We examined the effects of dietary n-<em>3</em> polyunsaturated and saturated fatty acids on the development of the atherogenic process in mice and on the macrophage ability to secrete several effector molecules that may be involved in the atherogenic process. The secretion of inflammatory proteins such as tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and interleukin-1 beta (IL-1 beta) and the production of lipoprotein lipase (LPL), nitrogen oxide (NO2), and prostaglandin E2 (PGE2) were evaluated in peritoneal macrophages isolated from atherosclerosis-susceptible C57BL/6J mice. The mice were assigned at random to three experimental groups: the first group was fed a semi-defined control diet (control diet); the second group was maintained on the control diet supplemented with 10% menhaden oil (menhaden diet); and the third group received the control diet supplemented with 10% palm oil plus 2% cholesterol (saturated fat diet). Macrophages derived from mice fed the menhaden diet showed a suppression of their basal TNF-<em>alpha</em> mRNA expression and production. They also presented a dramatically decreased ability to express TNF-<em>alpha</em> and IL-1 beta mRNAs in response to exposure to lipopolysaccharide (LPS) compared with the macrophages from the control group. LPL mRNA and protein expression were downregulated after 6 and 15 weeks of menhaden-diet feeding. Significantly higher NO2 production in response to <em>interferon</em> gamma was found, both after 6 and 15 weeks of diet feeding, in the menhaden group compared with the control group. In addition, prostaglandin production and macrophage tumoricidal activity in response to LPS were decreased in this group compared with the control group. Macrophages derived from the saturated fat group did not show any significant alterations in TNF-<em>alpha</em>, LPL, NO2, or PGE2 secretion compared with controls. Interestingly, we observed a progressive increase of the LPS-induced IL-1 beta gene expression and secretion among macrophages harvested from mice receiving the dietary supplement of saturated fatty acids. At 6 and 15 weeks histologic examination of the atherosclerotic lesions did not reveal any important lesions in the control and menhaden groups, whereas a gradual development of fatty streaks was observed in the menhaden experimental diets for 10 additional weeks resulted in a major development of lesions in the control group, whereas only slight lesions were observed in the menhaden group.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

The objective of the current study was to identify patient and disease related factors that influence response and survival for patients with unresectable hepatocellular carcinoma (HCC) who received a systemic combination chemotherapy consisting of cisplatin, alpha-interferon, doxorubicin, and 5-fluorouracil (PIAF).

METHODS

From July 1996 to February 1999, 149 patients with unresectable HCC were treated with PIAF: cisplatin (20mg/m2 intravenously, Days 1-4), doxorubicin (40mg/m2 intravenously, Day 1), 5-fluorouracil (400mg/m2 intravenously, Days 1-4), and alpha-interferon (5MU/m2 subcutaneously, Days 1-4), once every 3 weeks up to a maximum of six cycles. Univariate and multivariate analyses of patient and disease characteristics were used to identify factors predicting response and survival.

RESULTS

The objective response rate according to conventional criteria was 16.8% (complete response in 3 out of 149 patients, or 2%, 95% confidence interval [CI] 0-4.3%; partial response in 22 out of 149 patients, or 14.8%, 95% CI 9-20%). The median survival time was 30.9 weeks (95% CI 22.1 to 40). Significant independent predictors of an objective response were: absence of cirrhosis (P = 0.006), low bilirubin level (P = 0.006), and positive hepatitis C serology (P = 0.025). The following factors were related to a shorter survival time: high Okuda stage (P = 0.001), vascular involvement (P = 0.018), and cirrhosis (P = 0.008). Good risk patients (absence of cirrhosis and total bilirubin < or = 0.6mg/dL) had an objective response rate of 50%. CONCLUSIONS. Patients with unresectable HCC who also have normal total bilirubin and non-cirrhotic livers have a better chance of response and prolonged survival after treatment with systemic PIAF.

A recent report demonstrated that free human T-cell leukemia virus 1 (HTLV-1) could infect plasmacytoid dendritic cells (pDCs). The major role of pDCs is to secrete massive levels of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) upon virus exposure; however, the induction of IFN-<em>alpha</em> by HTLV-1 remains unknown. We demonstrate here that cell-free HTLV-1 generated a pDC innate immune response by producing massive levels of IFN-<em>alpha</em> that were inhibited by anti-HTLV-1 antibodies. HTLV-1 induced costimulatory molecules and rapid expression of the apoptotic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, HTLV-1 stimulated pDC-induced apoptosis of CD4(+) T cells expressing DR5, transforming pDCs into IFN-producing killer pDCs. We also observed that an endosomal acidification inhibitor and a Toll-like receptor-7 (TLR7)-specific blocker drastically inhibited pDC response to HTLV-1. Three-dimensional microscopy analysis revealed that unstimulated pDCs were "dormant" IFN-producing killer pDCs with high levels of intracellular TRAIL that could be rapidly mobilized to the surface in response to TLR7 activation. Inhibition of viral degradation in endosomes by chloroquine maintained viral integrity, allowing virus detection by <em>3</em>-dimensional microscopy. We demonstrate that pDCs respond to cell-free HTLV-1 by producing high levels of IFN-<em>alpha</em> and by mobilizing TRAIL on cell surface after TLR7 triggering. This is the first demonstration of an innate immune response induced by free HTLV-1.

