Vaccination with naked DNA elicits cellular and humoral immune responses that have a T helper cell type 1 bias. However, plasmid vectors expressing large amounts of gene product do not necessarily induce immune responses to the encoded antigens. Instead, the immunogenicity of plasmid DNA (pDNA) requires short immunostimulatory DNA sequences (ISS) that contain a CpG dinucleotide in a particular base context. Human monocytes transfected with pDNA or double-stranded oligonucleotides containing the ISS, but not those transfected with ISS-deficient pDNA or oligonucleotides, transcribed large amounts of interferon-alpha, interferon-beta, and interleukin-12. Although ISS are necessary for gene vaccination, they down-regulate gene expression and thus may interfere with gene replacement therapy by inducing proinflammatory cytokines.

Serum levels of interleukin-1 (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and C-reactive protein (CRP) were investigated in patients with chronic liver diseases (CLD) and correlated with the type of underlying disease and various clinical and laboratory parameters. Two hundred sixty-four patients suffering from various CLD were studied; 136 cases presented with liver cirrhosis, and 128 patients were in the noncirrhotic stage of their underlying liver diseases. Serum levels of IL-1 beta, IL-6, TNF-alpha, IFN-gamma, and CRP were elevated in patients with CLD. Endogenous cytokine patterns in CLD were stage dependent and only marginally affected by the type of underlying disease. The cirrhotic group of CLD patients showed higher serum levels in IL-1 beta, IL-6, TNF-alpha, and CRP than did noncirrhotic cases, and these differences reached the level of statistical significance. IL-1 beta and TNF-alpha values were closely correlated but did not correlate with IL-6 levels. Elevated concentrations of cytokines represent a characteristic feature of CLD regardless of underlying disease. This and the apparent stage-dependency suggest that enhanced endogenous cytokine levels represent a consequence of liver dysfunction rather than of inflammatory disease.

BACKGROUND

To characterize the role of intestinal epithelial cells in mucosal host defense, we have examined constitutive cytokine expression and regulated expression of interleukin (IL)-8 by human colonic epithelial cells.

METHODS

Cytokine expression by the human colonic epithelial cell lines, T84, Caco-2, SW620, and HT29 was assessed by using polymerase chain reaction amplification of reverse-transcribed RNA. Regulated IL-8 expression was analyzed by nuclear run-off assays, Northern blot analysis, and enzyme-linked immunosorbent assay.

RESULTS

The cell lines constitutively expressed messenger RNA (mRNA) for IL-8 and transforming growth factor beta 1. In addition, some cell lines expressed mRNA for IL-1 alpha, IL-1 beta, IL-10 and tumor necrosis factor alpha (TNF alpha). None of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, or interferon gamma. Cell lines secreted IL-8 either constitutively or after stimulation with the physiological agonists TNF alpha, IL-1 beta, or lipopolysaccharide. Increased IL-8 secretion after TNF alpha stimulation of T84 cells was accompanied by increased IL-8 mRNA levels and an increased transcription rate of the IL-8 gene. IL-8 was preferentially secreted at the basolateral surface of polarized T84 cells. In further studies, freshly isolated human colon epithelial cells also secreted IL-8.

CONCLUSIONS

These results support the notion of bidirectional communication between intestinal epithelial cells and mucosal immune and inflammatory cells.

