The ability of the human immunodeficiency virus (HIV) to replicate in CD+ T lymphocytes and mononuclear phagocytes(MP) is strongly influenced by immunoregulatory cytokines. In the T cell system, interleukin-2 (IL-2) provides a mitogenic signal leading to both cell proliferation and virus replication. Among other HIV-inductive cytokines, only tumor necrosis factor-<em>alpha</em> or -beta (TNF-<em>alpha</em>/-beta) have been shown thus far to trigger virus expression both in T cells and MP. The mechanism of action of TNF involves the activation of the cellular transcription factor NF-kB which binds to specific consensus sequences present in the enhancer region of the HIV proviral LTR. In addition, several other cytokines (including colony stimulating factors, IL-1, IL-<em>3</em>, and IL-6) have demonstrated upregulatory effects on HIV production in MP, whereas nonimmune <em>interferons</em> (INF-<em>alpha</em>/-beta) have been shown to suppress HIV replication in T cells and MP by acting at different phases in the virus life cycle. Finally, cytokines such as TGF-beta, IFN-gamma, and IL-4 have demonstrated either upregulatory or suppressive effects on virus expression depending on the experimental conditions. This scenario indicates that HIV expression is under the control of a complex network of immunoregulatory cytokines, in addition to its own endogenous regulatory proteins, suggesting that new pharmacologic strategies may be aimed at either mimicking or interrupting cytokine-dependent virus expression. In this regard, a number of different physiologic and pharmacologic agents capable of interfering with cytokine-mediated events, including glucocorticoids, anti-oxidants, such as N-Acetyl-L-Cysteine (NAC), and retinoic acid (RA) have already been shown to profoundly affect HIV replication in vitro.

BACKGROUND

<em>Interferons</em> (IFNs) are a class of cytokines which confer cellular resistance against viral infections. Type I (IFN-<em>alpha</em> and -beta) and type II (IFN-gamma) IFNs utilize distinct receptors, the stimulation of which results in the induction of downstream target genes. These target genes usually contain within their promoter region an IFN responsive element, termed ISRE (IFN stimulated response element) which binds a heterotrimeric transcription factor, ISGF<em>3</em> (IFN-stimulated gene factor <em>3</em>) consisting of p48 (ISGF<em>3</em> gamma), Stat1 (Signal transducers and activators of transcription-1; <em>alpha</em> or beta), and Stat2. The ISRE sequence overlaps with that of IRF-E which binds another IFN-inducible factor, IRF-1 (IFN regulatory factor-1).

RESULTS

We generated mice lacking p48 by gene targeting. We show that p48 plays an essential role in both type I and type II IFN responses; activation of IFN-inducible genes and establishment of the antiviral state by IFN-alpha or -gamma are both severely impaired, and ISRE-binding activities induced by both IFNs are absent in the p48-negative embryonic fibroblasts (EFs). Furthermore, we generated mice deficient for both p48 and IRF-1 and found that at least one IFN-inducible gene is dependent on both factors.

CONCLUSIONS

p48 and IRF-1 do not perform redundant functions in the cell, but rather complement one another in both type I and II IFN responses.

The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the <em>interferon</em> regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-<em>3</em>, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10. 6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-<em>3</em>-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-<em>3</em> mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-1<em>3</em>-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-<em>3</em> ORF encodes a 7<em>3</em>-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-<em>3</em> functioned as a dominant-negative mutant of both IRF-<em>3</em> and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-<em>3</em> in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active <em>interferons</em>. These results suggest that by targeting IRF-<em>3</em> and IRF-7, which play a critical role in the activation of <em>alpha</em>/beta <em>interferon</em> (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.