BACKGROUND

The eosinophilias encompass a broad range of nonhematologic (secondary or reactive) and hematologic (primary, clonal) disorders with potential for end-organ damage.

METHODS

Hypereosinophilia (HE) has generally been defined as a peripheral blood eosinophil count greater than 1,500/mm(<em>3</em>) and may be associated with tissue damage. After exclusion of secondary causes of eosinophilia, diagnostic evaluation of primary eosinophilias relies on a combination of morphologic review of the blood and marrow, standard cytogenetics, fluorescent in situ hybridization, flow immunocytometry, and T-cell clonality assessment to detect histopathologic or clonal evidence for an acute or chronic myeloid or lymphoproliferative disorder.

METHODS

Disease prognosis relies on identifying the subtype of eosinophilia. After evaluation of secondary causes of eosinophilia, the 2008 World Health Organization establishes a semimolecular classification scheme of disease subtypes including "myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1', chronic eosinophilic leukemia, not otherwise specified" (CEL, NOS), lymphocyte-variant HE, and idiopathic hypereosinophilic syndrome (HES), which is a diagnosis of exclusion.

METHODS

The goal of therapy is to mitigate eosinophil-mediated organ damage. For patients with milder forms of eosinophilia (e.g., <1,500/mm(<em>3</em>)) without symptoms or signs of organ involvement, a watch and wait approach with close-follow-up may be undertaken. Identification of rearranged PDGFRA or PDGFRB is critical because of the exquisite responsiveness of these diseases to imatinib. Corticosteroids are first-line therapy for patients with lymphocyte-variant HE and HES. Hydroxyurea and interferon-alpha have demonstrated efficacy as initial treatment and steroid-refractory cases of HES. In addition to hydroxyurea, second-line cytotoxic chemotherapy agents and hematopoietic cell transplant have been used for aggressive forms of HES and CEL with outcomes reported for limited number of patients. Although clinical trials have been performed with anti-IL-5 (mepolizumab) and anti-CD52 (alemtuzumab) antibodies, their therapeutic role in primary eosinophilic diseases and HES has yet to be established.

There is evidence that TNF-<em>alpha</em> contributes to the pathogenesis of chronic viral hepatitis. The cellular effects of this cytokine are regulated by two specific receptors, and membranous shedding of these receptors reflects activation of the TNF system. We performed a study of TNF-<em>alpha</em> and functionally active soluble TNF-receptors (TNFR-p55 and -p75) in 105 patients with chronic HCV infection. In HCV RNA-positive patients a significant enhancement of TNF-<em>alpha</em> and both receptor types was observed compared with controls (TNF-<em>alpha</em> 8<em>3</em>.8+/-91.7 pg/ml versus 18.8+/-8.4 pg/ml, P<0.001; TNFR-p55 1.4+/-0.4 ng/ml versus 0.9+/-0.2 ng/ml, P<0.0001; TNFR-p75 6.4+/-2.4 ng/ml versus 2.9+/-0.6 ng/ml, P<0.0001, respectively). The enhanced serum levels of TNF-<em>alpha</em> and TNFRs were reflected by a significant expression of TNFR-specific mRNA in peripheral mononuclear cells of HCV-infected patients (P<0.001). Serum aminotransferases correlated with soluble TNFR-p75 (P<0.001) but not with TNFR-p55 and TNF-<em>alpha</em>. We demonstrated an association of the degree of histological inflammation with both TNFRs (P<0.01). Furthermore, enhanced hepatocellular expression of TNF-<em>alpha</em> and TNFRs could be demonstrated by immunohistochemical staining in HCV-infected patients. Sixty-eight out of 105 patients were treated with <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) (<em>3</em>x10(6)U x <em>3</em>/week). Pretreatment levels of TNF-<em>alpha</em> and TNFRs did not differ between responders and non-responders. Our results demonstrate that TNF-<em>alpha</em> and TNFRs are enhanced in chronic HCV infection and reflect histological activity of the disease. This up-regulation of TNFRs might modify host response and potentially contribute to liver damage in chronic HCV infection.

