and abnormal serum aminotransferases, all within 1 year of therapy. The remaining 20 patients continued to have no detectable HBeAg or HBV DNA (using blot hybridization) in serum and to be asymptomatic for liver disease, although 3 had minimal elevations in serum aminotransferases. Thirteen patients (65%) became negative for HBsAg between 0.2 and 6 years (mean, 3 years) after loss of HBeAg. Although no patient had HBV DNA that was detectable by blot hybridization, the 7 patients who remained HBsAg positive all had HBV DNA in serum detected by polymerase chain reaction, but only 2 of 13 HBsAg-negative patients had viral genome using this method. Testing sequential samples indicated that HBV DNA detected by polymerase chain reaction usually disappeared at or around the time that test results for HBsAg became negative.

CONCLUSIONS

Remissions in chronic hepatitis B induced by alpha-interferon are of long duration and are followed, in most patients, by the loss of HBsAg and all evidence of residual virus replication.

For more than 50 years, experimental studies on fever have focused on a substance from leukocytes called leukocytic or endogenous pyrogen. Various investigators concluded that changes associated with infection--such as numbers of circulating leukocytes; levels of trace metals, amino acids, and hepatic proteins; and altered lymphocyte function--were also caused by endogenous leukocyte mediators. There was reasonable evidence that fever and these other changes were brought about through the action of a single endogenous pyrogen, now known as interleukin 1 (IL-1). Two forms of IL-1 have been cloned (IL-1 beta and IL-1 alpha), and studies of recombinant IL-1 preparations have confirmed that fever and the broad spectrum of host responses to infection and injury are indeed mediated by this substance. However, IL-1 is not the only leukocyte product that induces fever: tumor necrosis factor (cachectin) and interferon produce fever in humans and animals. Accordingly, the concept of a single endogenous pyrogen now requires modification. Nature has conferred the ability to produce fever on no fewer than three structurally distinct molecules. Investigators trying to determine what triggers the hypothalamus to initiate fever in a particular disease must now consider these three endogenous pyrogens, either alone or together, as mediators of fever.

Plasmacytoid dendritic cells (pDCs) competent to make type I interferon were rigorously defined as a Ly-6C(+) and CD11c(Lo) subset of the BBM) Fraction A. Otherwise similar Ly6C(-) cells expressed the natural killer (NK) markers DX5 and NK1.1. pDCs represented a stable, discrete, and long-lived population. Stem cells and early lymphoid progenitors (ELPs), but not prolymphocytes, were effective precursors of pDCs, and their differentiation was blocked by ligation of Notch receptors. Furthermore, pDCs were present in the BM of RAG1(-/-), CD127/IL-7Ra(-/-), and Pax5(-/-) mice. pDCs in RAG1/GFP knock-in mice could be subdivided, and immunoglobulin D(H)-J(H) rearrangements, as well as transcripts for the B-lineage-related genes Pax5, mb1/CD79a, ebf, and Bcl11a, were identified only in the green fluorescent protein-positive (GFP(+)) pDC1 subset. All pDCs expressed terminal deoxynucleotidyl transferase (TdT), the ETS transcription factor Spi-B, the nuclear factor-kappaB transcription factor RelB, toll-like receptor 9 (TLR9), and interferon consensus sequence binding protein (ICSBP)/interferon regulatory factor 8 (IRF-8) transcripts; lacked CD16 and granulocyte colony-stimulating factor receptor (G-CSFR); and were uniformly interleukin-7 receptor alpha (IL-7Ralpha(-)) AA4.1(Lo), CD27(-), Flk-2(Lo), c-Kit(-), DX-5(-), and CD11b(-), while CD4 and CD8alpha were variable. GFP(+) pDC1 subset was less potent than GFP(-) pDC2s in T allostimulation and production of tumor necrosis factor alpha (TNFalpha), interferon alpha (IFNalpha), and interleukin-6 (IL-6), while only pDC2s made IFNgamma and IL-12 p70. Thus, 2 functionally specialized subsets of pDCs arise in bone marrow from progenitors that diverge from B, T, and NK lineages at an early stage.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.