Uncomplicated influenza in humans, horses or swine is characterized by massive virus replication in respiratory epithelial cells, inflammation and an abrupt onset of general and respiratory disease. There is now growing evidence that the so-called early cytokines produced at the site of infection mediate many of the clinical and pathological manifestations. Among these cytokines are <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), interleukin-1 (IL-1) <em>alpha</em> and beta, interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte-attracting chemokines. This paper reviews: (1) in vivo examinations of the cytokine profiles during influenza in mice, humans or swine; (2) in vivo data on the probable role of these cytokines; and (<em>3</em>) selected in vitro data on cytokine induction by the influenza virus. Examination of respiratory secretions of experimentally infected humans or animals revealed a brisk and concurrent rise in several of the cytokines mentioned. Moreover, peak cytokine levels directly correlated with virus replication and disease. In the mouse model, specific anti-cytokine strategies have further confirmed the role of cytokines in body temperature changes, anorexia and lung inflammation. However, cytokines were clearly not the only factor contributing to disease, and they seemed to be essential for resolution of the infection. Though influenza virus was shown to induce cytokines in cell culture, in vitro experiments have also revealed conflicting data. Furthermore, the viral genes or products that are responsible for cytokine induction are unknown. Exactly this information would make important contributions to our understanding of the genetic basis of viral virulence.

The type I <em>interferons</em>--<em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) and <em>interferon</em>-beta (IFN-beta)--are critical for protection against viruses during the acute stage of viral infection [1,2]. Furthermore, type I <em>interferons</em> have been implicated as important mediators in the regulation of lymphocyte development [<em>3</em>], immune responses [4,5] and the maintenance of immunological memory of cytotoxic T cells [6,7]. The different IFN-<em>alpha</em> subtypes are encoded by 12 genes in the mouse [8] whereas IFN-beta is encoded by only one gene [9]. IFN-<em>alpha</em> and IFN-beta have a high degree of sequence homology and are thought to interact with the same surface receptor on target cells [10,11]. As an approach to analysing the different biological functions of IFN-<em>alpha</em> and IFN-beta, we have generated a mouse strain with an inactivated IFN-beta gene. We report here that embryonic fibroblasts from such mice produce neither IFN-beta nor IFN-<em>alpha</em> upon Sendal virus infection, whereas the production of IFN-<em>alpha</em> by leukocytes from the same strain of mice is intact. IFN-<em>alpha</em> production in embryonic fibroblasts from IFN-beta-/- mice could be rescued by 'priming' the cells using exogenous IFN-beta. These results imply a unique role for IFN-beta in the induction of type I <em>interferons</em> in peripheral tissues.

BACKGROUND

During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia.

METHODS

In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by <em>3</em> months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation.

RESULTS

Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,<em>3</em>-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation.

CONCLUSIONS

These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

BACKGROUND

Acute kidney injury (AKI) is associated with high mortality rates. New biomarkers that can identify subjects with early AKI (before the increase in serum creatinine) are needed to facilitate appropriate treatment. The purpose of this study was to test the role of serum cytokines as biomarkers for AKI and prolonged mechanical ventilation.

METHODS

This was a case-control study of children undergoing cardiac surgery. AKI was defined as a 50% increase in serum creatinine from baseline within <em>3</em> days. Levels of serum interleukin (IL)-1beta, IL-5, IL-6, IL-8, IL-10, IL-17, <em>interferon</em> (IFN)-gamma, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured using a bead-based multiplex cytokine kit in conjunction with flow-based protein detection and the Luminex LabMAP multiplex system in 18 cases and 21 controls. Levels of IL-6 and IL-8 were confirmed with single-analyte ELISA; IL-18 was also measured with single-analyte ELISA.

RESULTS

IL-6 levels at 2 and 12 hours after cardiopulmonary bypass (CPB) and IL-8 levels at 2, 12 and 24 hours were associated with the development of AKI using the Wilcoxon rank-sum test and after adjustment for age, gender, race, and prior cardiac surgery in multivariate logistic regression analysis. In patients with AKI, IL-6 levels at 2 hours had excellent predictive value for prolonged mechanical ventilation (defined as mechanical ventilation for more than 24 hours postoperatively) by receiver operator curve (ROC) analysis, with an area under the ROC curve of 0.95. IL-8 levels at 2 hours had excellent predictive value for prolonged mechanical ventilation in all patients. Serum IL-18 levels were not different between those with and without AKI.