With a new flow cytometric cytotoxicity assay, we examined the mechanism of action of chimeric mouse human anti-CD20 monoclonal antibody IDEC-C2B8. IDEC-C2B8 alone induced direct cytotoxicity in four of eight examined CD20-expressing lymphoma cell lines (RAJI, DAUDI, JOK-1, and WT100) at a concentration above 100 ng/ml. Moreover, after 4 h incubation in human serum, only a moderate complement-dependent cellular cytotoxicity (CDCC) was observed, whereas cytotoxicity increased markedly after <em>3</em> days of culture, indicating that combined direct cytotoxicity and CDCC were responsible. IDEC-C2B8 induced an effective antibody-dependent cellular cytotoxicity (ADCC) in seven of eight tested lymphoma cell lines when peripheral blood mononuclear cells were used as effector cells. ADCC was moderately enhanced by cytokine interleukin-2, whereas interleukin-12, <em>interferon</em>-<em>alpha</em>, and GM-CSF had no influence. Interestingly, we could demonstrate a correlation between CD<em>3</em>2 expression on lymphoma cell lines and IDEC-C2B8-induced direct cytotoxicity, indicating that crosslinking of CD20 with CD<em>3</em>2 may be involved in the mechanism of cytotoxicity. We propose that direct cytotoxicity, CDCC, and ADCC result in the marked elimination of CD20-expressing tumor cells observed after treatment with IDEC-C2B8.

OBJECTIVE

To clarify the effect of genetic polymorphisms on the response to interferon alfa (IFN-alpha) for metastatic renal cell carcinoma (MRCC), and to find a reliable molecular marker to select those patients with MRCC who would benefit from IFN-alpha immunotherapy.

METHODS

We carried out an association study in which 463 single nucleotide polymorphisms (SNPs) in 33 candidate genes were genotyped in 75 Japanese patients who had received IFN-alpha for MRCC.

RESULTS

After adjusting for lung metastasis, stepwise logistic regression analysis revealed that the SNPs in signal transducer and activator 3 (STAT3) were most significantly associated with better response to IFN-alpha. Linkage disequilibrium mapping revealed that the SNP in the 5' region of STAT3, rs4796793, was the most significant predictor of IFN-alpha response (odds ratio [OR] = 2.73; 95% CI, 1.38 to 5.78). The highest OR was shown in the CC genotype at rs4796793 compared to the GG + GC genotypes (OR = 8.38, 95% CI, 1.63 to 42.96). Genotype-dependent expressions of STAT3 in B lymphocyte cell lines and the enhanced growth inhibitory effects of IFN- by STAT3 suppression in an RCC cell line supported the results of the present association study.

CONCLUSIONS

The present study suggested that the STAT3 polymorphism is a useful diagnostic marker to predict the response to IFN-alpha therapy in patients with MRCC. An efficient response marker for IFN-alpha needs to be utilized to establish individual optimal treatment strategies, even when newer drug therapies are used as first line treatments for MRCC.

We defined the function of type I <em>interferons</em> (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyer's patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-<em>alpha</em>, IFN-beta, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFN<em>alpha</em>R1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor <em>3</em> or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.

Erythropoiesis is positively regulated by stem cell factor, interleukin <em>3</em>, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, <em>interferon</em> gamma, tumor necrosis factor <em>alpha</em>, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. <em>Interferon</em> gamma and <em>alpha</em> act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD<em>3</em>6(+) erythroid progenitors, stem cell factor, interleukin <em>3</em>, and erythropoietin allowed massive proliferation (x<em>3</em>00) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD<em>3</em>6(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

The prime anti-viral cytokine <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) has been implicated in several central nervous system (CNS) disorders in addition to its beneficial effects. Systemic IFN-<em>alpha</em> treatment causes severe neuropsychiatric complications in humans, including depression, anxiety and cognitive impairments. While numerous neuromodulatory effects by IFN-<em>alpha</em> have been described, it remains unresolved whether or not systemic IFN-<em>alpha</em> acts directly on the brain to execute its CNS actions. In the present study, we have analyzed the genes directly regulated in post-IFN-<em>alpha</em> receptor signaling and found that intraperitoneal administration of mouse IFN-<em>alpha</em>, but not human IFN-<em>alpha</em>, activated expression of several prototypic IFN-stimulated genes (ISGs), in particular signal transducers and activators of transcription (STAT1), IFN-induced 15 kDa protein (ISG15), ubiquitin-specific proteinase 18 (USP18) and guanylate-binding protein <em>3</em> (GBP<em>3</em>) in the brain. A similar temporal profile for the regulated expression of these IFN-<em>alpha</em>-activated ISG genes was observed in the brain compared with the peripheral organs. Dual labeling in situ hybridization combined with immunocytochemical staining demonstrated a wide distribution of the key IFN-regulated gene STAT1 transcripts in the different parenchyma cells of the brain, particularly neurons. The overall response to IFN-<em>alpha</em> challenge was abolished in STAT1 knockout mice. Together, our results indicate a direct, STAT1-dependent action of systemic IFN-<em>alpha</em> in the CNS, which may provide the basis for a mechanism in humans for neurological/neuropsychiatric illnesses associated with IFN-<em>alpha</em> therapy.

