Several NS1 mutant viruses of human influenza A/PR/8/34 (H1N1) virus were tested for their ability to induce pro-inflammatory cytokines in primary human macrophages. The findings revealed a pronounced difference in the virus-induced cytokine pattern, depending on the functionality of the NS1 protein-encoded domains. The PR8/NS1-125 mutant virus, which encodes the first 125 aa of the NS1 protein, thus lacking the C-terminal domains, induced significantly higher amounts of beta interferon, interleukin (IL) 6, tumour necrosis factor alpha and CCL3 (MIP-1alpha) when compared with the A/PR/8/34 wild-type virus. However, this mutant virus was as efficient as wild-type virus in the inhibition of IL1beta and IL18 release from infected macrophages. Another group of viral mutants either lacking or possessing non-functional RNA-binding and dimerization domains induced 10-50 times more biologically active IL1beta and five times more biologically active IL18 than the wild-type or PR8/NS1-125 viruses. The hallmark of infection with this group of mutant viruses was the induction of rapid apoptosis in infected macrophages, which correlated with the enhanced activity of caspase-1. These results indicated that the NS1 protein, through the function of its N-terminal domains, might control caspase-1 activation, thus repressing the maturation of pro-IL1beta-, pro-IL18- and caspase-1-dependent apoptosis in infected primary human macrophages.

How viral infections trigger autoimmunity is poorly understood. A role for Toll-like receptor 3 (TLR3) was hypothesized in this context as viral double-stranded RNA (dsRNA) activates dendritic cells to secrete type I interferons 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 TLR3 expression in infiltrating antigen-presenting cells as well as in glomerular mesangial cells. TLR3-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-alpha 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/TLR3-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.

BACKGROUND

T cells are implicated in atherosclerosis, but little is known about the genetic control or molecular pathways, especially under conditions of mild hypercholesterolemia.

RESULTS

BALB/c mice, making a CD4+ Th2 (IL-4+) cell response, express both MHC class II antigens (IA(d), IE(d)) and are atherosclerosis-resistant. C57Bl/6 mice produce a CD4+ Th1 (interferon [IFN]gamma+) response, express IA(b) but no IE, and are atherosclerosis-prone. To evaluate T helper-cell phenotype in fatty streak formation, wild-type C57Bl/6 mice (IA(b)+IE-) and transgenic mice, either AB(o), IA(b)-IE-; ABEalpha, IA-IE(k)+; or BL:TG:Ealpha, IA(b)+IE(k)+, were fed a high-cholesterol diet for 16 weeks and evaluated histomorphometrically for aortic lesions. Lesion size in AB(o), ABEalpha, and BL:TG:Ealpha strains was decreased by 54%, 79%, and 82%, respectively, compared with wild-type, correlating with decreased Th1 and increased Th2 expression and suggesting that T helper-cell phenotype is important in fatty lesion development. Decreasing Th1 cells by antibodies (alpha-CD4) or cytokines (IL-4) also caused>>/=80% reductions in lesion size. Immunohistology revealed IFN-gamma, but not IL-4, colocalized with activated macrophages. Confirming these findings in a different mouse strain, BALB/c Stat 6 knockout mice (Th2 cell-deficient) developed aortic lesions comparable to C57Bl/6 mice on the same diet.

CONCLUSIONS

In mildly hypercholesterolemic C57Bl/6 mice, presence of IA(b) and absence of IE regulated CD4+ T helper-cell phenotype; fatty lesions were proportional to IFNgamma+ Th1 cells in both C57Bl/6 and BALB/c strains. IFN-gamma may participate through macrophage activation, whereas IL-4 may act to limit Th1-cell response.

The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their interferon (IFN)-alpha/beta responder ability and in mutant mice lacking alpha/beta (IFN-alpha/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-alpha/beta responder phenotype. Whereas IFN-gamma R0/0 mice were comparable to wild-type mice, IFN-alpha/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 30-fold reduced in wild type and IFN-gamma R0/0, but remained unchanged in IFN-alpha/beta R0/0 mice. Viral titers in BM 3 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-alpha/beta R0/0 mice, LCMV was detected in>> 90% of megakaryocytes and 10-15% of myeloid precursors, but not in erythroblasts Although IFN-alpha/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-alpha/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 3 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-alpha/beta.

The pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are acute-phase proteins produced by liver epithelial cells. PTX3 was recently cloned as an interleukin-1 (IL-1)-inducible gene in endothelial cells, with structural similarities to pentraxins in the C-terminal half of the molecule. The present study was designed to investigate the expression of PTX3 in the human leukocyte populations. Human peripheral blood mononuclear cells exposed to lipopolysaccharide (LPS) or IL-1 beta expressed significant levels of PTX3 mRNA. Tumor necrosis factor-alpha (TNF-alpha) was a less-effective inducer of PTX3, whereas IL-6, monocyte chemotactic protein-1, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interferon-gamma were inactive. Among leukocytes, only monocytes exposed to inflammatory cytokines or LPS expressed the PTX3 transcript, which was undetectable in resting or stimulated polymorphonuclear cells, T or B lymphocytes, and natural killer cells. PTX3 mRNA was also inducible in in vitro monocyte-derived macrophages, in tumor-associated macrophages, and in the myelomonocytic cell lines HL60, U937, and THP1, but not in GFD8, with the latter possibly representative of earlier stages of myelomonocytic differentiation. T- and B-cell lines had no detectable PTX3. Inhibition of transcription by actinomycin D blocked induction of PTX3 in monocytes and nuclear run-on analysis showed that LPS induces the expression of the PTX3 gene at the transcriptional level in isolated monocytes. Cycloheximide had no effect on PTX3 induction in U937 cells, but was inhibitory on monocytes exposed to LPS or IL-1 beta. Monoclonal antibody against TNF and the IL-1 receptor antagonists did not inhibit induction of PTX3 in monocytes by LPS, thus excluding these cytokines as secondary stimulators of PTX3. IL-4, but not dexamethasone or transforming growth factor-beta, inhibited PTX3 expression in monocytes. Using a PTX3-specific antiserum, release of PTX3 protein was demonstrated for the first time in stimulated monocytes as well as in endothelial and fibroblastic cells. Thus, PTX3, unlike the classical pentraxins CRP and SAP, is expressed and released by cells of the monocyte-macrophage lineage exposed to inflammatory signals.

The inflamed liver in chronic hepatitis B virus (HBV) infection (CHB) is characterized by a large influx of non-virus-specific CD8 T cells. Little is known about the functional capacity of these lymphocytes, which could provide insights into mechanisms of failure of viral control and liver damage in this setting. We compared the effector function of total circulating and intrahepatic CD8 T cells in CHB patients and healthy donors. We demonstrated that CD8 T cells from CHB patients, regardless of their antigen specificity, were impaired in their ability to produce interleukin-2 and proliferate upon TCR-dependent stimulation. In contrast, these CD8 T cells had preserved production of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha. This aberrant functional profile was partially attributable to down-regulation of the proximal T cell receptor signaling molecule CD3zeta, and could be corrected in vitro by transfection of CD3zeta or replenishment of the amino acid arginine required for its expression. We provide evidence for depletion of arginine in the inflamed hepatic microenvironment as a potential mechanism for these defects in global CD8 T cell signaling and function. These data imply that polarized CD8 T cells within the HBV-infected liver may impede proliferative antiviral effector function, while contributing to the proinflammatory cytokine environment.

Interferons (IFNs) type-1 (IFN alpha/beta) and type-II (IFN-gamma) are the most pleiotropic molecules in the intricate cytokine network. This dominance arises from three crucial factors: (i) initiation of IFN-alpha/beta and IFN-gamma production at the inception of most innate immune responses, which primes for the ensuing adaptive immune responses, primarily through the sine qua non upregulation of major histocompatibility complex and costimulatory molecules; (ii) magnification of their production and signaling by cross-talk between themselves, and synergistic or antagonistic effects on other cytokines; and (iii) direct or indirect initiation of transcription of hundreds of immunologically relevant genes. Considering that aberrant immune responses against self-molecules seem to depend on the same constituents and pathways as those against exogenous antigens, it follows that IFNs are also major effectors in the pathogenesis of autoimmunity. Here, we review the diverse biological effects of IFNs on the immune system, discuss findings pertaining to the nature of exogenous and endogenous stimuli that might induce IFN production through the engagement of Toll-like receptors, and summarize the detrimental and, in some instances, beneficial effects of IFNs in systemic and organ-specific autoimmune diseases.

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-alpha (TNF-alpha)-protein by immunostaining. Rats were subjected to either permanent left anterior descending (LAD) occlusion or to 35 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-alpha, IL-1 beta, IL-2, IL-6, interferon-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 30 minutes of LAD occlusion and increased levels were generally sustained for 3 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-alpha, 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-alpha protein revealed two patterns of reactivity: 1) microvascular staining for both IL-1 beta and TNF-alpha 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 (25-35), (1-39), 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; 3) 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 3-kinase and by dibutyryl cyclic AMP, and is potentiated by interferon-gamma or tumor necrosis factor-alpha.