CONCLUSIONS

Serum IL-6 and IL-8 values identify AKI early in patients undergoing CPB surgery. Furthermore, among patients with AKI, high IL-6 levels are associated with prolonged mechanical ventilation, suggesting that circulating cytokines in patients with AKI may have deleterious effects on other organs, including the lungs.

OBJECTIVE

Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy resembling congenital viral infection that is characterized by basal ganglia calcifications, loss of white matter, cerebrospinal fluid (CSF) lymphocytosis, and elevated <em>interferon</em>-<em>alpha</em> levels in the CSF. Studies have shown that AGS is an autosomal-recessive disease linked to mutations in 5 genes, encoding the <em>3</em>'-repair DNA exonuclease 1 (TREX1), the <em>3</em> subunits of ribonuclease H2 (RNASEH2A-C), and sterile <em>alpha</em> motif domain and HD domain-containing protein 1 (SAMHD1). In this study we further characterized the phenotypic spectrum of this disease.

METHODS

Clinical and laboratory data were obtained from 26 patients fulfilling the clinical diagnostic criteria for AGS. Genomic DNA was screened for mutations in all 5 AGS genes by direct sequencing, and sera were analyzed for autoantibodies.

RESULTS

In 20 patients with AGS, 20 mutations, 12 of which were novel, were identified in all 5 AGS genes. Clinical and laboratory investigations revealed a high prevalence of features (some not previously described in patients with AGS) that are commonly seen in patients with systemic lupus erythematosus (SLE), such as thrombocytopenia, leukocytopenia, antinuclear antibodies, erythematous lesions, oral ulcers, and arthritis, which were observed in 12 (60%) of 20 patients with AGS. Moreover, the coexistence of AGS and SLE, was for the first time, demonstrated in 2 patients with molecularly proven AGS.

CONCLUSIONS

These findings expand the phenotypic spectrum of lupus erythematosus in AGS and provide further insight into its disease mechanisms by showing that activation of the innate immune system as a result of inherited defects in nucleic acid metabolism could lead to systemic autoimmunity.

BACKGROUND

Natural killer (NK) cells are highly efficient in the cellular immune response against malignant tumors without restriction of major histocompatibility complex. However clinical studies using autologous NK cells have been reported in only a very limited number of cases, due to the fact that selective NK expansion is difficult to achieve in this patient population. Here, we report the results of adoptive immunotherapy in patients with recurrent malignant gliomas using autologous NK cells that were expanded ex vivo by a novel method.

METHODS

Peripheral blood mononuclear cells (PBMCs) were prepared from patients with malignant gliomas, and were co-cultured with an irradiated human feeder cell line (HFWT) in RHAM-<em>alpha</em> medium supplemented with 5% autologous plasma and interleukin-2. The resulting NK cell-rich effector cells were injected into 9 patients (16 courses) with recurrent malignant glioma (6 cases of WHO grade-<em>3</em> glioma and <em>3</em> cases of grade-4 glioma).

RESULTS

The mean frequency of NK cells among lymphocytes was 82.2 +/- 10.5%. A combination of focal and intravenous injections was peformed in 10 courses. Intravenous injection alone was performed in 6 courses. Further, intravenous injection of low-dose interferon beta (6x10(6) IU/week) was performed as an adjuvant therapy in all courses to achieve maximum benefit for enrolled patients. Clinical evaluation demonstrated <em>3</em> PR, 2 MR, 4 NC and 7 PD in a total of 16 courses of treatment. Severe neurological toxicity was not observed in any of the patients.

CONCLUSIONS

It was demonstrated that NK cell-rich effector cells were expanded ex vivo from PBMCs in all nine cases of recurrent malignant glioma and that NK cell therapy was safe and partially effective in patients with recurrent malignant gliomas.