Mammary gland factor (MGF) is a transcription factor discovered initially in the mammary epithelial cells of lactating animals. It confers the lactogenic hormone response to the milk protein genes. We reported recently the isolation of the cDNA encoding MGF. MGF is a novel member of the cytokine-regulated transcription factor gene family. Members of this gene family mediate interferon alpha/beta and interferon gamma induction of gene transcription, as well as the response to epidermal growth factor and interleukin-6, and have been named signal transducers and activators of transcription (Stat). The name Stat5 has been assigned to MGF. We studied the mechanisms involved in the prolactin activation of Stat5 in COS cells co-transfected with cDNA encoding Stat5 and the prolactin receptor. Prolactin treatment of the transfected cells caused activation of Stat5 within 5-10 min. This activation does not require ongoing protein synthesis. Tyrosine kinase inhibitors prevent Stat5 activation in transfected COS cells. Treatment of recombinant Stat5 with a tyrosine-specific protein phosphatase in vitro abolishes its DNA binding activity. Prolactin stimulation of transfected cells induces Stat5 phosphorylation on tyrosine. Phosphorylation of in vitro transcribed and translated Stat5 with the Jak2 tyrosine kinase, but not with fyn, lyn or lck, confers DNA binding activity. The prolactin response of the beta-casein milk protein gene promoter can be observed in COS cells transfected with cDNA vectors encoding Stat5 and the long form of the prolactin receptor. The short form of the prolactin receptor is unable to promote Stat5 phosphorylation and confer transcriptional induction in COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In humans, the type I interferon (IFN) family consists of 13 IFN-alpha subtypes, IFN-beta and IFN-omicron the newly discovered IFN-like family consists of IFN-lambda1, -lambda2 and -lambda3. We have investigated the expression of type I and lambda IFN genes following virus infections or Toll-like receptor (TLR) triggering in monocyte-derived DC (MDDC) and plasmacytoid DC (pDC). We found that all IFN-alpha, -beta, -omicron and -lambda subtypes are expressed in influenza-virus-infected MDDC or pDC. Conversely, differential type I IFN gene transcription was induced in MDDC and pDC stimulated by specific TLR agonists. TLR-9 stimulation by CpG DNA induced the expression of all IFN-alpha, -beta, -omicron and -lambda subtypes in pDC, whereas TLR-4 stimulation by LPS, or TLR-3 stimulation by poly I:C, induced only IFN-beta and IFN-lambda gene expression in MDDC. The expression pattern of IFN regulatory factor (IRF)-5 and IRF-7 in MDDC and pDC was also determined. IRF-5 was constitutively expressed in the two DC subsets whereas IRF-7 was constitutive in pDC but its expression was induced along MDDC maturation. Overall, our data indicate that the coordinated expression of IFN-lambda with IFN-beta would be of crucial importance for the maturation of DC.

Toll-like receptors (TLR) play a key role in innate immunity. To examine the ability of diverse TLRs to modulate hepatitis B virus (HBV) replication, HBV transgenic mice received a single intravenous injection of ligands specific for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. All of the ligands except for TLR2 inhibited HBV replication in the liver noncytopathically within 24 h in a alpha/beta interferon-dependent manner. The ability of these TLR ligands to induce antiviral cytokines at the site of HBV replication suggests that TLR activation could represent a powerful and novel therapeutic strategy for the treatment of chronic HBV infection.

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.

OBJECTIVE

To evaluate the ability of mesenchymal stem cells (MSCs), a subset of adult stem cells from bone marrow, to cure experimental autoimmune encephalomyelitis.

METHODS

The outcome of the injection of MSCs, in mice immunized with the peptide 139-151 of the proteolipid protein (PLP), was studied analyzing clinical and histological scores of treated mice. The fate of MSCs labeled with the green fluorescent protein was tracked in vivo by a photon emission imaging system and postmortem by immunofluorescence. The modulation of the immune response against PLP was studied through the analysis of in vivo T- and B-cell responses and by the adoptive transfer of MSC-treated encephalitogenic cells.

RESULTS

MSC-treated mice showed a significantly milder disease and fewer relapses compared with control mice, with decreased number of inflammatory infiltrates, reduced demyelination, and axonal loss. In contrast, no evidence of green fluorescent protein-labeled neural cells was detected inside the brain parenchyma, thus not supporting the hypothesis of MSCs transdifferentiation. In vivo, PLP-specific T-cell response and antibody titers were significantly lower in MSC-treated mice. When adoptively transferred, encephalitogenic T cells activated against PLP(139-151) in the presence of MSCs induced a milder disease compared with that induced by untreated encephalitogenic T cells. These cells showed decreased production of interferon-gamma and tumor necrosis factor-alpha and did not proliferate on antigen recall, and thus were considered anergic.

CONCLUSIONS

Overall, these findings suggest that the beneficial effect of MSCs in experimental autoimmune encephalomyelitis is mainly the result of an interference with the pathogenic autoimmune response.

