Interferon (IFN) regulatory factor 1 (IRF-1) and IRF-2 were originally identified as transcription factors involved in the regulation of the IFN system. IRF-1 functions as a transcriptional activator, while IRF-2 represses IRF-1 function. More recently, evidence has been provided that IRF-1 and IRF-2 manifest antioncogenic and oncogenic properties, respectively, and that loss of one or both of the IRF-1 alleles may be critical for the development of human hematopoietic neoplasms. Both factors show a high degree of structural similarity in their N-terminal DNA-binding domains, and previous studies suggested that IRF-1 and IRF-2 bind to similar or identical cis elements within type I IFN (IFN-alpha and -beta) and IFN-inducible genes. However, the exact recognition sequences of these two factors have not yet been determined; hence, the spectrum of the IRF-responsive genes remains unclear. In this study, we determined the DNA sequences recognized by IRF-1 and IRF-2, using a polymerase chain reaction-assisted DNA-binding site selection method. We report that sequences selected by this method and the affinities for each sequence were virtually indistinguishable between IRF-1 and IRF-2. We confirm the presence of two contiguous IRF recognition sequences within the promoter region of the IFN-beta gene and of at least one such sequence in all of the IFN-inducible genes examined. Furthermore, we report the presence of potential IRF sequences in the upstream region of several genes involved in cell growth control.

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.

ISG15 is an interferon (IFN)-alpha/beta-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections. A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-beta, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.

TAK-242, a small-molecule antisepsis agent, has shown to suppress lipopolysaccharide (LPS)-induced inflammation. In this study, we demonstrate that TAK-242 is a selective inhibitor of Toll-like receptor (TLR)-4 signaling. TAK-242 almost completely suppressed production of nitric oxide (NO) or tumor necrosis factor (TNF)-alpha induced by a TLR4-specific ligand, ultra-pure LPS, in mouse RAW264.7, human U-937 and P31/FUJ cells, whereas this agent showed little effect on other TLR ligands, Pam(3)CSK(4) (TLR1/2), peptidoglycan (TLR2/6), double strand RNA (TLR3), R-848 (TLR7) and CpG oligonucleotide (TLR9). Furthermore, TAK-242 potently inhibited nuclear factor (NF)-kappaB activation induced by ultra-pure LPS in HEK293 cells transiently expressing TLR4 and co-receptors, myeloid differentiation protein-2 (MD2) and CD14, whereas this agent showed little effect on other TLRs, TLR1/2, TLR2/6, TLR3, TLR5, TLR7 and TLR9. TAK-242 also inhibited ligand-independent NF-kappaB activation resulting from over-expression of TLR4. Although chimera receptors, which are consist of the extracellular domain of CD4 and the intracellular domain of human or mouse TLR4, showed constitutive NF-kappaB activation, TAK-242 potently inhibited the signaling from CD4-TLR4 chimera receptors. In contrast, the NF-kappaB activation mediated by TLR4 adaptors, myeloid differentiation factor 88 (MyD88), TIR-associated protein (TIRAP), Toll/IL-1R homology (TIR)-domain-containing adaptor protein-inducing interferon-beta (TRIF) or TRIF-related adaptor molecule (TRAM) was not affected by TAK-242. TAK-242 is therefore a selective inhibitor of signaling from the intracellular domain of TLR4 and represents a novel therapeutic approach to the treatment of TLR4-mediated diseases.

Innate cytokine responses are important mediators of early defense against infections. Certain of their effects can be delivered directly to activate protective mechanisms in infected cells. Others activate innate immune cells, including natural killer (NK) cells and macrophages, to mediate defense. Still others shape adaptive immune responses. The compositions and magnitudes of innate cytokine responses are modulated, by the nature of the infectious agent, to facilitate accessing of the anti-microbial defense functions most beneficial in defense against the particular infection. In the context of viral infections, interferons alpha and beta (IFN-alpha/beta) are induced to high levels, and help to mediate and regulate immune responses most effective against this class of agents. The cytokines induce anti-viral mechanisms in infected cells, negatively regulate interleukin 12 expression, and activate NK cell-mediated lysis. Protective development of adaptive immunity to viral infections includes prominent CD8 T cell expansion and activation, and IFN-alpha/beta can mediate functions with the potential to promote these T cell responses. Together, the characteristics define regulation of unique or unique prominent defense mechanisms in place to fight off viral infections.