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-<em>3</em>, <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>), and IFN-gamma. Upon the stimulation of IL-6 or IL-<em>3</em>, Gab1 was found to form a complex with phosphatidylinositol (PI)-<em>3</em> kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp1<em>3</em>0, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp1<em>3</em>0 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp1<em>3</em>0-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp1<em>3</em>0, abrogated the interactions of Gab1 with SHP-2 and PI-<em>3</em> kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-<em>3</em> kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp1<em>3</em>0 and that SHP-2, PI-<em>3</em> kinase, and Ras are involved in Gab1-mediated ERK activation.

Calcium and its major downstream effector, calcium/calmodulin-dependent protein kinase II (CaMKII), are found to be important for the functions of immune cells. Lipopolysaccharide (LPS) has been shown to induce intracellular calcium release in macrophages; however, whether and how CaMKII is required for Toll-like receptor (TLR) signaling remain unknown. Here we demonstrate that TLR 4, 9, and <em>3</em> ligands markedly induce intracellular calcium fluxes and activate CaMKII-<em>alpha</em> in macrophages. Selective inhibition or RNA interference of CaMKII significantly suppresses TLR4, 9, <em>3</em>-triggered production of interleukin-6 (IL-6), tumor necrosis factor-<em>alpha</em>, and <em>interferon</em>-<em>alpha</em>/beta (IFN-<em>alpha</em>/beta) in macrophages. Coincidently, overexpression of constitutively active CaMKII-<em>alpha</em> significantly enhances production of the above cytokines. In addition to the activation of mitogen-activated protein kinase and nuclear factor kappaB pathways, CaMKII-<em>alpha</em> can directly bind and phosphorylate transforming growth factor beta-activated kinase 1 (TAK1) and IFN regulatory factor <em>3</em> (IRF<em>3</em>; serine on <em>3</em>86) via the N-terminal part of its regulatory domain. Therefore, CaMKII can be activated by TLR ligands, and in turn promotes both myeloid differentiating factor 88 and Toll/IL-1 receptor domain-containing adaptor protein-inducing IFN-beta-dependent inflammatory responses by directly activating TAK1 and IRF<em>3</em>. The cross-talk with the calcium/CaMKII pathway is needed for full activation of TLR signaling in macrophages.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, <em>3</em>, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1<em>3</em>74 genes were significantly upregulated and that <em>3</em>44<em>3</em> genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin <em>3</em> (PTX<em>3</em>), IL6, and <em>interferon</em>-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor <em>alpha</em>-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

The present study was undertaken to elucidate the mechanism for the antifibrotic effect of <em>interferon</em> gamma (IFN-gamma) in the bleomycin (BL)-mouse model of lung fibrosis. The expression of transforming growth factor (TGF-beta) and procollagen I and III and their mRNAs was investigated in the BL-mouse model of lung fibrosis with and without IFN-gamma treatment by Northern and slot blot analyses. Temporal changes in the content of procollagen and TGF-beta mRNAs in the lungs of mice receiving saline or BL by intratracheal route, with and without IFN-gamma treatment by intramuscular route, were quantitated. The level of TGF-beta mRNA increased rapidly and peaked at day 5, whereas the levels of mRNAs for procollagens <em>alpha</em> 1(I) and <em>alpha</em> 1(III) peaked at 10 days after BL instillation. The peak levels of these mRNAs in BL-treated animals were five- to sevenfold higher than those of the control. The increase in TGF-beta mRNA in the lungs of BL-treated mice preceded the increase in the synthesis of type I and type III procollagen mRNAs. BL treatment also increased the hydroxyproline content significantly from <em>3</em> to 14 days as compared to the corresponding saline control groups. A maximal increase to 447 micrograms/lung from 22<em>3</em> micrograms/lung in saline control was obtained at 10 days after instillation. Daily treatment with IFN-gamma markedly reduced the BL-induced increases in the mRNA levels of TGF-beta, and procollagen <em>alpha</em> 1(I) and <em>alpha</em> 1(III) without any effect on the lung level of beta-actin mRNA. IFN-gamma treatment also caused significant reduction in the BL-induced increase in the lung hydroxyproline content from 417 to 28<em>3</em> micrograms/lung at 7 days and from 447 to 264 micrograms/lung at 10 days. It may be concluded from the findings of the present study that the cellular mechanisms for the antifibrotic effect of IFN-gamma in the BL-mouse model of lung fibrosis are to initially downregulate the BL-induced overexpression of TGF-beta mRNA, and subsequently procollagen mRNAs, leading to a decreased collagen content.