Type 2 nitric oxide synthase (iNOS or NOS2) was originally described as an enzyme that is expressed in activated macrophages, generates nitric oxide (NO) from the amino acid L-arginine, and thereby contributes to the control of replication or killing of intracellular microbial pathogens. Since interferon (IFN)-gamma is the key cytokine for the induction of NOS2 in macrophages and the prototypic product of type 1 T-helper cells, high-level expression of NOS2 has been regarded to be mostly restricted to the adaptive phase of the immune response. In this review, we summarize data that demonstrate a prominent role of NOS2/NO also during innate immunity. During the early phase of infection with the intracellular pathogen Leishmania major, focally expressed NOS2/NO not only exerts antimicrobial activities but also controls the function of natural killer cells and the expression of cytokines such as IFN-gamma or transforming growth factor-beta. Some of these effects result from the function of NOS2/NO as an indispensable co-factor for the activation of Tyk2 kinase and, thus, for interleukin-12 and IFN-alpha/beta signaling in natural killer cells.

An adjuvant role for certain short bacterial immunostimulatory DNA sequences (ISSs) has recently been proposed on the basis of their ability to stimulate T helper-1 (Th1) responses in gene-vaccinated animals. We report here that noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens. The ISS-DNAs suppress IgE synthesis, but promote IgG and interferon-gamma (IFN-gamma) production. They furthermore initiate the production of IFN-gamma, IFN-alpha, IFN-beta, and interleukins 12 and 18, all of which foster Th1 responses and enhance cell-mediated immunity. Consideration should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.

Several dengue (DEN) virus vaccines are in development; however, the lack of a reliable small animal model in which to test them is a major obstacle. Because evidence suggests that interferon (IFN) is involved in the human anti-DEN virus response, we tested mice deficient in their IFN functions as potential models. Intraperitoneally administered mouse-adapted DEN 2 virus was uniformly lethal in AG129 mice (which lack alpha/beta IFN and gamma IFN receptor genes), regardless of age. Immunized mice were protected from virus challenge, and survival times increased following passive transfer of anti-DEN polyclonal antibody. These results demonstrate that AG129 mice are a promising small animal model for DEN virus vaccine trials.

Dermatomyositis has been modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B and T helper cell muscle infiltration, antibody and complement-mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. To further understand the pathophysiology of dermatomyositis, we used microarrays, computational methods, immunohistochemistry and electron microscopy to study muscle specimens from 67 patients, 54 with inflammatory myopathies, 14 with dermatomyositis. In dermatomyositis, genes induced by interferon-alpha/beta were highly overexpressed, and immunohistochemistry for the interferon-alpha/beta inducible protein MxA showed dense staining of perifascicular, and, sometimes all myofibers in 8/14 patients and on capillaries in 13/14 patients. Of 36 patients with other inflammatory myopathies, 1 patient had faint MxA staining of myofibers and 3 of capillaries. Plasmacytoid dendritic cells, potent CD4+ cellular sources of interferon-alpha, are present in substantial numbers in dermatomyositis and may account for most of the cells previously identified as T helper cells. In addition to an adaptive immune response, an innate immune response characterized by plasmacytoid dendritic cell infiltration and interferon-alpha/beta inducible gene and protein expression may be an important part of the pathogenesis of dermatomyositis, as it appears to be in systemic lupus erythematosus.

BACKGROUND

By using a novel experimental system for the study of reactive fibroblast differentiation at the molecular level, we describe the induction of the alpha-isoform of smooth muscle actin by transforming growth factor-beta (TGF-beta 1) in human normal breast gland fibroblasts.

METHODS

The experimental system allowed fibroblasts to plate and remain quiescent and nonreactive but at the same time stay sensitive to environmental cues for more than 2 weeks after explantation. Most important, the biological activity of growth factors and cytokines could be studied without cells entering the cell cycle, thus serving as a model for stromal reactions with little cell turnover.

RESULTS

By use of double-labeling immunocytochemistry, isoelectric focusing, two-dimensional gel electrophoresis, and fluorography, evidence is presented that the effect of TGF-beta 1 is dose-dependent with a maximal response above 80 pg/ml and a course of 6 days. No other growth factor/cytokine tested (platelet-derived growth factor, interleukin-1, insulin-like growth factor-1, acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, and interferon-gamma) could induce alpha-smooth muscle actin on their own or potentiate the effect of TGF-beta 1. In an inhibitory assay, only basic fibroblast growth factor was found to prevent the action of TGF-beta 1. The relative contribution of TGF-beta 1-like activity to carcinoma cell induced alpha-smooth muscle actin in fibroblasts was deciphered using TGF-beta neutralizing antibodies and medium conditioned by two different breast carcinoma cell lines. Conditioned medium elicited a fibroblast response indistinguishable from that obtained with exogenously added TGF-beta 1, and indeed neutralization attributed the entire response to the sole effect of secreted TGF-beta 1-like activity.