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the effects of human recombinant <em>interferon</em>-<em>alpha</em>2a and human recombinant <em>interferon</em>-beta on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative effects of IFN-beta than of IFN-<em>alpha</em>. Analysis of the early signals triggered by IFN-<em>alpha</em> and IFN-beta demonstrated that the two IFNs were similarly effective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-beta treatment, with concomitant activation of p<em>3</em>8 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-beta was found to be impaired by p<em>3</em>8 MAPkinase inhibitor (SB20<em>3</em>580). IFN-beta induced the formation of the <em>Interferon</em> Stimulated Gene Factor <em>3</em> complex more efficiently than IFN-<em>alpha</em>, as well as sustained induction of IRF-1, which may account for its greater induction of 2'5'oligo(A)synthetase and greater inhibition of cell proliferation. IFN-beta, but not IFN-<em>alpha</em>, induced apoptosis in wild-type p5<em>3</em> EW-7 and COH cell lines, but not in the mutated p5<em>3</em> EW-1 or ORS cell lines. The apoptosis induced by IFN-beta in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-beta-induced apoptosis in parental EW-7 and COH cell lines.

The effects of prostaglandin E2 (PGE2), cyclic nucleotides, leukotriene B4 (LTB4), and <em>interferons</em> on interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated C<em>3</em>H/HeNCrl mouse peritoneal macrophages were studied. IL 1 production was inhibited by PGE2, the adenosine <em>3</em>':5'-monophosphate analog dibutyryl cAMP, the cAMP agonist isoproterenol, and the phosphodiesterase inhibitor isobutylmethylxanthine. These agents were more inhibitory when added early in the latent phase of IL 1 synthesis following stimulation with LPS rather than just prior to release of IL 1 into the medium. Production of both the intracellular and extracellular forms of IL 1 was blocked by PGE2 and cAMP. Suppression of LPS-induced IL 1 production by PGE2 was prevented by leukocyte <em>alpha</em>-<em>interferon</em>. Moreover, <em>alpha</em>-<em>interferon</em> augmented LPS-induced IL 1 production but did not stimulate IL 1 production in the absence of LPS. Immune gamma-<em>interferon</em> markedly inhibited LPS-stimulated IL 1 production. The lipoxygenase inhibitor eicosa-5,8,11,14-tetraynoic acid suppressed, whereas <em>3</em>-amino-1-(<em>3</em>-trifluoromethylphenyl)-2-pyrazoline augmented, LPS-induced IL 1 production. The opposing effects of these agents suggested that lipoxygenase metabolites do not act as inducers of IL 1 production. Purified LTB4 did not stimulate base-line or augment LPS-induced IL 1 production (both intracellular and extracellular forms). Moreover, calcium ionophore A2<em>3</em>187 (a lipoxygenase activator) did not stimulate IL 1 production, alone or in combination with LTB4. Thus, net IL 1 production by macrophages may be regulated by a balance between the effects of PGE2, cAMP, <em>alpha</em>-<em>interferon</em>, and gamma-<em>interferon</em>, but not LTB4.

The medical treatment of neuroendocrine GEP tumours must be based on the growth properties of the tumour. Medical treatment includes chemotherapy, somatostatin analogues and <em>alpha</em> <em>interferons</em>. Chemotherapy has been particularly active in patients with high proliferating neuroendocrine tumours such as endocrine pancreatic tumours and lung carcinoids. Streptozotocin-based combinations including 5-flourouracil and doxorubicin have generated partial remissions in 40%-60% of the patients giving a median survival of about two years in patients with advanced disease. Cisplatinum plus etoposide have demonstrated significant antitumour effects in anaplastic endocrine pancreatic tumours and lung carcinoids. However, in low proliferating tumours such as classical midgut carcinoids the response rates with the same combinations of cytotoxic agents have only generated short lasting responses in less than 10% of patients. In these patients, biological treatment has been of benefit. <em>Alpha</em> <em>interferon</em> at doses of <em>3</em>-9 million units three to seven times per week subcutaneously, has given biochemical response rates of 50% and significant tumour reduction in about 15% of patients with long duration, up to three years. Somatostatin analogues have been widely used in the treatment of neuroendocrine gut and pancreatic tumours. The currently available somatostatin analogues particularly bind somatostatin receptor 2 and 5 and with low affinity also receptor subtype <em>3</em>. Octreotide is registered in most countries for the treatment of patients with carcinoid syndrome and also VIP and glucagon producing tumours. Regular octreotide at standard doses of 100-<em>3</em>00 microg/day gives symptomatic responses in a medium of 60% of patients and biochemical responses in up to 70% of patients. Significant tumour responses are rare, less than 5%. Long-acting formulations of somatostatin analogues have been of significant benefit for the patients with similar response rates as for regular formulations. The quality of life has been significantly improved by using the long-acting formulations.