To generate an ''off the shelf'' tissue-engineered heart valve, the cells would need to be of allogeneic origin. Here, we report the possibility of using human bone marrow-derived mesenchymal stem cells (MSCs) as a suitable allogeneic cell source for tissue-engineered heart valves. Proliferative responses of primary and primed CD4+ T cells to allogeneic MSCs were examined. A protein microarray system was used to detect soluble factors from supernatants collected from the T cell assays. MSCs are poor stimulators of primary and primed CD4+ T cell proliferation, despite provision of B7-1 trans-co-stimulation. MSCs not only directly inhibited primary and primed T cell responses to allogeneic peripheral blood mononuclear cells (PBMCs), but 24-h pre-culture of T cells with MSCs suppressed subsequent T cell proliferative responses to allogeneic PBMCs in a contact-dependent manner. Analysis of supernatants revealed a distinctly different cytokine profile after co-culture of T cells with MSCs than with PBMCs or endothelial cells. Pro-inflammatory Th1 cytokines interleukin (IL)-1<em>alpha</em> and beta, <em>interferon</em> (IFN)gamma, and tumor necrosis factor (TNF)<em>alpha</em> were downregulated, whereas, anti-inflammatory Th2 cytokines IL-<em>3</em>, IL-5, IL-10, and IL-1<em>3</em> and the Th2 chemokine I-<em>3</em>09, a chemoattractant for regulatory T cells, were upregulated. Further analysis revealed that after co-culture with MSCs, the T cells exhibited a regulatory phenotype (CD4+ CD25(lo) CD69(lo) FoxP<em>3</em>+). MSCs downregulate T cell responses through direct contact and secretion of anti-inflammatory and tolerogenic cytokines, which may involve the recruitment of regulatory T cells. This implies that allogeneic MSCs could be a suitable cell source for tissue engineering a heart valve.

Transcriptional regulation of the gene encoding a guanylate-binding protein (GBP) by the two <em>interferon</em> (IFN) types was studied. GBP gene transcription was regulated by <em>alpha</em> IFN in a manner identical to that of previously described IFN-stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis-dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the <em>alpha</em> IFN-induced active repression and reverses the <em>alpha</em> IFN-induced repressed state. Moreover, the <em>alpha</em> IFN-mediated repression of ISG54, a gene normally responsive to only <em>alpha</em> IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator <em>Interferon</em> Stimulated Gene Factor <em>3</em> (ISGF<em>3</em>) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types.

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I <em>interferon</em> (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and IRF-7 and in mice lacking the type I <em>alpha</em>/beta <em>interferon</em> receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I <em>interferon</em> response.

