Cytokines have been recognized to play an important role both in normal development of the brain, when they act as neurotrophic factors, as well as following injury. While both the cytokines and their receptors are synthesized and expressed in the brain normally (albeit at low levels), it has become clear that elevated levels are associated with many neurological disorders. In this review, we have chosen to present the data for only a few of the cytokines, including interleukin-1beta, interleukin-<em>3</em>, interleukin-6, <em>interferon</em>-gamma, transforming growth factor-beta, and tumor necrosis factor-<em>alpha</em>. Data are presented that suggest roles they may play in human disorders, including stroke, multiple sclerosis, Alzheimer's disease, and several psychiatric disorders. The results in human disease are compared with results obtained in a variety of transgenic animal models. The mouse models have very different disorders depending on whether a cytokine is overexpressed either peripherally or in either astrocytes or neurons. The potential significance of this to the understanding of human disease is discussed.

<em>Interferon</em>-gamma (IFN-gamma)-induced indoleamine 2,<em>3</em>-dioxygenase (IDO) activity inhibits the growth of susceptible intracellular pathogens by catalyzing the oxidative cleavage of the indole ring of L-tryptophan and depleting pools of the essential amino acid. Tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) synergistically enhances the IDO activity induced by IFN-gamma at the level of transcription in human epithelial cells. The purpose of this study was to characterize the molecular mechanisms responsible for synergistic gene expression in response to IFN-gamma and TNF-<em>alpha</em>. It was found that IFN-gamma-induced mechanisms, such as the binding of Stat1 to gamma activation sequences (GAS) and IFN regulatory factor-1 (IRF-1) to IFN-stimulated response elements (ISREs), are more highly activated following treatment with IFN and TNF-<em>alpha</em>. This enhanced signal transduction may be due to the increase in IFN-gamma receptor (IFNGR) expression following combined cytokine stimulation and is a likely contributor to the synergy. Additionally, the contribution of a third previously uncharacterized GAS element that forms a complex with Stat1 was investigated using a plasmid reporter system that controls for copy number. When the GAS-<em>3</em> sequence was included in the regulatory region, gene expression was significantly increased relative to a region containing the mutated GAS-<em>3</em>. This suggests that GAS-<em>3</em> is transcriptionally active and contributes to IFN-gamma-induced regulation of the IDO gene.

beta-Glucans derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are highly purified Saccharomyces cerevisiae cell walls composed of beta1,6-branched beta1,<em>3</em>-d-glucan and free of mannans. YGPs stimulated secretion of the proinflammatory cytokine tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) in wild-type murine bone marrow-derived myeloid dendritic cells (BMDCs) but did not stimulate interleukin-12p70 (IL-12p70) production. A purified soluble beta1,6-branched beta1,<em>3</em>-d-glucan, scleroglucan, also stimulated TNF-<em>alpha</em> in BMDCs. These two beta-glucans failed to stimulate TNF-<em>alpha</em> in Dectin-1 (beta-glucan receptor) knockout BMDCs. Costimulation of wild-type BMDCs with beta-glucans and specific Toll-like receptor (TLR) ligands resulted in greatly enhanced TNF-<em>alpha</em> production but decreased IL-12p70 production compared with TLR agonists alone. The upregulation of TNF-<em>alpha</em> and downregulation of IL-12p70 required Dectin-1, but not IL-10. Gamma <em>interferon</em> (IFN-gamma) priming did not overcome IL-12p70 reduction by beta-glucans. Similar patterns of cytokine regulation were observed in human monocyte-derived dendritic cells (DCs) costimulated with YGPs and the TLR4 ligand lipopolysaccharide. Finally, costimulation of BMDCs with YGPs and either the TLR9 ligand, CpG, or the TLR2/1 ligand, Pam(<em>3</em>)CSK(4), resulted in upregulated secretion of IL-1<em>alpha</em> and IL-10 and downregulated secretion of IL-1beta, IL-6, and IFN-gamma-inducible protein 10 but had no significant effects on IL-12p40, keratinocyte-derived chemokine, monocyte chemotactic protein 1, or macrophage inflammatory protein <em>alpha</em>, compared with the TLR ligand alone. Thus, beta-glucans have distinct effects on cytokine responses following DC stimulation with different TLR agonists. These patterns of response might contribute to the skewing of immune responses during mycotic infections and have implications for the design of immunomodulators and vaccines containing beta-glucans.