We examined the effects of <em>interferon</em>-<em>alpha</em> and -gamma, which are known to have psychiatric side effects including depression, on the transcriptional regulation of the serotonin transporter and the uptake activity of the serotonin transporter in order to clarify the involvement of the serotonin transporter in the pathogenesis of <em>interferon</em>-induced depression. In human placental choriocarcinoma cells (BeWo cells), both messenger RNA (mRNA) for the serotonin transporter and the imipramine-sensitive uptake of serotonin were detected. The levels of serotonin transporter mRNA were increased by treatment with <em>interferon</em>-<em>alpha</em> and -gamma for <em>3</em> h. The increase in serotonin transporter mRNA elicited by the <em>interferons</em> was inhibited by treatment with actinomycin D, an inhibitor of transcription. Treatment with <em>interferon</em>-<em>alpha</em> or -gamma for <em>3</em>-6 h, but not for <em>3</em>0 min, increased the uptake activity of the serotonin transporter. Treatment with dibutyryl cAMP (Dib-cAMP) which was reported to up-regulate the transcription of the serotonin transporter, also increased the mRNA levels and the activity of serotonin transporter in BeWo cells. The levels of serotonin transporter mRNA gradually increased after treatment with Dib-cAMP over 24 h, while the maximal increase in serotonin transporter mRNA elicited by the <em>interferons</em> was detected <em>3</em> h after the treatment. The level of serotonin transporter mRNA was increased both in the midbrain and adrenal glands of mice which were treated with <em>interferons</em> for <em>3</em> h. These results suggest that the <em>interferon</em>-induced psychiatric side effects arise through regulation of serotonin transporter transcription and that the transcriptional regulation of the serotonin transporter is a possible neurochemical mechanism of affective disorders.

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor <em>3</em> (CXCR<em>3</em>) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR<em>3</em> or TLR4 ligands, respectively) synergized with <em>interferon</em>-gamma (IFN-gamma) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-gamma was combined with TLR<em>3</em> ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-gamma-dependent CXCR<em>3</em> ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) or interleukin-1beta (IL-1beta), in combination with IFN-gamma, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG(1)Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-<em>alpha</em> plus IFN-gamma, but not synergy between IFN-gamma and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the physiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.

Patients with polycythemia vera (PV) are most often treated with phlebotomy-only (PHL-O) or phlebotomy plus hydroxyurea (PHL + HU). Such treatment is often unsatisfactory because of persistent susceptibility to thrombosis owing to inadequate control of abnormal erythropoiesis and thrombopoiesis. Recombinant <em>interferon</em>-<em>alpha</em> (rIFN <em>alpha</em>) inhibits erythroid progenitors and affects megakaryocyte function and thus may be a more effective treatment, but reports of its use have been of relatively short duration. The long-term use (median, 1<em>3</em> years) of rIFN <em>alpha</em> in 55 patients previously treated with PHL alone or with PHL + HU was studied. Data pertaining to the natural history of the disease were also examined. Patients achieved partial response of their disease by 6 months, and complete response by 1-2 years (phlebotomy-free, HCT < or =45%, platelets < or =600,000/microL); spleen size was reduced in 27 of <em>3</em>0 patients with prior splenomegaly. The initial dose of rIFN <em>alpha</em> was 1 mega unit <em>3</em> times a week (1 MU/tiw) for the majority of patients, with periodic dose increases as required and as tolerated. The maintenance dose, usually <em>3</em> MU/tiw, could be decreased after the second year of treatment in half the patients. Toxicity was acceptable. Disease-free survival was marked by no thrombohemorrhagic complications reflecting both the effect of rIFN <em>alpha</em> and total patient care. Evidence is presented indicating that rIFN <em>alpha</em> effectively reduces PHL requirements, thrombocythemia, splenomegaly, and thrombohemorrhagic events. It is an effective drug for treating PV with acceptable toxicity.

OBJECTIVE

We evaluated the role of type I <em>interferons</em> (IFNs) and IFN receptors in the regulation of cell growth in <em>3</em> human pancreatic adenocarcinoma cell lines (BxPC-<em>3</em>, MiaPaCa-2, and Panc-1).

BACKGROUND

Chemotherapy and radiotherapy have a marginal role in the management of pancreatic adenocarcinoma. The addition of IFN-alpha showed promising results in early clinical trials.

METHODS

Cell proliferation and apoptosis were evaluated by DNA measurement and DNA fragmentation, respectively. Type I IFN receptor (IFNAR-1 and IFNAR-2 subunits) was determined by quantitative RT-PCR and immunocytochemistry. Cell cycle distribution was evaluated by propidium iodide staining and flow-cytometric analysis.