An upstream inverted repeat (IR) element mediates transcriptional activation of the <em>interferon</em> response factor-1 gene (IRF-1) by <em>interferon</em> (IFN)-<em>alpha</em> and IFN-gamma. IFN-<em>alpha</em> and IFN-gamma fail to induce IRF-1 in cells that lack signal transducer and activator of transcription 1 (STAT1), and STAT1 homodimers bind to IR elements in extracts of IFN-<em>alpha</em>-treated cells. We now report that STAT2 also plays an important role in the IFN-<em>alpha</em>-mediated transcriptional activation of the IRF-1 gene. A new factor, most likely a STAT1-STAT2 heterodimer, was detected with an IR probe in extracts of IFN-<em>alpha</em>-treated cells. STAT1 and STAT2 are already known to combine with p48, a DNA-binding protein, to form IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>), which binds to IFN-stimulated response elements (ISREs) distinct from the IR of the IRF-1 gene. In extracts of U2A cells, which lack p48, STAT1-STAT2 heterodimers were still formed, indicating that they do not contain p48. We manipulated the intracellular levels of STAT1-STAT2 heterodimers and STAT1 homodimers to examine their roles in the induction of IRF-1 by IFN-<em>alpha</em>. Although both dimers can induce IRF-1 transcription, the heterodimers are more potent and thus may be the major activators in vivo. Deletion analysis reveals that the C-terminal domain of STAT2 is important for transcriptional activation mediated by both STAT1-STAT2 heterodimers and ISGF<em>3</em>.

OBJECTIVE

After liver transplantation (LT) infection of the graft with the hepatitis C virus (HCV) is almost universal and chronic hepatitis and cirrhosis develop in a significant proportion of patients. One of the possible strategies to prevent HCV infection recurrence is to eradicate HCV before LT.

METHODS

We evaluated the efficacy and safety of antiviral therapy to prevent HCV recurrence in <em>3</em>0 HCV-cirrhotic patients awaiting LT. At the time of inclusion 15 patients were Child-Pugh A and 15 Child-Pugh B/C. The infecting genotype was 1b in 25 patients. Treatment with <em>interferon</em> <em>alpha</em>-2b <em>3</em> MU/day and ribavirin 800 mg/day was initiated when the expected time for LT was less than 4 months and continued until LT. The median duration of treatment was 12 weeks.

RESULTS

Nine patients (<em>3</em>0%) achieved a virological response and 21 did not respond to therapy. In nine (4<em>3</em>%) of the 21 non-responders viral load decreased>> or =2 log10 during treatment. A viral load decrease>> or = 2 log10 at week 4 of treatment was the strongest predictor of virological response. All nine virological responders have already undergone LT; six patients remain free of infection after a median follow-up of 46 weeks and HCV infection recurred in three patients after LT. In one of these patients HCV-RNA was still detectable in the explanted liver. Side effects were frequent and dose reduction was necessary in 19 (6<em>3</em>%) of the <em>3</em>0 patients; no patient died while on therapy.

CONCLUSIONS

Our data support the utilization of antiviral therapy in HCV-infected patients awaiting LT as one of the strategies to prevent hepatitis C recurrence after transplantation.

BACKGROUND

Patients with malignant endocrine pancreatic tumors (EPTs) are responsive to combinations of chemotherapy with streptozotocin and 5-fluorouracil/doxorubicin, whereas patients with malignant carcinoids are not. For both categories of patients, alpha-interferon and/or somatostatin analogs can produce long-lasting responses. Cisplatin in combination with etoposide has been suggested to be effective in patients with malignant neuroendocrine carcinomas. The authors used this therapy as second-line or third-line treatment in patients with poorly differentiated and/or rapidly progressing disease.

METHODS

Thirty-six patients with histopathologically verified malignant neuroendocrine tumors were included: Eighteen tumors were of foregut origin, of which 5 were atypical, and 15 tumors were EPTs, of which 4 were poorly differentiated endocrine carcinomas. Three tumors were of midgut origin. The median patient age was 47.5 years. The median duration of disease from the time of diagnosis was 12 months. All patients had metastatic disease. Thirty of 36 patients had received previous treatment. Etoposide was given at a dose of 100 mg/m(2) per day for 3 days, and cisplatin was given at a dose of 45 mg/m(2) on Days 2 and 3 as a continuous intravenous infusion that was repeated every 4 weeks.

RESULTS

Ten of 18 patients with foregut carcinoids (56%) responded radiologically and/or biochemically, with a median duration of 9 months; and 7 of 14 patients with EPTs (50%) responded radiologically and/or biochemically, with a median duration of 9 months. No difference in response was seen between patients with atypical or typical foregut carcinoids or between patients with well differentiated or poorly differentiated endocrine pancreatic carcinoma. Nineteen of 36 patients (53%) experienced World Health Organization (WHO) Grade 1-2 nephrotoxicity, and 23 patients (64%) suffered from WHO Grade 3-4 neutropenia.