The interferons (IFNs) and their receptors represent a subset of the class 2 alpha-helical cytokines that have been in chordates for millions of years. This brief review focuses on the discovery and purification of interferons, cloning of human IFN-alpha and IFN-beta, interferon receptors, activities and therapeutic uses of interferons, and the side effects of interferons.

Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3-kb RNA designated IFN-beta 2 and distinct from the 0.9-kb IFN-beta 1 mRNA. In vitro transcription--translation mapping of the full-length IFN-beta 2 cDNA sequence, shows that it encodes a 23.7-kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN-beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN-beta 2. Specific immunoassays show that IFN-beta 2 secreted by DNA-transformed rodent cells is a processed 21-kd protein, whose activity is cross-neutralized by antibodies to human IFN-beta 1 but not to IFN-alpha or gamma. The immunoassay also demonstrates the induction of IFN-beta 2 secretion by fibroblasts in response to growth-regulatory cytokines, such as interleukin-1 and tumor necrosis factor. The function of this IFN-beta 2 as an autoregulatory inhibitor of cell growth is discussed.

OBJECTIVE

Data from clinical trials suggest a potential role for on-treatment monitoring of serum HBsAg titres during interferon-alpha (pegIFN) therapy in predicting virological responses. However, baseline HBsAg titres during the natural history of chronic hepatitis B (CHB) have not been well-characterized. We aimed to define the serum HBsAg titres during the different phases of CHB in a cohort of Asian patients infected with either genotype B or C HBV.

METHODS

Two-hundred and twenty patients were classified into immune-tolerant (IT), immune-clearance (IC), non/low-replicative (LR) or hepatitis B e antigen negative hepatitis (ENH) phases. Serum HBsAg was quantified using the ARCHITECT platform (Abbott Laboratories, Chicago, USA). Correlation of HBsAg titre with HBV DNA and serum ALT within each phase of infection was performed.

RESULTS

Median HBsAg titres were different between each phase of CHB (p=0.001): IT (4.53 log(10)IU/ml), IC (4.03 log(10)IU/ml), LR (2.86 log(10)IU/ml), and ENH (3.35 log(10)IU/ml). HBsAg titres were highest in the IT phase, and lowest in the LR phase. In general, median HBsAg titres were similar between genotypes B and C HBV. Serum HBsAg titres only correlated with HBV viral load in the IC phase. No correlation between the serum HBsAg level and ALT was observed.

CONCLUSIONS

This study demonstrated significant differences in median baseline serum HBsAg titres across the different phases of CHB. These results provide further insight into the HBV viral life cycle in the setting of the various phases of CHB. Baseline HBsAg quantification may help refine future treatment algorithms for both immune-modulator therapy and oral nucleos(t)ide analogue therapy.

Cytokines play an important role in controlling the homoeostasis of the immune system. Infection with HIV results in dysregulation of the cytokine profile in vivo and in vitro. During the course of HIV-1 infection secretion of T-helper type 1 (Th1) cytokines, such as interleukin (IL)-2, and antiviral interferon (IFN)-gamma, is generally decreased, whereas production of T helper type 2 (Th2) cytokines, IL-4, IL-10, proinflammatory cytokines (IL-1, IL-6, IL-8) and tumour necrosis factor (TNF)-alpha, is increased. Such abnormal cytokine production contributes to the pathogenesis of the disease by impairing cell-mediated immunity. A number of cytokines have been shown to modulate in vitro HIV-1 infection and replication in both CD4 T lymphocytes and cells of macrophage lineage. HIV-inductive cytokines include: TNF-alpha, TNF-beta, IL-1 and IL-6, which stimulate HIV-1 replication in T cells and monocyte-derived macrophages (MDM), IL-2, IL-7 and IL-15, which upregulate HIV-1 in T cells, and macrophage-colony stimulating factor, which stimulates HIV-1 in MDM. HIV-suppressive cytokines include: IFN-alpha, IFN-beta and IL-16, which inhibit HIV-1 replication in T cells and MDM, and IL-10 and IL-13, which inhibit HIV-1 in MDM. Bifunctional cytokines such as IFN-gamma, IL-4 and granulocyte-macrophage colony-stimulating factor have been shown to have both inhibitory and stimulatory effects on HIV-1. The beta-chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES are important inhibitors of macrophage-tropic strains of HIV-1, whereas the alpha-chemokine stromal-derived factor-1 suppresses infection of T-tropic strains of HIV-1. This review outlines the interactions between cytokines and HIV-1, and presents clinical applications of cytokine therapy combined with highly active antiretroviral therapy or vaccines.

The genes encoding nearly all of the serologically defined class II antigens of the major histocompatibility complex have been isolated. Three class II loci have been studied in great detail. The DR region contains a single alpha gene and 3 beta chain genes, 1 of which is a pseudogene. The DR alpha chain gene has been linked to a DR beta gene which encodes a beta protein which contains the serological determinant MT3. A second cosmid cluster contains 2 beta genes, 1 of which encodes the DR4 allospecificity. The identification of these genes has been made by the comparison of amino terminal sequences of DR molecules obtained from a DR4 cell line and the deduced protein sequences of the beta 1 exons from cosmid and phage clones. A conserved element including the promoter and signal sequence is found at the 5' end of each of the 3 DR beta genes. Additionally, this element occurs three more times in the DR region, raising the question of whether additional beta chain genes might be found. The DQ region contains 2 pairs of genes, 1 of which encodes the DQ antigen. The 2nd pair of genes, called DX alpha and beta, appears to be capable of expressing a DQ-related product, although, to date, there is no evidence for its expression. The DP region also contains 2 pairs of genes. One pair encodes the DP antigen while the 2nd alpha-beta pair is shown to be composed of pseudogenes. The location of polymorphic regions in these genes and aspects of their relationship to the serology, evolution, and function of the class II MHC are discussed. The control of expression of class II genes by gamma-interferon has been examined. The promoters of class II genes are characterized by two conserved sequences common to all alpha and beta chain genes as well as by conserved sequences specific for either alpha or beta chain genes. In addition to studies of expression by DNA-mediated gene transformation, a system for the gene transfer of MHC antigens utilizing transmissible retrovirus vectors is described. Retrovirus vectors have been used to transmit DR alpha, DR beta, and the invariant chain (gamma) sequences to recipient cells with resultant expression of these proteins.

We have previously shown that Toll-like receptor (TLR)-activated murine nonparenchymal liver cells [(NPC); Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)] can suppress hepatitis B virus (HBV) replication. Therefore, the aim of this study was to investigate whether HBV has the ability to counteract the TLR-mediated control of its replication. Freshly purified murine hepatocytes and NPCs obtained from C57BL6 mice were stimulated by TLR 1-9 ligands in the presence or absence of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV virions, or supernatants from HBV-producing HBV-Met cells, and HBV replication was suppressed by anti- hepatitis B virus X protein (HBx) small interfering RNA (siRNA) in HBV-Met cells. Supernatants were collected and tested for antiviral cytokines by viral protection assay. HBV gene expression and replication was analyzed by southern blot. RNA and proteins were analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) or western blot and enzyme-linked immunosorbent assay, respectively. Pretreatment of hepatocytes and NPCs with HBV-Met cells supernatants, HBsAg, HBeAg, or HBV virions almost completely abrogated TLR-induced antiviral activity, which correlated with suppression of interferon beta (IFN-beta) production and subsequent interferon-stimulated gene induction as well as suppressed activation of interferon regulatory factor 3 (IRF-3), nuclear factor kappa B (NF-kappaB), and extracellular signal-regulated kinase (ERK) 1/2. In HBV-infected HBV-Met cells, TLR stimulation did not induce antiviral cytokines in contrast to primary hepatocytes. TLR-stimulated expression of proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6)], and activation of IRF-3 was suppressed after up-regulation of HBV replication in HBV-Met cells. Accordingly, suppression of HBV replication by siRNA led to activation or expression of proinflammatory transcription factors and cytokines.

CONCLUSIONS

Our data indicate that HBV can suppress the TLR-induced antiviral activity of liver cells. This has major implications for the interaction between HBV and the immune system.

We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-alpha/beta) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-alpha/beta inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA-containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-alpha/beta triggers intracellular events that either inhibit the assembly of pgRNA-containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte.