CONCLUSIONS

These results provide a strategy for the molecular characterization of epithelial-stromal interaction in breast neoplasia.

Human immunodeficiency virus (HIV) infection of brain macrophages and astroglial proliferation are central features of HIV-induced central nervous system (CNS) disorders. These observations suggest that glial cellular interactions participate in disease. In an experimental system to examine this process, we found that cocultures of HIV-infected monocytes and astroglia release high levels of cytokines and arachidonate metabolites leading to neuronotoxicity. HIV-1ADA-infected monocytes cocultured with human glia (astrocytoma, neuroglia, and primary human astrocytes) synthesized tumor necrosis factor (TNF-alpha) and interleukin 1 beta (IL-1 beta) as assayed by coupled reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and biological activity. The cytokine induction was selective, cell specific, and associated with induction of arachidonic acid metabolites. TNF-beta, IL-1 alpha, IL-6, interferon alpha (IFN-alpha), and IFN-gamma were not produced. Leukotriene B4, leukotriene D4, lipoxin A4, and platelet-activating factor were detected in large amounts after high-performance liquid chromatography separation and correlated with cytokine activity. Specific inhibitors of the arachidonic cascade markedly diminished the cytokine response suggesting regulatory relationships between these factors. Cocultures of HIV-infected monocytes and neuroblastoma or endothelial cells, or HIV-infected monocyte fluids, sucrose gradient-concentrated viral particles, and paraformaldehyde-fixed or freeze-thawed HIV-infected monocytes placed onto astroglia failed to induce cytokines and neuronotoxins. This demonstrated that viable monocyte-astroglia interactions were required for the cell reactions. The addition of actinomycin D or cycloheximide to the HIV-infected monocytes before coculture reduced,>> 2.5-fold, the levels of TNF-alpha. These results, taken together, suggest that the neuronotoxicity associated with HIV central nervous system disorders is mediated, in part, through cytokines and arachidonic acid metabolites, produced during cell-to-cell interactions between HIV-infected brain macrophages and astrocytes.

Alpha/beta interferon (IFN) and gamma IFN exert widely overlapping biological effects. Still, mice with individually inactivated alpha/beta or gamma receptors exhibit variably severely reduced resistance to infection and altered immune responses. To investigate to what extent the two IFN systems are functionally redundant, we generated mice with a combined receptor defect (AG129 mice). Like mice with individual mutations, AG129 mice had no apparent anomalies, confirming that in the mouse the IFN system is not essential for normal development. These mice showed an additive phenotype with respect to antiviral defense and exhibited an increased susceptibility to lymphocytic choriomeningitis virus (LCMV) and notably vaccinia virus infection. Because of unlimited replication and subsequent rapid exhaustion of cytotoxic T lymphocyte (CTL) precursors, these mice were unable to mount a CTL response to LCMV. CD8(+)-mediated immunopathology was absent in LCMV-infected mice, and virus persisted. Vaccinia virus replicated much faster in AG129 mice, and a 10(4)-fold lower dose of vaccinia virus was sufficient to prime these mice. With the normal priming dose of 10(6) PFU, cytopathic effects and overwhelming infection possibly causing partial exhaustion of CTL interfered with the anti-vaccinia virus response. Even though global antiviral immunoglobulin G (IgG) titers were within normal ranges, the IgG subclass distribution was heavily biased toward IgG1.

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1beta, TNF-alpha, and IFN-gamma in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.