RESULTS

The incubation with IFN-beta for 6 days showed a potent inhibitory effect on the proliferation of BxPC-<em>3</em> (IC(50), 14 IU/mL) and MiaPaCa-2 (IC(50), 64 IU/mL). The inhibitory effect of IFN-beta was stronger than IFN-alpha in all <em>3</em> cell lines and mainly modulated by the stimulation of apoptosis, although cell cycle arrest was induced as well. The expression of the type I IFN receptors was significantly higher in BxPC-<em>3</em> (the most sensitive cell line to IFN) and mainly localized on the membrane, whereas in Panc-1 (the most resistant cell line) about 60% to 70% of cells were negative for IFNAR-2c with a mainly cytoplasmic staining for IFNAR-2c.

CONCLUSIONS

The antitumor activity of IFN-beta is more potent than IFN-alpha in pancreatic cancer cell lines through the induction of apoptosis. Further studies should investigate in vivo whether the intensity and distribution of IFNAR-1 and IFNAR-2c may predict the response to therapy with IFN-alpha and IFN-beta in pancreatic cancer.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly discovered Phlebovirus causing an emerging hemorrhagic fever in East Asia, with reported case fatality rates up to <em>3</em>0%. Despite the high case fatality rate and large number of persons at risk of infection, the pathobiology of the disease is unknown, and no effective animal model has been available for investigating its pathogenesis. We have studied mice and hamsters as potential small-animal models of SFTSV infection following subcutaneous, intraperitoneal, or intracerebral inoculation. Animal tissues were processed for viral load determination, histopathology, immunohistochemistry, and confocal microscopic studies. We found that immunocompetent adult mice and hamsters did not become ill after SFTSV infection. However, <em>alpha</em>/beta <em>interferon</em> receptor knockout (IFNAR(-/-)) mice were highly susceptible to SFTSV infection, and all mice died within <em>3</em> to 4 days after subcutaneous inoculation of 10(6) focus-forming units of SFTSV. Histologic examination of tissues of IFNAR(-/-) mice infected with SFTSV showed no detectable lesions. In contrast, by immunohistochemistry virus antigen was found in liver, intestine, kidney, spleen, lymphoid tissue, and brain, but not in the lungs. Mesenteric lymph nodes and spleen were the most heavily infected tissues. Quantitative reverse transcription-PCR (RT-PCR) confirmed the presence of virus in these tissues. Confocal microscopy showed that SFTSV colocalized with reticular cells but did not colocalize with dendritic cells, monocytes/macrophages, neutrophils, or endothelium. Our results indicate that SFTSV multiplied in all organs except for lungs and that mesenteric lymph nodes and spleen were the most heavily infected tissues. The major target cells of SFTSV appear to be reticular cells in lymphoid tissues of intestine and spleen.

OBJECTIVE

Heatstroke, characterized by hyperthermia and neurologic abnormalities, can cause shock, adult respiratory distress syndrome, and multiorgan failure culminating in death. The mediation of metabolic changes and tissue damage is not fully understood. Recent evidence suggests the involvement of endotoxin, tumor necrosis factor alpha (TNF-alpha), and interleukin 1 alpha (IL-1 alpha) and we hypothesized that other pyrogenic cytokines may be implicated.

METHODS

Prospective analysis.

METHODS

Heatstroke Center in Makkah (Mecca), Saudi Arabia.

RESULTS

We measured plasma IL-1 beta, IL-6, and interferon gamma (INF-gamma) concentrations by enzyme-linked immunosorbent assay in 28 heatstroke patients at the time of hospital admission (precooling) and after complete cooling (postcooling), and in 10 normal control subjects. We measured C-reactive protein (CRP) as a marker of acute phase response and calculated severity of illness using the simplified acute physiology score. Twenty-five male and 3 female subjects had mean (+/- SEM) rectal temperature of 41.2 +/- 0.2 degrees C. IL-6, IL-1 beta, and INF-gamma concentrations were elevated in 100 percent, 39 percent, and 50 percent of patients to (mean +/- SEM) 220 +/- 44 pg/ml, 42 +/- 14 pg/ml, and 1,180 +/- 879 pg/ml, respectively (normal control values: < 3.5 pg/ml, < 4.5 pg/ml, < 20 pg/ml). The CRP value was elevated in 72 percent of patients to 152 +/- 40 mg/L (control value: 0 to 17 mg/L). The IL-6 concentrations correlated with severity of illness (r = 0.516, p = 0.03); two patients with the highest concentrations died. There was no significant correlation between circulating levels of IL-6, IL-1 beta, INF-gamma, and temperature, or between IL-6, IL-1 beta, and CRP. Postcooling, IL-6, and IL-1 beta were still above normal control values; INF-gamma could be detected in one patient only.

CONCLUSIONS

Our findings of elevated circulating IL-6, IL-1 beta, and INF-gamma in the presence of acute phase response, and correlation with severity of illness, suggest that these cytokines have a role in the pathogenesis of heatstroke, which could lead to new therapeutic strategies.