Intrapulmonary activation of leukocytes and release of cellular mediators and enzymes are involved in the pathophysiology of the adult respiratory distress syndrome (ARDS). To investigate a possible role of local cytokines, we measured bronchoalveolar fluid (BALF) and plasma levels of tumor necrosis factor alpha (TNF-alpha) and its soluble inhibitors (sTNF-RI + RII), interleukin-1 beta (IL-1 beta), interferon-alpha (IFN-alpha), and granulocyte elastase in 14 patients at risk for ARDS and in 35 patients developing ARDS after trauma, sepsis, or shock. During clinical development of severe ARDS, BALF cytokines increased markedly: TNF-alpha from 116 +/- 36 to 10,731 +/- 5,048 pg/ml (mean +/- SEM), p = 0.001; sTNF-RI + RII from 3.7 +/- 1.4 to 24.6 +/- 2.6 ng/ml, p less than 0.05; and IL-1 beta from 7,746 +/- 5,551 to 42,255 +/- 19,176 pg/ml, p = 0.01. Plasma cytokines were not increased in most patients, nor were they correlated with the development or severity of ARDS. BALF elastase was higher in patients developing ARDS than in those at risk but not going into pulmonary failure (0.97 +/- 0.26 versus 0.28 +/- 0.13 U/ml, p = 0.026), and the highest values were observed in the early stages of severe ARDS (1.85 +/- 0.39 U/ml). BALF elastase levels correlated with IFN-alpha (r = 0.72, p less than 0.001). In conclusion, local release of TNF-alpha and IL-1 beta, possibly by pulmonary macrophages or other cells, and/or accumulation in the lung is associated with the development of ARDS.(ABSTRACT TRUNCATED AT 250 WORDS)

A huge amount of evidence has implicated amyloid beta (A beta) peptides and other derivatives of the amyloid precursor protein (beta APP) as central to the pathogenesis of Alzheimer's disease (AD). It is also widely recognized that age is the most important risk factor for AD and that the innate immune system plays a role in the development of neurodegeneration. Little is known, however, about the molecular mechanisms that underlie age-related changes of innate immunity and how they affect brain pathology. Aging is characteristically accompanied by a shift within innate immunity towards a pro-inflammatory status. Pro-inflammatory mediators such as tumour necrosis factor-alpha or interleukin-1 beta can then in combination with interferon-gamma be toxic on neurons and affect the metabolism of beta APP such that increased concentrations of amyloidogenic peptides are produced by neuronal cells as well as by astrocytes. A disturbed balance between the production and the degradation of A beta can trigger chronic inflammatory processes in microglial cells and astrocytes and thus initiate a vicious circle. This leads to a perpetuation of the disease.

In Fanconi anemia (FA) C mice tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) have key roles in the pathogenesis of bone marrow failure. In FA subjects TNF-alpha was found to be increased in the serum and overproduced by patient-derived B-cell lines. In acquired aplastic anemia, a disease in which, similarly to FA, marrow failure occurs, TNF-alpha and IFN-gamma act as late mediators of the stem cell damage and are overexpressed in patient marrow lymphocytes. This study evaluated in marrow mononuclear cells (MNCs) of patients with FA, the expression of negative modulators of the hematopoiesis, such as TNF-alpha, IFN-gamma, macrophage inflammatory protein 1alpha (MIP-1alpha), and surface Fas ligand, and the role of TNF-alpha on FA erythropoiesis in vitro. TNF-alpha and IFN-gamma were significantly overexpressed in stimulated marrow MNCs of FA patients as compared to healthy controls. MIP-1alpha and Fas ligand were undetectable in patients and controls. In bone marrow cultures, the addition of anti-TNF-alpha increased the size and significantly increased the number of erythroid colony-forming units and erythroid burst-forming units grown from FA patients but not from healthy controls. This indicates that FA subjects have a marrow TNF-alpha activity that inhibits erythropoiesis in vitro. TNF-alpha has a relevant role in the pathogenesis of erythroid failure in FA patients.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.