Experimental autoimmune encephalomyelitis (EAE) in the Lewis rat is a self-limited inflammatory process localized to the central nervous system that is induced by the injection of myelin basic protein (MBP) in adjuvant. Oral administration of MBP suppresses EAE, and this suppression is mediated by CD8+ T cells that adoptively transfer protection and suppress both in vitro and in vivo by the release of transforming growth factor (TGF) beta after antigen-specific triggering. Furthermore, oral tolerance to MBP is enhanced by the concomitant oral administration of lipopolysaccharide (LPS). The present study was undertaken to determine whether the disease course in EAE and its suppression by oral tolerization to MBP is associated with distinct patterns of cytokine expression in the target organ. Detailed immunohistology of the brain was performed at the peak of clinical disease (day 14 after immunization) and after recovery (day 18) in control (ovalbumin [OVA]-fed), MBP-fed, and MBP plus LPS-fed animals. Brains from OVA-fed animals at the peak of disease showed perivascular infiltration with activated mononuclear cells which secreted the inflammatory cytokines interleukins (IL) 1, 2, 6, 8, TNF-alpha, and interferon gamma. The inhibitory cytokines TGF-beta and IL-4, and prostaglandin E2 (PGE2) were absent. In MBP orally tolerized animals there was a marked reduction of the perivascular infiltrate and downregulation of all inflammatory cytokines. In addition, there was upregulation of the inhibitory cytokine TGF-beta. In MBP plus LPS orally tolerized animals, in addition to upregulation of TGF-beta and reduction of inflammatory cytokines, there was enhanced expression of IL-4 and PGE2, presumably secondary to activation of an additional population of immunoregulatory cells. In OVA-fed animals that had recovered (day 18), staining for inflammatory cytokines diminished, and there was the appearance of TGF-beta and IL-4. These results suggest that suppression of EAE, either induced by oral tolerization or that which occurs during natural recovery is related to the secretion of inhibitory cytokines or factors that actively suppress the inflammatory process in the target organ.

A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-alpha, -beta, and -gamma for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-alpha and -beta protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-alpha or -beta decreases viral RNA levels greater than 1, 000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-gamma has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA.

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.

Virus-specific CD4 T cells are endowed with multiple functions, such as cytokine production, CD40 ligand (CD40L) expression (associated with the costimulation of CD8 and B cells), and degranulation (associated with cytotoxic potential). Here, we used antiviral CD4 T cells present in human blood to evaluate the relationship between cytokine production and other functions of CD4 T cells. Antiviral CD4 T cells specific for a virus causing persistent infection, cytomegalovirus (CMV), and two viruses causing nonpersistent infections, influenza virus and the smallpox vaccine virus (vaccinia virus), were studied. CD4 T cells specific for each of the viruses produced all seven possible combinations of the cytokines gamma interferon (IFN-gamma), interleukin-2, and tumor necrosis factor alpha. Cells producing three or two cytokines (triple producers and double producers) represented nearly 50% of the total response to each of the viruses. Triple producers expressed the highest levels of cytokines per cell, and single producers expressed the lowest. Following stimulation, higher frequencies of triple producers than single producers expressed CD40L. Only CMV-specific CD4 T cells underwent degranulation. However, higher frequencies of CMV-specific triple producers than single producers showed this functional characteristic. In contrast to the functional phenotypes, the memory phenotypes of triple producers and IFN-gamma single producers did not differ. These results demonstrate a strong positive association between the cytokine coproduction capacity of a virus-specific CD4 T cell and its other functional characteristics and suggest that vaccines should aim to elicit T cells that coproduce more than one cytokine.

Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-alpha and IFN-beta, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti-IFN-alpha/beta antibody to purified splenic DCs in vitro partially blocked the "spontaneous" activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-gamma, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators.

Worldwide, chronic hepatitis B virus (HBV) infection is the primary cause of cirrhosis and hepatocellular carcinoma and is one of the ten leading causes of death. Traditionally, people with chronic HBV infection have been identified with blood tests for HBV antigens and antibodies. Recently, another group of patients with chronic HBV infection has been identified by sensitive, molecular testing for HBV DNA. Members of this group are often referred to as having occult hepatitis B because they are HBV-DNA positive, but hepatitis B surface antigen negative. Occult hepatitis B occurs in a number of clinical settings. In this review, we examine occult hepatitis B in people co-infected with hepatitis C, in whom occult hepatitis B has been associated with advanced fibrosis and diminished response to interferon alpha. Although much more research is needed, existing reports justify a heightened awareness of the medical importance and means of testing for occult hepatitis B.

Rat astrocytes, immunologically competent glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharide-stimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and alpha- and beta-interferons. Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems.