Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V<em>3</em>000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide <em>3</em> (nt <em>3</em>) of the 5' untranslated region (UTR) of the V<em>3</em>000 genome resulted in a virus (V<em>3</em>04<em>3</em>) that was avirulent in mice. The mechanism of attenuation by the V<em>3</em>04<em>3</em> mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V<em>3</em>04<em>3</em> replication was reduced in peripheral organs compared to that of V<em>3</em>000, titers in serum also were lower, and V<em>3</em>04<em>3</em> was cleared more rapidly from the periphery than V<em>3</em>000. Because clearance of V<em>3</em>04<em>3</em> from serum began 1 to 2 days prior to clearance of V<em>3</em>000, we examined the involvement of <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) activity in VEE pathogenesis. In IFN-<em>alpha</em>/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of <em>3</em>0 h compared to 7.7 days in the wild-type mice). The mutant V<em>3</em>04<em>3</em> was as virulent as the wild type (100% mortality, average survival time of <em>3</em>0 h). Virus titers in serum, peripheral organs, and the brain were similar in V<em>3</em>000- and V<em>3</em>04<em>3</em>-infected IFN-<em>alpha</em>/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-<em>alpha</em>/beta pathway. In cells derived from IFN-<em>alpha</em>/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-<em>alpha</em>/beta plays a major role in the attenuation of V<em>3</em>04<em>3</em> compared to V<em>3</em>000. There were no differences in the induction of IFN-<em>alpha</em>/beta between V<em>3</em>000 and V<em>3</em>04<em>3</em>, but the mutant virus was more sensitive than V<em>3</em>000 to the antiviral actions of IFN-<em>alpha</em>/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-<em>alpha</em>/beta plays a major role in the in vivo attenuation of V<em>3</em>04<em>3</em>.

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10(8) CFU and observed for 2 days. No clinical signs of illness developed in animals given AS- EBS- organisms, and all survived. All rabbits given AS- EBS+ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+ EBS- organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+ EBS+ organisms developed signs of illness and died. None of the rabbits receiving AS+ EBS+ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+ EBS+ organisms. In contrast, cell extracts from AS- EBS- organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+ enterococci contained high levels of gamma <em>interferon</em> and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and TNF-beta, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 x 10(9) CFU of test organisms. None of the animals given AS- EBS- organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS- EBS+ or AS+ EBS- organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+ EBS+ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for <em>3</em> days revealed that all rabbits given AS- EBS- or AS+ EBS+ organisms developed vegetations, but animals given AS+ EBS+ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.

Dengue viruses (DV), composed of four distinct serotypes (DV1 to DV4), cause 50 to 100 million infections annually. Durable homotypic immunity follows infection but may predispose to severe subsequent heterotypic infections, a risk conferred in part by the immune response itself. Antibody-dependent enhancement (ADE), a process best described in vitro, is epidemiologically linked to complicated DV infections, especially in Southeast Asia. Here we report for the first time the ADE phenomenon in primary human dendritic cells (DC), early targets of DV infection, and human cell lines bearing Fc receptors. We show that ADE is inversely correlated with surface expression of DC-SIGN (DC-specific intercellular adhesion molecule-<em>3</em>-grabbing nonintegrin) and requires Fc gamma receptor IIa (FcgammaRIIa). Mature DC exhibited ADE, whereas immature DC, expressing higher levels of DC-SIGN and similar FcgammaRIIa levels, did not undergo ADE. ADE results in increased intracellular de novo DV protein synthesis, increased viral RNA production and release, and increased infectivity of the supernatants in mature DC. Interestingly, tumor necrosis factor <em>alpha</em> and interleukin-6 (IL-6), but not IL-10 and gamma <em>interferon</em>, were released in the presence of dengue patient sera but generally only at enhancement titers, suggesting a signaling component of ADE. FcgammaRIIa inhibition with monoclonal antibodies abrogated ADE and associated downstream consequences. DV versatility in entry routes (FcgammaRIIa or DC-SIGN) in mature DC broadens target options and suggests additional ways for DC to contribute to the pathogenesis of severe DV infection. Studying the cellular targets of DV infection and their susceptibility to ADE will aid our understanding of complex disease and contribute to the field of vaccine development.

The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor <em>3</em> and <em>alpha</em>/beta <em>interferons</em> as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1<em>3</em>98-nucleotide <em>3</em>'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>) or interleukin (IL)-1beta but not with IL-4 or <em>interferon</em> gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNF<em>alpha</em>, IFNgamma inhibited the TNF<em>alpha</em>-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.

Identification of human CD1d-restricted T-cell receptor (TCR)-invariant natural killer T (iNKT) cells has been dependent on utilizing combinations of monoclonal antibodies or CD1d tetramers, which do not allow for the most specific analysis of this T-cell subpopulation. A novel monoclonal antibody (clone 6B11), specific for the invariant CDR<em>3</em> loop of human canonical V<em>alpha</em>24J<em>alpha</em>18 TCR <em>alpha</em> chain, was developed and used to specifically characterize iNKT cells. In healthy individuals studied for up to 1 year, a wide but stable frequency of circulating iNKT cells (range: 0.01-0.92%) was observed, with no differences in frequency by gender. Four stable iNKT cell subsets were characterized in peripheral blood based on the expression of CD4 and CD8, with CD8(+) iNKT cells being a phenotypic and functionally different subset from CD4(+) and double negative iNKT cells; in particular, LAG-<em>3</em> was preferentially expressed on CD8(+) iNKT cells. In addition, a strong negative linear correlation between the frequency of total iNKT cells and percentage of the CD4(+) subset was observed. In terms of their potential association with disease, patients at risk for type 1 diabetes had significantly expanded frequencies of double negative iNKT cells when compared to matched controls and first-degree relatives. Moreover, peripheral blood CD4(+) iNKT cells were the highest producers of interleukin-4, while the production of <em>interferon</em>-gamma and tumour necrosis factor-<em>alpha</em> was similar amongst all iNKT cell subsets. These differences in iNKT cell subsets suggest that in humans the relative ratio of iNKT cell subsets may influence susceptibility vs. resistance to immune-mediated diseases.

Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear. We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release. Sprague-Dawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 microg/kg; protective antigen 200 microg/kg) that produced death beginning at 9 h and a 7-day mortality rate of 5<em>3</em>%. By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P < or = 0.05 for each comparing the <em>3</em> groups). Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-<em>alpha</em>, IL-beta, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls. Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls. In additional experiments, although 24-h infusions of LeTx and Escherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (1<em>3</em>.2 +/- 0.8 vs. 11.0 +/- 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-<em>alpha</em>, IL-1 <em>alpha</em> and -1 beta, IL-6, IL-10, <em>interferon</em>-gamma, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1 <em>alpha</em>, -2, and -<em>3</em>, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P < or = 0.05 for the effects of LPS). Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this at model. Although therapies to modulate these host mediators may be applicable fo shock caused by LPS or other bacterial toxins, they may not with LeTx.

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta), and IFN-like interleukin-28A/B (IFN-lambda2/<em>3</em>) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-<em>alpha</em>, IFN-<em>alpha</em>/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-<em>alpha</em>, IFN-<em>alpha</em>, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-<em>alpha</em>. IFN-<em>alpha</em> priming led to increased expression of Toll-like receptor <em>3</em> (TLR<em>3</em>), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF<em>3</em>. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-<em>alpha</em> or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.

Hepatitis C virus genotype 4 (HCV-4) is the most common variant of the hepatitis C virus (HCV) in the Middle East and Africa, particularly Egypt. This region has the highest prevelance of HCV worldwide, with more than 90% of infections due to genotype 4. HCV-4 has recently spread in several Western countries, particularly in Europe, due to variations in population structure, immigration, and routes of transmission. The features of HCV-4 infection and the appropriate therapeutic regimen have not been well characterized. This review discusses the virology, epidemiology, natural history, histology, clinical data, and treatment options for patients with HCV-4 infections. Early reports on the treatment of patients with chronic HCV-4 with conventional interferon (IFN)-alpha monotherapy indicated poor rates of sustained viral response (SVR), which improved slightly when combined with ribavirin. Pegylated IFN and ribavirin combination therapy has dramatically improved the response rates, with recent clinical trials showing rates that exceed 60%. These data can now be used as a platform for further research to define optimal treatment duration and predictors of SVR in patients with HCV-4 infection.

CONCLUSIONS

HCV-4 infection is spreading beyond its strongholds in Africa and the Middle East. Recent clinical trials show that HCV-4 is not difficult to treat, as the response to treatment may be at an intermediate level compared with genotype 1 and genotypes 2 or 3. Tailored treatment options that are comparable to the treatment approaches for genotype 1, 2, and 3 patients to optimize treatment for each patient are now being developed.

Structurally, the monocyte chemotactic proteins MCP-1, -2, and -<em>3</em> form a subfamily of the C-C or beta-chemokines. Like other chemokines, MCPs are produced by a variety of cells on stimulation with cytokines (interleukin-1, tumor necrosis factor-<em>alpha</em>, <em>interferon</em>-gamma), bacterial and viral products or mitogens. MCP-1 levels are enhanced during infection and inflammation, which are characterized by leukocyte infiltration. In vitro, MCPs are chemotactic for a distinct spectrum of target cells and show different specific biological activities depending on the cell type and the chemokine tested. MCP-<em>3</em> has the broadest range in that it activates monocytes, dendritic cells, lymphocytes, natural killer cells, eosinophils, basophils, and neutrophils. The most sensitive cells to all three MCPs are lymphocytes and monocytes. MCP-1 is a potent basophil activator but does not attract eosinophils, whereas, at higher concentrations, MCP-2 also stimulates both eosinophils and basophils. The signal transduction of MCPs on monocytes involves at least two G protein-linked C-C chemokine receptors: C-C CKR-1 binds MCP-<em>3</em> and C-C CKR-2 binds MCP-1 and MCP-<em>3</em> but not MCP-2. Receptor binding leads to enhanced [Ca2+]i for all chemokines except for MCP-2.

Since the first identification of interleukin (IL)-6 as a myeloma cell growth factor by Dr. Kawano's and Dr. Klein's groups 14 years ago, numerous studies have emphasized its major roles in the emergence of malignant plasma cells in vivo and in the generation of normal plasma cells. Four transcription factors control B-cell differentiation into plasma cells. The B-cell transcription factor pax-5 is mainly responsible for a B-cell phenotype, and bcl-6 represses the plasma cell transcription factor blimp-1 and plasma cell differentiation. bcl-6 expression is triggered by CD40 and IL-4 activation. A lack of CD40 and IL-4 activation yields a down-regulation of bcl-6 expression, and IL-6 stimulation yields an up-regulation of blimp-1, mainly through STAT<em>3</em> activation. Blimp-1 further down-regulates bcl-6 and pax-5 expression and makes plasma cell differentiation possible. IL-6 as well as IL-10 up-regulate XBP-1. XBP-1 is another transcription factor that is involved in plasma cell differentiation and whose gene expression is shut down by pax-5. The plasma cell transcription factors blimp-1 and XBP-1 are up-regulated, and the B-cell transcription factors bcl-6 and pax-5 are down-regulated, in malignant cells compared to B-cells. Apart from the recent identification of these 4 transcription factors, the factors involved in normal plasma cell generation are mostly unknown. Regarding malignant plasma cells, <em>3</em> categories of growth factors have been identified: (1) the IL-6 family cytokines, IL-10, and <em>interferon</em> <em>alpha</em> that activate the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase pathways; (2) growth factors activating the phosphatidylinositol (PI)-<em>3</em> kinase/AKT and MAP kinase pathways, unlike the JAK/STAT pathway (insulin-like growth factor 1, hepatocyte growth factor, and members of the epidermal growth factor family able to bind syndecan-1 proteoglycan); and (<em>3</em>) B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL) that activate the nuclear factor KB and PI-<em>3</em> kinase/AKT pathways. BAFF and APRIL bind to BAFF receptor and TACI and are major B-cell survival factors. Recent data indicate that these various growth factors may cooperate to provide optimum signaling because they are localized together and with cytoplasmic transduction elements in caveolinlinked membrane caveolae. The identification of these myeloma cell growth factors and of the associated transduction pathways should provide novel therapeutic targets in multiple myeloma.

<em>Interferons</em> are pleiotropic cytokines that exhibit negative regulatory effects on the growth of normal and malignant hematopoietic cells in vitro and in vivo. There are two different classes of <em>interferons</em>, Type I (<em>alpha</em>, beta, and omega) and Type II (gamma) <em>interferons</em>. Although the precise mechanisms by which these cytokines exhibit their potent effects on hematopoiesis remain unknown, there has been considerable progress in our understanding of the cellular changes that occur upon engagement of <em>interferon</em> receptors. It is now well established that Type I <em>interferons</em> activate multiple signaling pathways in hematopoietic cells, a finding consistent with their pleiotropic biological effects. One major pathway in Type I IFN signaling involves activation of Stat- proteins and formation of complexes that translocate to the nucleus and bind to specific elements to regulate gene transcription. The activation of this pathway (Jak-Stat pathway) is apparently regulated by members of the Jak-family of kinases, which are constitutively associated with the Type I IFN receptor. In addition to the Jak-Stat pathway, multiple other Jak-kinase-dependent signaling cascades are activated, including the IRS-PI <em>3</em>'-kinase pathway, a pathway involving the vav proto-oncogene product, and a pathway involving adaptor proteins of the Crk-family (CrkL and CrkII). The only Type II <em>interferon</em>, IFNgamma, also activates multiple Jak-kinase-dependent signaling cascades, including the Stat and Crk pathways. Recent evidence suggests that non-Stat pathways play a critical role in the generation of signals for both Type I and Type II <em>interferons</em> and may be the primary mediators of their growth inhibitory effects on hematopoietic cells.

OBJECTIVE

To elucidate the role of activated microglia in the photoreceptor apoptosis of rd mice by identifying sequential events and factors associated with microglial activation, migration, and cytotoxicity during retinal degeneration.

METHODS

Photoreceptor apoptosis in rd mice at postnatal days (P)8, 10, 12, 14, 16, and 18 was detected by terminal dUTP transferase nick end labeling (TUNEL). Retinal microglia were identified by CD11b antibody. Expression of chemokine mRNA, including monocyte chemoattractant protein (MCP)-1, MCP-<em>3</em>, macrophage inflammatory protein (MIP)-1<em>alpha</em>, MIP-1beta, regulated on activation normal T-cell expressed and secreted (RANTES), <em>interferon</em>-gamma-inducible 10-kDa protein (IP-10), and fractalkine in the retina were examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. Production of tumor necrosis factor (TNF)-<em>alpha</em> in the dystrophic retina was studied by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry analysis. Microglial expression of TNF-<em>alpha</em> was determined by double immunolabeling.

RESULTS

Whereas photoreceptor apoptosis in the rd mice started at P10 and reached a peak at P16, activation and migration of microglial cells were observed at P10 and peaked at P14. The expression of MCP-1, MCP-<em>3</em>, MIP-1<em>alpha</em>, MIP-1beta, and RANTES transcripts were noted at P8 and reached a peak at P12. Production of TNF-<em>alpha</em> was noted in the outer nuclear layer (ONL) of the rd mice at P8 and reached a peak at P12. At the peak of microglial activity, TNF-<em>alpha</em> was predominantly expressed in the activated microglial cells in the ONL.

CONCLUSIONS

Activation of microglia, as well as expression of their signaling molecules (chemokines) and microglia-derived toxic factor (TNF-<em>alpha</em>), coincides with or precedes the occurrence of photoreceptor apoptosis, suggesting activated microglia play a major role in retinal degeneration in rd mice. The chemokines MCP-1, MCP-<em>3</em>, MIP-1<em>alpha</em>, MIP-1beta, and RANTES are involved in activation and recruitment of the microglia to the degenerating photoreceptor cell layer. TNF-<em>alpha</em>, produced by the activated microglia, may accentuate the photoreceptor cell death.

<em>Interferon</em> establishes an antiviral state in numerous cell types through the induction of a set of immediate-early response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the <em>alpha</em>/beta-<em>interferon</em>-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced <em>interferon</em>-stimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of <em>interferon</em>-stimulated gene factor <em>3</em>. IRF<em>3</em>, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p<em>3</em>00, to form CIF. Activating <em>interferon</em>-stimulated genes without the need for prior synthesis of <em>interferons</em> might provide the host cell with a potential shortcut in the activation of its antiviral defense.

Murine hybridomas producing monoclonal antibodies (mAb) specific to human interleukin 6 (IL 6/BSF-2) were established. One of these hybridomas (MH60.BSF2) was found to be dependent on IL 6 for its in vitro growth. None of the known biological factors tested, such as recombinant (r) human (Hu) IL 1 <em>alpha</em>, rHuIL 1 beta, rHuIL 2, rHuIL <em>3</em>, rHuIL 4, rHu <em>interferon</em> (IFN)-gamma, HuIFN-beta, rHuG-CSF, or recombinant murine (Mu) IL <em>3</em>, MuIL 4, rMuIL 5, could induce the in vitro growth of MH60.BSF2 cells. The half-maximum tritiated thymidine uptake by MH60.BSF2 cells could be achieved by picogram amounts of rIL 6, making this hybridoma clone an indicator cell for specific and sensitive detection of the IL 6 activity in test samples. The MH166.BSF2 clone was found to produce IgG1,chi type mAb (<em>alpha</em> BSF2-166) capable of neutralizing IL 6 activity. The other clone, MH60.BSF2, produced IgM,chi type mAb (<em>alpha</em> BSF2-60) unable to neutralize IL 6 activity. Both mAb specifically reacted with IL 6 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis. An enzyme-linked immunosorbent assay (ELISA) utilizing <em>alpha</em> BSF2-166 and rabbit anti-IL 6 antibodies was established which could detect as low as 50 pg/ml of IL 6. Since both the ELISA and MH60.BSF2 hybridoma could detect small amounts of IL 6 in biological fluids, they constitute powerful tools in exploring the presence or the role of IL 6 in various immunological disorders.

In patients with chronic hepatitis C virus (HCV) infection scheduled for a 48-week treatment period, premature discontinuation of treatment was previously recommended if HCV-RNA levels remained detectable at week 24 of therapy. Considering the number of side effects and treatment costs, measurement of initial viral decline during therapy may identify virologic nonresponse earlier than 24 weeks. We retrospectively analyzed 260 European patients treated with standard or pegylated <em>interferon</em> alfa (IFN-<em>alpha</em>) and ribavirin for 24 to 48 weeks. Early prediction of virologic response by HCV-RNA decline at weeks 4 and 12 (Versant Quantitative [branched DNA (bDNA) <em>3</em>.0]; Bayer Diagnostics, Emeryville, CA; and Qualitative [transcription-mediated amplification (TMA)] HCV RNA assay; Bayer Diagnostics) as well as clinical, biochemical, virologic, and histologic baseline parameters were analyzed by logistic regression and receiver operating characteristic (ROC) curves. A viral load at treatment week 4 above 450,000 IU/mL and at week 12 above <em>3</em>0,000 IU/mL was 100% predictive for virologic nonresponse in all patients. From multivariate logistic regression analysis of all patients, independent predictors for sustained virologic response were: genotypes 2 and <em>3</em> (P <.0001), a low baseline gamma-glutamyltransferase (GGT) level (P <.0001), a high baseline alanine aminotransferase level (P =.002), and a low baseline viral load (P =.04). None of the latter <em>3</em> factors were predictive for sustained virologic response when analysis was restricted to the subgroup of genotypes 2- and <em>3</em>-infected patients. In conclusion, virologic nonresponse can be predicted early at week 12 of treatment independent from the applied therapeutic regimen based on a cutoff level for HCV RNA of <em>3</em>0,000 IU/mL. This algorithm recognizes 5<em>3</em>.7% of nonresponders previously identified at week 24 of treatment.

The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause frequently lethal viral hemorrhagic fever. These infections induce potent cytokine production, yet these host responses fail to prevent systemic virus replication. Consistent with this, filoviruses have been found to encode proteins VP<em>3</em>5 and VP24 that block host <em>interferon</em> (IFN)-<em>alpha</em>/beta production and inhibit signaling downstream of the IFN-<em>alpha</em>/beta and the IFN-gamma receptors, respectively. VP<em>3</em>5, which is a component of the viral nucleocapsid complex and plays an essential role in viral RNA synthesis, acts as a pseudosubstrate for the cellular kinases IKK-epsilon and TBK-1, which phosphorylate and activate <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and <em>interferon</em> regulatory factor 7 (IRF-7). VP<em>3</em>5 also promotes SUMOylation of IRF-7, repressing IFN gene transcription. In addition, VP<em>3</em>5 is a dsRNA-binding protein, and mutations that disrupt dsRNA binding impair VP<em>3</em>5 IFN-antagonist activity while leaving its RNA replication functions intact. The phenotypes of recombinant EBOV bearing mutant VP<em>3</em>5s unable to inhibit IFN-<em>alpha</em>/beta demonstrate that VP<em>3</em>5 IFN-antagonist activity is critical for full virulence of these lethal pathogens. The structure of the VP<em>3</em>5 dsRNA-binding domain, which has recently become available, is expected to provide insight into how VP<em>3</em>5 IFN-antagonist and dsRNA-binding functions are related. The EBOV VP24 protein inhibits IFN signaling through an interaction with select host cell karyopherin-<em>alpha</em> proteins, preventing the nuclear import of otherwise activated STAT1. It remains to be determined to what extent VP24 may also modulate the nuclear import of other host cell factors and to what extent this may influence the outcome of infection. Notably, the Marburg virus VP24 protein does not detectably block STAT1 nuclear import, and, unlike EBOV, MARV infection inhibits STAT1 and STAT2 phosphorylation. Thus, despite their similarities, there are fundamental differences by which these deadly viruses counteract the IFN system. It will be of interest to determine how these differences influence pathogenesis.

How viral infections trigger autoimmunity is poorly understood. A role for Toll-like receptor <em>3</em> (TLR<em>3</em>) was hypothesized in this context as viral double-stranded RNA (dsRNA) activates dendritic cells to secrete type I <em>interferons</em> and cytokines that are known to be associated with the disease activity in systemic lupus erythematosus (SLE). Immunostaining of nephritic kidney sections of autoimmune MRL(lpr/lpr) mice revealed TLR<em>3</em> expression in infiltrating antigen-presenting cells as well as in glomerular mesangial cells. TLR<em>3</em>-positive cultured mesangial cells that were exposed to synthetic polyinosinic-cytidylic acid (pI:C) RNA in vitro produced CCL2 and IL-6. pI:C RNA activated macrophages and dendritic cells, both isolated from MRL(lpr/lpr) mice, to secrete multiple proinflammatory factors. In vivo, a single injection of pI:C RNA increased serum IL-12p70, IL-6, and IFN-<em>alpha</em> levels. A course of 50 microg of pI:C RNA given every other day from weeks 16 to 18 of age aggravated lupus nephritis in pI:C-treated MRL(lpr/lpr) mice. Serum DNA autoantibody levels were unaltered upon systemic exposure to pI:C RNA in MRL(lpr/lpr) mice, as pI:C RNA, in contrast to CpG-DNA, failed to induce B cell activation. It therefore was concluded that viral dsRNA triggers disease activity of lupus nephritis by mechanisms that are different from those of bacterial DNA. In contrast to CpG-DNA/TLR9 interaction, pI:C RNA/TLR<em>3</em>-mediated disease activity is B cell independent, but activated intrinsic renal cells, e.g., glomerular mesangial cells, to produce cytokines and chemokines, factors that can aggravate autoimmune tissue injury, e.g., lupus nephritis.

The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their <em>interferon</em> (IFN)-<em>alpha</em>/beta responder ability and in mutant mice lacking <em>alpha</em>/beta (IFN-<em>alpha</em>/beta R0/0) or gamma IFN (IFN-gamma R0/0) receptors. During the first week of infection, wild type mice demonstrated a transient pancytopenia. Within a given genetic background, the extent of the blood cell abnormalities did not correlate with the virulence of the LCMV isolate but variations were detected between different mouse strains: they were found to depend on their IFN-<em>alpha</em>/beta responder phenotype. Whereas IFN-gamma R0/0 mice were comparable to wild-type mice, IFN-<em>alpha</em>/beta R0/0 mice exhibited unchanged peripheral blood values during acute LCMV infection. In parallel, the bone marrow (BM) cellularity, the pluripotential and committed progenitor compartments were up to <em>3</em>0-fold reduced in wild type and IFN-gamma R0/0, but remained unchanged in IFN-<em>alpha</em>/beta R0/0 mice. Viral titers in BM <em>3</em> d after LCMV infection were similar in these mice, but antigen localization was different. Viral antigen was predominantly confined to stromal BM in normal mice and IFN-gamma R0/0 knockouts, whereas, in IFN-<em>alpha</em>/beta R0/0 mice, LCMV was detected in>> 90% of megakaryocytes and 10-15% of myeloid precursors, but not in erythroblasts Although IFN-<em>alpha</em>/beta efficiently prevented viral replication in potentially susceptible hematopoietic cells, even in overwhelming LCMV infection, unlimited virus multiplication in platelet and myeloid precursors in IFN-<em>alpha</em>/beta R0/0 mice did not interfere with the number of circulating blood cells. Natural killer (NK) cell expansion and activity in the BM was comparable on day <em>3</em> after infection in mutant and control mice. Adaptive immune responses did not play a major role because comparable kinetics of LCMV-induced pancytopenia and transient depletion of the pluripotential and committed progenitor compartments were observed in CD8(0/0) and CD4(0/0) mice, in mice depleted of NK cells, in lpr mice, and in perforin-deficient (P0/0) mice lacking lytic NK cells. Thus, the reversible depression of hematopoiesis during early LCMV infection was not mediated by LCMV-WE-specific cytotoxic T lymphocyte, cytolysis, or secreted IFN-gamma from virally induced NK cells but was a direct effect of IFN-<em>alpha</em>/beta.

While the role of cytokines in mediating injury during hind limb skeletal muscle ischemia followed by reperfusion has recently been described, the role of cytokines in myocardial infarction and ischemia/reperfusion have remained relatively unexplored. We hypothesize that cytokines play an important role in the regulation of postischemic myocardial inflammation. This study reports the temporal sequence of proinflammatory cytokine gene expression in postischemic/reperfused myocardium and localizes interleukin-1 beta (IL-1 beta) and tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>)-protein by immunostaining. Rats were subjected to either permanent left anterior descending (LAD) occlusion or to <em>3</em>5 minutes of LAD occlusion followed by reperfusion and sacrificed up to 7 days later. Rat-specific oligonucleotide probes were used to semiquantitatively assess the relative expression of mRNA for TNF-<em>alpha</em>, IL-1 beta, IL-2, IL-6, <em>interferon</em>-gamma (IFN-gamma), and transforming growth factor-beta 1 (TGF-beta 1) utilizing the reverse transcriptase-polymerase chain reaction amplification technique. Increased cardiac mRNA levels for all cytokines except IL-6 and IFN-gamma were measurable within 15 to <em>3</em>0 minutes of LAD occlusion and increased levels were generally sustained for <em>3</em> hours. During early reperfusion, mRNA levels for IL-6 and TGF-beta 1 were significantly reduced compared with permanent LAD occlusion. In both groups, cytokine mRNA levels all returned to baseline levels at 24 hours, while IL-1 beta, TNF-<em>alpha</em>, and TGF-beta 1 mRNA levels again rose significantly at 7 days only in animals with permanent LAD occlusion. Immunostaining for IL-1 beta and TNF-<em>alpha</em> protein revealed two patterns of reactivity: 1) microvascular staining for both IL-1 beta and TNF-<em>alpha</em> protein only in postischemic reperfused myocardium in early post-reperfusion time points; and 2) staining of infiltrating macrophages in healing infarct zones which was most prominent at 7 days after permanent LAD occlusion. These results provide evidence for local expression of cytokine mRNA in postischemic myocardium and suggest that regulation of local cytokine release is altered during the postischemic period.

The deposition of beta-amyloid in the brain is the key pathogenetic event in Alzheimer's disease. Among the various mechanisms proposed to explain the neurotoxicity of beta-amyloid deposits, a new one, recently identified in our and other laboratories, suggests that beta-amyloid is indirectly neurotoxic by activating microglia to produce toxic inflammatory mediators such as cytokines, nitric oxide, and oxygen free radicals. Three findings presented here support this mechanism, showing that beta-amyloid peptides (25-<em>3</em>5), (1-<em>3</em>9), and (1-42) activated the classical NADPH oxidase in rat primary culture of microglial cells and human phagocytes: 1) The exposure of the cells to beta-amyloid peptides stimulates the production of reactive oxygen intermediates; 2) the stimulation is associated with the assembly of the cytosolic components of NADPH oxidase on the plasma membrane, the process that corresponds to the activation of the enzyme; <em>3</em>) neutrophils and monocytes of chronic granulomatous disease patients do not respond to beta-amyloid peptides with the stimulation of reactive oxygen intermediate production. Data are also presented that the activation of NADPH oxidase requires that beta-amyloid peptides be in fibrillary state, is inhibited by inhibitors of tyrosine kinases or phosphatidylinositol <em>3</em>-kinase and by dibutyryl cyclic AMP, and is potentiated by <em>interferon</em>-gamma or tumor necrosis factor-<em>alpha</em>.