OBJECTIVE

To report on the results of interferon-alpha 2a (IFNalpha) treatment in patients with Behçet uveitis unresponsive to conventional immunosuppressive therapy.

METHODS

We retrospectively analyzed the medical records of 44 patients who had been treated with IFNalpha between September 2001 and May 2005. The initial dose of IFNalpha was 6 MU/day in 37 patients (84.1%) and 3 MU/day in 7 patients (15.9%), and was gradually tapered after ocular inflammation was suppressed. Immunosuppressive agents were discontinued. Oral corticosteroids were discontinued or maintained at a dosage of less than 10 mg prednisone equivalent per day. Main outcome measures were recurrence of posterior or panuveitis attacks and changes in visual acuity.

RESULTS

Sixteen patients (36.4%) remained relapse free during treatment, whereas 28 (63.6%) patients had recurrent uveitis attacks. Four of these were considered treatment failures and were switched to other treatments. In the remaining 40 patients, the mean duration of treatment was 12.4+/-10.8 months (range 3-45 months). In 9 of 40 patients (22.5%) treatment could be discontinued 22.2+/-13.4 months after therapy, and 8 (20%) of these patients had sustained remission for up to 24 months. Three patients (7.5%) were switched to other therapies because of side effects. The frequency of uveitis attacks per 6 months was reduced from 1.6+/-1.2 to 0.8+/-0.9 in 26 patients who had a minimum follow-up of 6 months before and after IFNalpha therapy (p<0.05). There was a significant improvement in visual acuity and this was preserved throughout follow-up in 38 (95%) of 40 patients.

CONCLUSIONS

A partial or complete response was obtained with IFNalpha therapy in 91% of Turkish patients with Behçet uveitis refractory to conventional immunosuppressive treatment. Our results suggest that there may be differences in therapeutic efficacy and side-effect profile of IFNalpha in different patient populations. Comparative studies are needed to investigate this hypothesis.

OBJECTIVE

Giant cell tumors are classified and treated based on their biologic behavior. We hypothesize that they are proliferative vascular lesions and would be expected to respond to antiangiogenic therapy. The purpose of this report is to present a treatment protocol consisting of enucleation, with preservation of vital structures, followed by subcutaneous interferon alpha.

METHODS

Patients with a biopsy-confirmed giant cell lesion satisfying criteria for "aggressive giant cell tumor" were included. Instead of wide en bloc resection, lesions were enucleated and the patients started on interferon alpha-2 or beta (3,000,000 units/m(2)) 48 to 72 hours postoperatively. The subjects were followed by clinical examination and radiography, immediately after surgery and every 3 months until the bone cavity completely healed. Thereafter, follow-up was every 6 months.

RESULTS

Eight patients (7 females), with a mean age of 18.7 +/- 11.1 years, have been enrolled. Six tumors were in the posterior mandible, and 2 were in the anterior maxilla. The mean size was 29.0 mm (range, 15 to 70 mm). All patients underwent enucleation. There were no postoperative complications, and all patients tolerated interferon. There was no evidence of tumor growth during treatment. Seven of 8 patients have completed interferon therapy, and there have been no recurrences during 1 to 6 years of follow-up. The other patient continues on treatment with no evidence of disease.

CONCLUSIONS

Antiangiogenic therapy, in combination with curettage, is a promising strategy for treatment of aggressive giant cell tumors. Combined treatment results in a high rate of tumor control with decreased operative morbidity compared with conventional treatment.

During experimental infection of pigs with swine influenza virus (SIV), there is a strong temporal correlation between peak virus titers in the lungs, levels of different proinflammatory cytokines in bronchoalveolar lavage (BAL) fluids, and disease. Vaccination against SIV can greatly reduce or prevent virus replication after challenge and the resulting disease. Here, we took advantage of pigs from vaccination-challenge experiments, with different degrees of virological and clinical protection, to further correlate SIV replication with cytokines and disease. Forty-nine pigs were vaccinated twice with a commercial inactivated SIV vaccine or with experimental vaccines, and <em>3</em>5 control pigs were not vaccinated. Between 2 and 4 weeks after the last vaccination, all pigs were challenged intratracheally with SIV. Twenty-four hours after the challenge, we determined body temperatures, respiratory scores, lung virus titers, and neutrophils and cytokines in BAL fluids. <em>Interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), tumor necrosis factor (TNF-<em>alpha</em>), interleukin-1 (IL-1), and -6 (IL-6) were determined by bioassay, and IL-8 by a commercial ELISA. The results were analyzed for three comparison groups. The unvaccinated control pigs (group 1, n = <em>3</em>5) were positive for all or most parameters examined. Vaccinated pigs with challenge virus replication in the lungs (group 2, n = 28) had slightly lower virus titers than the challenge control pigs, and clear reductions in disease severity and mean titers of all five cytokines, but neutrophil numbers were not affected. Vaccinated pigs without detectable virus replication (group <em>3</em>, n = 21) were largely protected against clinical signs and neutrophil infiltration. Mean levels of IFN-<em>alpha</em>, TNF-<em>alpha</em>, and IL-6, but not IL-1 or IL-8, were lower than in both other groups. Virus titers in the lungs of individual pigs showed highly significant correlations with IFN-<em>alpha</em> and IL-6, and lower correlations with TNF-<em>alpha</em> and IL-8. Clinical signs were most closely associated with IFN-<em>alpha</em>, IL-6, and TNF-<em>alpha</em>. The relationship between disease and IL-8 or IL-1 was much weaker. Our data provide further evidence for a role of IFN-<em>alpha</em>, TNF-<em>alpha</em>, and IL-6 in the pathogenesis of SIV. The similarities with cytokine profiles during human influenza virus infection are discussed.