Cells respond to contact with human cytomegalovirus (HCMV) virions by initiating intracellular signaling and gene expression characteristic of the interferon (IFN)-responsive pathway. Herein, we demonstrate that a principal mechanism of HCMV-induced signal transduction is via an interaction of the primary viral ligand, glycoprotein B (gB), with its cellular receptor. Cells incubated with a purified, soluble form of gB resulted in the transcriptional upregulation of IFN-responsive genes OAS and ISG54 (encoding 2'-5' oligoadenylate synthetase and an IFN-stimulated gene product of 54 kDa) to a comparable level as virions or IFN. Gene induction was an immediate and direct response to gB which did not require de novo protein synthesis. Neither the initial virus attachment site, heparan sulfate proteoglycans, nor the IFN-alpha/beta or IFN-gamma receptors are involved in the response. Pleotropic protein phosphorylation was required for cellular gene induction, and the mitogen-activated protein kinases ERK1 and ERK2 were activated in response to the ligand. Together these data indicate that a principal means by which cytomegalovirus induces intracellular signaling and activation of the interferon-responsive pathway is via an interaction of gB with an as yet unidentified, likely novel cellular receptor that interfaces with the IFN signaling pathway.

The relationship between malaria-related outcomes and cytokine production in whole blood cultures associated with cellular immune responses and immunity to Plasmodium falciparum malaria was examined in a study in southern Ghana. Production of malaria-specific interferon (IFN)-gamma was associated with reduced risk of fever and clinical malaria. Protective IFN-gamma responses were induced by live schizonts but not by dead parasites. Production of malaria-specific tumor necrosis factor (TNF)-alpha was associated with reduced risk of fever during follow-up. Baseline levels of TNF-alpha and phytohemagglutinin (PHA)-induced interleukin (IL)-10 were positively associated with hemoglobin concentration. IL-12 production was associated with reduced risk of parasitemia. PHA-induced transforming growth factor-beta production was associated with reduced risk of fever during follow-up. High ratios of proinflammatory to anti-inflammatory cytokines were associated with increased risk of fever and higher hemoglobin concentrations. Thus, absolute levels and ratios of proinflammatory and anti-inflammatory cytokines influence susceptibility to infection, clinical disease, and anemia. These data contradict data from cross-sectional clinical studies and indicate a need for detailed analysis of the relationship between cellular immunity to malaria and resistance to disease.

An effective type I interferon (IFN-alpha/beta) response is critical for the control of many viral infections. Here we show that in vesicular stomatitis virus (VSV)-infected mouse embryonic fibroblasts (MEFs) the production of IFN-alpha is dependent on type I IFN receptor (IFNAR) triggering, whereas in infected mice early IFN-alpha production is IFNAR independent. In VSV-infected mice type I IFN is produced by few cells located in the marginal zone of the spleen. Unlike other dendritic cell (DC) subsets, FACS((R))-sorted CD11c(int)CD11b(-)GR-1(+) DCs show high IFN-alpha expression, irrespective of whether they were isolated from VSV-infected IFNAR-competent or -deficient mice. Thus, VSV preferentially activates a specialized DC subset presumably located in the marginal zone to produce high-level IFN-alpha largely independent of IFNAR feedback signaling.

OBJECTIVE

Standard therapy of patients with chronic hepatitis C virus (HCV) infected with HCV genotype-2 or -3 is the combination of pegylated interferon-alpha and ribavirin for 24 weeks. Whether shorter treatment durations are possible for these patients without compromising sustained virologic response rates is unknown.

METHODS

Patients chronically infected with HCV-2 (n = 39), HCV-2/3 (n = 1), or HCV-3 (n = 113) were treated with peginterferon-alpha-2a (180 microg/wk) plus ribavirin 800-1200 mg/day. HCV RNA was quantitatively assessed after 4 weeks. Patients with a rapid virologic response (HCV RNA below 600 IU/mL) were randomized for a total treatment duration of 16 (group A) or 24 weeks (group B). All patients with HCV RNA>> or =600 IU/mL at week 4 (group C) were treated for 24 weeks. End-of-treatment and sustained virologic response were assessed by qualitative RT-PCR (sensitivity 50 IU/mL).

RESULTS

Only 11 of 153 patients (7%) were allocated to group C. End-of-treatment and sustained virologic response rates were 94% and 82%, (group A), 85% and 80% (group B), and 73% and 36% (group C), respectively. In patients infected with genotype HCV-3 and high viral load (>800,000 IU/mL), a significant lower sustained virologic response rate was found than in patients infected with HCV-3 and a viral load lower or equal to 800,000 IU/mL (59% vs 85%, respectively; P = .003).