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, 3, 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, 3, 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 interferon (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,3-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 [3-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-alpha 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 interferon, interferon alpha (IFN-alpha) B/D, and a mismatched double-stranded (ds) RNA interferon (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-alpha B/D once daily for 3 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 32,000 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, 3-deazaneplanocin A and Alferon (human leukocyte IFN-alpha-n3) 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-alpha can inhibit SARS-CoV replication in the lungs of mice.

The basic feature in the pathogenesis of abdominal aortic aneurysm (AAA) is the degradation of extracellular matrix components. This process is induced partly by cytokines secreted from inflammatory and mesenchymal cells. Circulating levels of inflammatory cytokines were studied in AAA patients and compared with subjects suffering from atherosclerotic disease only. Furthermore, the predictive value of cytokine concentrations was evaluated for aneurysm expansion rate. Circulating levels of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were measured in 50 AAA patients (40 men, 10 women), 42 patients with coronary heart disease (CHD) (23 men, 19 women), and 38 controls whose angiogram was normal (17 men, 21 women). No differences in cytokine concentrations were found between the CHD patients and the controls. AAA disease was found to be associated with significantly higher IL-1 beta and IL-6 concentrations in both male patients (median concentrations of 19.40 pmol/L and 6.45 pmol/L, respectively) and female patients (19.26 pmol/L and 7.99 pmol/L) than in either the CHD patients or the controls (P < .005). TNF-alpha levels were slightly higher in the AAA patients (1.64 pmol/L in the males and 1.59 pmol/L in the females) than in the other groups (P < .05). IFN-gamma levels were elevated significantly in the female AAA patients (3.75 pmol/L) compared with levels found in the other female (P < .05) or male (P < .01) patient groups. The measured cytokine concentrations were not related to the size of the aneurysm or the maximal thickness of the thrombus within the aneurysm. IFN-gamma concentration showed a significant positive correlation to the aneurysm expansion (R = .37, P < .02) and negative correlation to the concentration of aminoterminal propeptide of type III procollagen during 6-month follow up (R = -.42, P < .005). The results show that circulating levels of inflammatory cytokines are elevated in patients with AAA disease, suggesting that the production of these cytokines is increased in these patients compared with CHD patients and controls. Elevated INF-gamma concentrations seem to predict an increased rate of expansion in AAA.

Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene in endothelial cells is induced by lipopolysaccharide and the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA-protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3' of the TATA element. We have identified heterodimeric p65 and p50 as the NF-kappa B species binding to the VCAM1 promoter in TNF-alpha-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and high-mobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assay in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1.

A human gene (termed ISG-54K) that is induced from near undetectable levels to high transcriptional activity by alpha- and beta-interferons has been cloned. The genomic structure and nucleotide sequence of the coding region were determined and the RNA initiation site was identified. The 5' portion of the gene was fused with a heterologous gene lacking an active promoter in recombinant plasmid and adenoviral vectors. These fusion genes were used to assess the activity of the ISG-54K promoter in response to interferon. RNA was formed in HeLa cells from recombinant plasmids only in response to interferon. Furthermore, in human diploid fibroblasts, infection with the recombinant adenovirus vector resulted in a 50-fold increase in specific RNA in response to interferon, followed by a subsequent decrease, imitating the natural regulated transcriptional cycle of the endogenous gene.

Sendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-alpha/beta). However, in human cells, IFN-alpha/beta did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-alpha/beta-responsive promoter and on that of the IFN-beta promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-alpha/beta-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-alpha/beta-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-beta promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.

We have used a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-alpha in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-beta was detected at comparable levels in both groups, while IFN-gamma was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-alpha cDNAs generated by the RT-PCR were cloned and sequenced to determine which alpha-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-alpha or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-alpha was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.

An assay was developed to detect the cytotoxic effects of cytokines on rat pancreatic islet cells in monolayer culture. Cell lysis was detected by a 51Cr-release assay after 4 days of incubation with various cytokines. When tested alone, murine (rat and mouse) interferon-gamma (mIFN-gamma) produced a small dose-dependent lysis of islet cells; human IFN-gamma, mouse IFN-alpha/beta, interleukins 1 and 2 (IL-1 and IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) were inactive. When added together, the following combinations of cytokines showed synergistic cytotoxic effects: TNF (or LT) plus IL-1, TNF (or LT) plus mIFN-gamma, and IL-1 plus mIFN-gamma. These results indicate that the cytokine products of mononuclear cells of the immune system, IFN-gamma, TNF, LT, and IL-1 have strong synergistic cytotoxic effects on islet cells and therefore may act as direct chemical mediators of islet beta-cell destruction in type I (insulin-dependent) diabetes.