BACKGROUND

Regulation of gene expression by signal transducer and activator of transcription 1 (STAT1) within host tissues mediates the antitumor effects of interferon alfa (IFN alpha). We used a novel flow cytometric assay to examine phosphorylation-mediated activation of STAT1 within immune effector cell subsets following in vitro or in vivo IFN alpha treatments.

METHODS

Peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 17) or melanoma patients (n = 19) were treated in vitro with interferon alfa-2b (IFN alpha-2b) or phosphate-buffered saline (PBS) and subjected to multiparametric flow cytometry to measure the levels of phosphorylated STAT1 (P-STAT1) within immune cell subsets. We similarly analyzed PBMCs isolated from melanoma patients before and 1 hour after immunotherapy with IFN alpha-2b. All statistical tests were two-sided.

RESULTS

P-STAT1 levels in all major immune cell subsets increased within 15 minutes of in vitro IFN alpha-2b treatment of PBMCs; the increase was most pronounced in T lymphocytes and monocytes. Relatively low doses of IFN alpha-2b (i.e., 10(2)-10(3) IU/mL) induced maximal STAT1 activation in vitro. Compared with melanoma patients, healthy donors had higher basal levels of P-STAT1 (specific fluorescence [Fsp]; i.e., Fsp(PBS), the level of P-STAT1 in PBS-treated cells) in total PBMCs, natural killer (NK) cells, and T cells (mean Fsp(PBS) in total PBMCs: 5.5 in healthy donors versus 1.6 in patients, difference = 3.9, 95% confidence interval [CI] = 1.4 to 6.5, P =.004; mean Fsp(PBS) in NK cells: 4.6 in healthy donors versus 0.9 in patients, difference = 3.7, 95% CI = 1.7 to 5.7, P =.001; mean Fsp(PBS) in T cells: 6.8 in healthy donors versus 0.9 in patients, difference = 5.9, 95% CI = 2.5 to 9.3, P =.002). P-STAT1 was detected in the NK and T cells of two patients who received IFN alpha-2b immunotherapy (20 MU/m2 [MU = million units], administered by intravenous injection). P-STAT1 levels in the PBMCs of a patient treated sequentially with 5 MU/m2 and 10 MU/m2 IFN alpha-2b (administered by subcutaneous injection) also increased in response to treatments with IFN alpha-2b but did not increase further with the increased dosage of IFN alpha-2b.

CONCLUSIONS

This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.

Both neonatal and C57BL/6 gamma <em>interferon</em> (IFN-gamma) knockout (C57BL/6-GKO) mice are susceptible to Cryptosporidium parvum, but the course of infection is different. Neonatal mice are able to clear the parasite within <em>3</em> weeks, whereas C57BL/6-GKO mice, depending on age, die rapidly or remain chronically infected. The mechanism by which IFN-gamma leads to a protective immunity is yet poorly understood. In order to investigate the effect of IFN-gamma on other cytokines expressed in the intestinal mucosa during C. parvum infection, we studied cytokine mRNA expression in the neonates and GKO (neonatal and adult) mice by quantitative reverse transcription-PCR (RT-PCR) at 4 and 9 days after infection. IFN-gamma mRNA levels were quickly and strongly up-regulated in the mucosa of neonatal mice. In GKO mice, the Th1-type response was dramatically altered during the infection, whereas the mRNA expression levels of the Th2-type cytokines interleukin 4 (IL-4) and IL-10 were increased in both mouse models. In the absence of IFN-gamma, the adult knockout mice up-regulated the mRNA levels of inflammatory cytokines, such as IL-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor, in the mucosa, but not tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>), whereas all these cytokines were up-regulated in the infected neonatal mice. Further experiments indicated that injections of TNF-<em>alpha</em> into GKO adult mice significantly reduced oocyst shedding. The results of the present study indicate that the resolution of infection is dependent on the expression of Th1-type cytokines in the mucosa of C57BL/6 mice and that TNF-<em>alpha</em> may participate in the control of parasite development.