CONCLUSIONS

The combination of cisplatin and etoposide can produce significant responses in patients with heavily pretreated and poorly differentiated/rapidly progressing neuroendocrine tumors. The toxicity is considerable, and nephrotoxicity is the dose limiting factor.

Four plasma proteins, referred to as positive acute phase proteins because of increases in concentration following inflammatory stimuli, are reviewed: C-reactive protein (CRP), serum amyloid A protein (SAA), <em>alpha</em> 1-acid glycoprotein (AAG), and fibrinogen. The CRP and SAA may increase in concentration as much as 1000-fold, the AAG and fibrinogen approximately twofold to fourfold. All are synthesized mainly in the liver, but each may be produced in a number of extrahepatic sites. The role of cytokines in induction of the acute phase proteins is discussed, particularly the multiple functional capabilities of interleukin-6 (IL-6). Other cytokines that regulate acute phase gene expression and protein synthesis include IL-1, tumor necrosis factor <em>alpha</em>, <em>interferon</em> gamma, as well as other stimulatory factors and cofactors. The physicochemical characteristics of each protein are reviewed together with the molecular biology. For each protein, the known biological effects are detailed. The following functions for CRP have been described: reaction with cell surface receptors resulting in opsonization, enhanced phagocytosis, and passive protection; activation of the classical complement pathway; scavenger for chromatin fragments; inhibition of growth and/or metastases of tumor cells; modulation of polymorphonuclear function; and a few additional diverse activities. The role of plasma SAA is described as a precursor of protein AA in secondary amyloidosis; other functions are speculative. AAG may play an immunoregulatory role as well as a role in binding a number of diverse drugs. In addition to clot formation, new data are described for binding of fibrinogen and fibrin to complement receptor type <em>3</em>. Finally, the concentration of each protein is discussed in a wide variety of noninfectious and infectious disease states, particularly in connective tissue diseases. The quantification of the proteins during the course of various acute and chronic inflammatory disorders is useful in diagnosis, therapy, and in some cases, prognosis.

Persistent infection with hepatitis C virus (HCV) can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All current therapies of hepatitis C include <em>interferon</em>-<em>alpha</em> (IFN-α). Moreover, IFN-gamma (IFN-γ), the only type II IFN, strongly inhibits HCV replication in vitro and is the primary mediator of HCV-specific antiviral T-cell responses. However, for both cytokines the precise set of effector protein(s) responsible for replication inhibition is not known. The aim of this study was the identification of IFN-α and IFN-γ stimulated genes (ISGs) responsible for controlling HCV replication. We devised an RNA interference (RNAi)-based "gain of function" screen and identified, in addition to known ISGs earlier reported to suppress HCV replication, several new ones with proven antiviral activity. These include IFIT<em>3</em> (IFN-induced protein with tetratricopeptide repeats <em>3</em>), TRIM14 (tripartite motif containing 14), PLSCR1 (phospholipid scramblase 1), and NOS2 (nitric oxide synthase 2, inducible). All ISGs identified in this study were up-regulated both by IFN-α and IFN-γ, demonstrating a substantial overlap of HCV-specific effectors induced by either cytokine. Nevertheless, some ISGs were more specific for IFN-α or IFN-γ, which was most pronounced in case of PLSCR1 and NOS2 that were identified as main effectors of IFN-γ-mediated anti-HCV activity. Combinatorial knockdowns of ISGs suggest additive or synergistic effects demonstrating that with either IFN, inhibition of HCV replication is caused by the combined action of multiple ISGs.

CONCLUSIONS

Our study identifies a number of novel ISGs contributing to the suppression of HCV replication by type I and type II IFN. We demonstrate a substantial overlap of antiviral programs triggered by either cytokine and show that suppression of HCV replication is mediated by the concerted action of multiple effectors.