We performed immunoperoxidase stains on skin biopsies taken from nine patients with recurrent peripheral herpes simplex lesions at 12 h to 6 d after onset of signs of symptoms to phenotype the inflammatory infiltrate, to detect cells producing interferons alpha and gamma, and to locate herpes simplex virus antigen-containing cells. Viral glycoprotein antigen was located in the nuclei and cytoplasm of necrotic epidermal cells, often within vesicles, in biopsies taken between the first and third day. Histologically, biopsies of all stages showed intradermal focal perivascular and diffuse mononuclear inflammatory infiltrates. The cells constituting the infiltrates were predominantly T lymphocytes with lesser numbers of histiocytes; Leu 7+ (most natural killer/killer) cells and B cells were rare in the biopsy specimens. Leu 3a+ ("helper") T lymphocytes predominated in both subepidermal and perivascular regions of early lesions (12-24 h). Tissue helper/suppressor ratios ranged from 6.3 to 3.4 compared with 1.9-1.0 in blood. In later lesions (after 2 d), monocytes/macrophages were more prominent in tissue sections and the helper/suppressor ratios (2.3-2.5) more nearly approximated those of blood (1.6-2.7). The negative correlation of tissue ratios with time was significant (P less than or equal to 0.02). A large proportion of the infiltrated T lymphocytes expressed DR antigens. There was also diffuse strong DR expression on epidermal cells in five cases (all of two or more days). In six biopsies, scattered macrophages and small cells, presumably lymphocytes, demonstrated cytoplasmic or membrane staining for a substance which copurifies with interferon gamma. We identified such stained cells within vessels, suggesting that these cells circulate. Gamma interferon might have an important role within the herpetic lesions, possibly inducing macrophage activation and cytotoxic T lymphocytes and increasing DR expression on monocyte and epidermal cells.

Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alphaalphaalphaalphaalpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alphaalphaalphabeta, and limitin) showed higher biological activity levels than others, whereas IFN-alphaalpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I*C) transfection or to viral infection were, however, similar.

The human MxA gene belongs to the class of interferon (IFN)-stimulated genes (ISGs) involved in antiviral resistance against influenza viruses. Here, we studied the requirements for MxA induction by influenza A virus infection. MxA is transcriptionally upregulated by type I (alpha and beta) and type III (lambda) IFNs. Therefore, MxA is widely used in gene expression studies as a reliable marker for IFN bioactivity. It is not known, however, whether viruses can directly activate MxA expression in the absence of secreted IFN. By using an NS1-deficient influenza A virus and human cells with defects in IFN production or the STAT1 gene, we studied the induction profile of MxA by real-time reverse transcriptase PCR. The NS1-deficient virus is known to be a strong activator of the IFN system because NS1 acts as a viral IFN-antagonistic protein. Nevertheless, MxA gene expression was not inducible by this virus upon infection of IFN nonproducer cells and STAT1-null cells. Likewise, neither IFN-alpha nor IFN-lambda had a sizeable effect on the STAT1-null cells, indicating that MxA expression requires STAT1 signaling and cannot be triggered directly by virus infection. In contrast, the expression of the IFN-stimulated gene ISG56 was induced by influenza virus in these cells, confirming that ISG56 differs from MxA in being directly inducible by viral triggers in an IFN-independent way. In summary, our study reveals that MxA is a unique marker for the detection of type I and type III IFN activity during virus infections and IFN therapy.

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-alpha/beta]) and type III (IFN-lambda1 to -lambda3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-alpha) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-alpha and IFN-beta inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-lambda3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-lambda3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.

The phase 3 trial HOVON-50 was designed to evaluate the effect of thalidomide during induction treatment and as maintenance in patients with multiple myeloma who were transplant candidates. A total of 556 patients was randomly assigned to arm A: 3 cycles of vincristine, adriamycin, and dexamethasone, or to arm B: thalidomide 200 mg orally, days 1 to 28 plus adriamycin and dexamethasone. After induction therapy and stem cell mobilization, patients were to receive high-dose melphalan, 200 mg/m(2), followed by maintenance with alpha-interferon (arm A) or thalidomide 50 mg daily (arm B). Thalidomide significantly improved overall response rate as well as quality of the response before and after high dose melphalan. Best overall response rate on protocol was 88% and 79% (P = .005), at least very good partial remission 66% and 54% (P = .005), and complete remission 31% and 23% (P = .04), respectively, in favor of the thalidomide arm. Thalidomide also significantly improved event-free survival from median 22 months to 34 months (P < .001), and prolonged progression free from median 25 months to 34 months (P < .001). Median survival was longer in the thalidomide arm, 73 versus 60 months; however, this difference was not significant (P = .77). Patients randomized to thalidomide had strongly reduced survival after relapse. This trial was registered on www.controlled-trials.com as ISRCTN06413384.