CONCLUSIONS

In HCV-2 and -3 (low viral load)-infected patients who have a rapid virologic response, treatment for 16 weeks with peginterferon-alpha-2a and ribavirin is sufficient. In patients infected by HCV-3 (high viral load), longer treatment may be necessary.

Cytokines are thought to cause the depression of cytochrome P-450 (CYP)-associated drug metabolism in humans during inflammation and infection. We have examined the role of five cytokines, i.e., interleukin-1 beta, interleukin-4, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma, on the expression of CYP1A2, CYP2C, CYP2E1, CYP3A, and epoxide hydrolase in primary human hepatocyte cultures. Steady state P-450 and epoxide hydrolase mRNA levels, as well as ethoxyresorufin-O-deethylase and nifedipine oxidation activities, which are mainly supported by CYP1A1/1A2 and CYP3A, respectively, were measured. Interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha were found to be the most potent depressors of P-450 enzymes. After 3 days of treatment, both mRNA levels and enzyme activities were depressed, typically by at least 40%, whatever the cytokine and the enzyme considered. Interferon-gamma also suppressed CYP1A2 and CYP2E1 mRNA levels and ethoxyresorufin-O-deethylase activity but had no effect on CYP3A and epoxide hydrolase mRNAs. In addition, interleukin-4 had the opposite effect, compared with other cytokines, on CYP2E1 mRNA, which was increased up to 5-fold; ethoxyresorufin-O-deethylase and nifedipine oxidation activities were not significantly affected. These results provide the first demonstration that various cytokines act directly on human hepatocytes to affect expression of major P-450 genes and that a wide range of responses can be observed among the enzymes for a given cytokine, suggesting that different regulatory mechanisms may be involved.

Interferons (IFNs) have established antitumor action; the mechanism underlying this effect is, however, not yet clear. To probe the possible contribution of inhibition of angiogenesis, we have assessed angiogenesis in the mouse initiated by either human or murine tumor cell lines. Whether test cells were inoculated in the dermis or tumor fragments were grafted onto the cornea, tumor-induced angiogenesis (TIA) was inhibited by IFNs. TIA was also inhibited by the potent IFN inducer polyriboinosinic-polyribocytidylic acid. The effect of IFN was species specific; human IFNs inhibited human tumors and mouse IFNs inhibited murine tumors. This effect suggested that in contrast to other angiogenesis inhibitors, IFNs modulated the signal for angiogenesis produced by the tumor cells. Tumor cells treated in vitro with homologous IFN were significantly (P less than 0.005) less competent to initiate angiogenesis than were untreated cells. Inhibition of angiogenesis was achieved whether vascular response was assessed 1 or 3 days after tumor cell inoculation, suggesting that antiangiogenesis activity was independent of the antiproliferative effects of IFNs. To further substantiate this, L1210 leukemia cells, resistant to the antiproliferative effects of IFNs, were treated with 500 units/ml IFN-beta. IFN had no effect on their proliferation, but in four separate experiments, L1210R cells were impaired in their ability to induce angiogenesis. Thus, inhibition of TIA by IFNs was species specific, occurred at least partly by modulation of the signal inducing angiogenesis, and was expressed in the absence of antiproliferative effects. IFNs also inhibited immunologically induced angiogenesis, whether initiated by allogeneic lymphocytes (LIA) or by the mouse's own T-cells in response to an exogenous antigen (sheep RBC). LIA was markedly suppressed by treatment of host mice with homologous IFN-beta. For example, mean vessel counts induced by allogeneic mouse lymphocytes were decreased from 22.8 +/- 1.4 (SE) to 12.5 +/- 0.8 (P less than 0.0001); mouse IFN-beta had no corresponding effect on xenogeneic human lymphocytes (mean vessel counts decreased to 21.7 +/- 2.6 from 22.7 +/- 2.0). Treatment with human IFN-alpha, -beta, or -gamma in vitro or host mice in vivo reduced the ability of inoculated human peripheral blood lymphocytes to initiate xenogeneic LIA. Inhibition of LIA required a lower dose and/or a shorter incubation period than that needed to modulate TIA. Treatment of the donor of the allogeneic spleen cells in vivo with murine IFN or inducers also resulted in lesser LIA.(ABSTRACT TRUNCATED AT 400 WORDS)

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.