Jak tyrosine kinases have a unique domain structure containing a kinase domain (JH1) adjacent to a catalytically inactive pseudokinase domain (JH2). JH2 is crucial for inhibition of basal Jak activity, but the mechanism of this regulation has remained elusive. We show that JH2 negatively regulated Jak2 in bacterial cells, indicating that regulation is an intrinsic property of Jak2. JH2 suppressed basal Jak2 activity by lowering the V(max) of Jak2, whereas JH2 did not affect the K(m) of Jak2 for a peptide substrate. Three inhibitory regions (IR1-<em>3</em>) within JH2 were identified. IR<em>3</em> (residues 758-807), at the C terminus of JH2, directly inhibited JH1, suggesting an inhibitory interaction between IR<em>3</em> and JH1. Molecular modeling of JH2 showed that IR<em>3</em> could form a stable <em>alpha</em>-helical fold, supporting that IR<em>3</em> could independently inhibit JH1. IR2 (725-757) in the C-terminal lobe of JH2, and IR1 (619-670), extending from the N-terminal to the C-terminal lobe, enhanced IR<em>3</em>-mediated inhibition of JH1. Disruption of IR<em>3</em> either by mutations or a small deletion increased basal Jak2 activity, but abolished <em>interferon</em>-gamma-inducible signaling. Together, the results provide evidence for autoinhibition of a Jak family kinase and identify JH2 regions important for autoregulation of Jak2.

Toll-like receptor 4 (TLR4) initiates both myeloid differentiation factor 88 (MyD88)-dependent and Toll/interleukin (IL)-1R domain-containing adapter, inducing <em>interferon</em> (IFN)-beta-dependent signaling, leading to production of proinflammatory mediators and type I <em>interferon</em> (IFN) to eliminate pathogens. However, uncontrolled TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. TLR4 is transported from the plasma membrane to the endosome for ubiqutination and to the lysosome for degradation, and downregulation of TLR4 expression or promotion of TLR4 degradation are important ways for negative regulation of TLR4 signaling. We previously identified a lysosome-associated small guanosine triphosphatase (GTPase) Rab7b that may be involved in lysosomal trafficking and degradation of proteins. Here we demonstrate that Rab7b can negatively regulate lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-<em>alpha</em>, IL-6, nitric oxide, and IFN-beta, and potentiate LPS-induced activation of mitogen-activated protein kinase, nuclear factor kappaB, and IFN regulatory factor <em>3</em> signaling pathways in macrophages by promoting the degradation of TLR4. Rab7b is localized in LAMP-1-positive subcellular compartments and colocalized with TLR4 after LPS treatment and can decrease the protein level of TLR4. Our findings suggest that Rab7b is a negative regulator of TLR4 signaling, potentially by promoting the translocation of TLR4 into lysosomes for degradation.

Fragments of the 5'-flanking sequence of a human 2-5A synthetase gene were assayed for their ability to respond to <em>interferon</em>-<em>alpha</em> (IFN). Transient transfection assays in monkey cells demonstrated that the 5' boundary of the sequence required for IFN-regulated transcription is, at most, 155 nucleotides upstream from the presumed translational initiation codon. The <em>3</em>' boundary of this sequence lies within a region of multiple transcription start sites preceded by no obvious TATA box. Binding assays, using a 40-bp probe derived from this IFN-responsive sequence, demonstrated the presence of three IFN-modulated, DNA-factor band shifts using nuclear extracts prepared from human and monkey cells. The induction of these complexes in human cells by IFN occurs with kinetics which closely parallel those previously observed for the transcriptional activation of the 2-5A synthetase gene by IFN. In vivo competition assays showed that the same 40-bp region which bound IFN-modulated factors could decrease the IFN-induced activity of a co-transfected 2-5A synthetase promoter; this fragment, regardless of its orientation, could confer IFN-inducibility on a heterologous promoter.