BACKGROUND

During wound healing, keratinocytes detach from the basement membrane and migrate to cover the exposed connective tissue. Subsequently, the wound clot is degraded gradually and replaced by the epithelial cells and the granulation tissue. Both of these processes are likely to be affected by matrix-modifying enzymes. Type IV collagenases are members of the matrix metalloproteinase family (MMP), which are known to degrade several matrix components. The aim of this study was to investigate the expression of MMP-2 and MMP-9 (72-kd and 92-kd type IV collagenases, respectively) during early human wound healing.

METHODS

Experimental wounds were created in human oral mucosa and biopsies were taken 1, <em>3</em>, and 7 days after wounding. In situ hybridization on paraffin sections was used for the detection of messenger RNAs coding for MMP-2 and MMP-9, and the secretion of MMPs into the oral cavity after wounding was followed by zymography. Regulation of MMP-2 and MMP-9 expression by cytokines was studied using cultured mucosal keratinocytes, gingival fibroblasts, and wound granulation tissue fibroblasts.

RESULTS

By in situ hybridization, the expression of MMP-2 was localized in the connective tissue fibroblasts and endothelial cells during all phases of wound healing. Mucosal epithelium was practically negative for MMP-2 expression. MMP-9 messenger RNA was found in mucosal epithelium on days 1, <em>3</em>, and 7. A strong signal was localized in basal and suprabasal cell layers in the nonwounded area, while only the basal cell layer was MMP-9 positive in the migrating epithelial sheet. Seven days after wounding, granulation tissue exhibited an unusually strong signal for MMP-9 messenger RNA. Wound fluid contained mainly MMP-9, the amount of which was highest in two- to four-day-old secretions. None of the cytokines tested (transforming growth factor beta-1, interleukin-1 beta, basic fibroblast growth factor, tumor necrosis factor-alpha, interferon-gamma) were able to regulate MMP-2 expression in cultured wound fibroblasts. However, keratinocyte MMP-9 production was enhanced by interleukin-1 beta, transforming growth factor beta-1, and tumor necrosis factor-alpha.

CONCLUSIONS

During wound healing, MMP-9 is suggested to be involved in keratinocyte migration and granulation tissue remodelling. Expression of MMP-2 remains stable during wound healing.

To determine whether extracellular tryptophan degradation represents an oxygen-independent antimicrobial mechanism, we examined the effect of exogenous tryptophan on the intracellular antimicrobial activity of gamma <em>interferon</em> (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,<em>3</em>-dioxygenase (IDO) activity and stimulated MDM, alveolar macrophages, and oxidatively deficient chronic granulomatous disease MDM to degrade tryptophan. All IFN-gamma-activated, tryptophan-degrading macrophages killed or inhibited Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani. Although exogenous tryptophan partially reversed this activity, the increases in intracellular replication were variable for normal MDM (T. gondii [5-fold], C. psittaci [<em>3</em>-fold], L. donovani [2-fold]), chronic granulomatous disease MDM (T. gondii [2.5-fold], C. psittaci [5-fold]), and alveolar macrophages (T. gondii [1.5-fold], C. psittaci [1.5-fold]). In addition, IFN-<em>alpha</em> and IFN-beta also stimulated normal MDM to express IDO and degrade tryptophan but failed to induce antimicrobial activity, and IFN-gamma-treated mouse macrophages showed neither IDO activity nor tryptophan degradation but killed T. gondii and L. donovani. These results suggest that while tryptophan depletion contributes to the oxygen-independent antimicrobial effects of the activated human macrophage, in certain cytokine-stimulated cells, tryptophan degradation may be neither sufficient nor required for antimicrobial activity.

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid <em>interferon</em>, <em>interferon</em> <em>alpha</em> (IFN-<em>alpha</em>) B/D, and a mismatched double-stranded (ds) RNA <em>interferon</em> (IFN) inducer, Ampligen (poly I:poly C124), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-<em>alpha</em> B/D once daily for <em>3</em> days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log10 at 10,000 and <em>3</em>2,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, beta-D-N4-hydroxycytidine, calpain inhibitor VI, <em>3</em>-deazaneplanocin A and Alferon (human leukocyte IFN-<em>alpha</em>-n<em>3</em>) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-<em>alpha</em> can inhibit SARS-CoV replication in the lungs of mice.