We measured the levels of <em>interferon</em> <em>alpha</em> (IFN <em>alpha</em>) in the sera of Thai children hospitalized with dengue hemorrhagic fever (DHF) or dengue fever (DF) to examine the role of IFN <em>alpha</em> in dengue virus infections of humans. The percentage of patients who had detectable levels of IFN <em>alpha</em> >> or = <em>3</em> U/ml) was higher in patients with DHF (80%, P < 0.001) and in patients with DF (60%, P < 0.001) than in healthy Thai children (7%). The levels of IFN <em>alpha</em> were higher in patients with DHF and in patients with DF on the first few days after the onset of fever than in healthy Thai children. The average levels of IFN <em>alpha</em> in patients with DHF were high two days before defervescence, decreasing gradually until the day of defervescence. There was a subset of patients with DHF who had increasing levels of IFN <em>alpha</em> after defervescence. However, the levels of IFN <em>alpha</em> in patients with DF were not high after fever subsided. The levels of IFN <em>alpha</em> were not different among children with DHF grades 1, 2 and <em>3</em>. Among patients with DHF, T lymphocytes were activated to a higher degree in high IFN <em>alpha</em> producers than in low IFN <em>alpha</em> producers. These results indicate that similarly high levels of IFN <em>alpha</em> are produced in vivo during the acute stages of DHF and DF, and that high levels of IFN <em>alpha</em> remain after fever subsides in some patients with DHF, but not in patients with DF.

This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, <em>interferon</em> (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-<em>alpha</em>, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-<em>3</em>, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.

Granulysin is a cytolytic and proinflammatory molecule first identified by a screen for genes expressed 'late' (<em>3</em>-5 days) after activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy termini. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule exocytosis. Nine kiloDalton granulysin is broadly cytolytic against tumors and microbes, including gram-positive and gram-negative bacteria, fungi/yeast and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes and other inflammatory cells and activates the expression of a number of cytokines, including regulated upon activation T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, MCP-<em>3</em>, macrophage inflammatory protein (MIP)-1 <em>alpha</em>, interleukin (IL)-10, IL-1, IL-6 and <em>interferon</em> (IFN)-<em>alpha</em>. Granulysin is implicated in a myriad of diseases including infection, cancer, transplantation, autoimmunity, skin and reproductive maladies. Small synthetic forms of granulysin are being developed as novel antibiotics. Studies of the full-length forms may give rise to new diagnostics and therapeutics for use in a wide variety of diseases.

The critical importance of plasmacytoid dendritic cells (pDCs) in viral infection, autoimmunity, and tolerance has focused major attention on these cells that are rare in blood and immune organs of humans and mice. The recent development of an Flt-<em>3</em> ligand (FL) culture system of bone marrow cells has led to the simple generation of large numbers of pDCs that resemble their in vivo steady-state counterparts. The FL system has allowed unforeseen insight into the biology of pDCs, and it is assumed that FL is the crucial growth factor for these cells. Surprisingly we have found that a cell type with high capacity for <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) production in response to CpG-containing oligonucleotides, a feature of pDCs, develop within macrophage-colony-stimulating factor (M-CSF)-generated bone marrow cultures. Analysis of this phenomenon revealed that M-CSF is able to drive pDCs as well as conventional DCs (cDCs) from BM precursor cells in vitro. Furthermore, application of M-CSF to mice was able to drive pDCs and cDCs development in vivo. It is noteworthy that using mice deficient in FL indicated that the M-CSF-driven generation of pDCs and cDCs in vitro and in vivo was independent of endogenous FL.

The therapeutic use of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), a proinflammatory cytokine, is known to cause various neuropsychiatric adverse effects. In particular, depression occurs in <em>3</em>0-45% of patients, frequently interrupting treatment. IFN-<em>alpha</em>-treated animals also show depression-like behaviors. However, mechanisms underlying the depression caused by IFN-<em>alpha</em> remain to be defined. Recently, a decrease in adult hippocampal neurogenesis was revealed as a possible neuropathological mechanism of depression. Therefore, we investigated the effect of subchronic IFN-<em>alpha</em> treatment on neurogenesis in the adult rat dentate gyrus (DG). Immediately after the administration of IFN-<em>alpha</em> for 1 week, a decrease in the number of 5-bromo-deoxyuridine-labeled proliferating cells was observed in the DG; however, no effect was detected on the expression of mature neuronal phenotype in the newly formed cells <em>3</em> weeks later. Also, an increase in the level of interleukin-1beta (IL-1beta), a major proinflammatory cytokine, was observed in the hippocampus following the administration of IFN-<em>alpha</em>. Furthermore, coadministration of an IL-1 receptor antagonist completely blocked the IFN-<em>alpha</em>-induced suppression of the cell-proliferative activity in the DG. Our results indicate that IFN-<em>alpha</em> suppresses neurogenesis in the DG, and that IL-1beta plays an essential role in the suppression. The decreased cell proliferation caused by IFN-<em>alpha</em>-induced IL-1beta may be responsible, at least in part, for IFN-<em>alpha</em>-induced depression.

All <em>alpha</em>-<em>interferons</em> (IFN<em>alpha</em>) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN<em>alpha</em>2. Here, we constructed a phage display library by randomizing three positions on IFN<em>alpha</em>2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN<em>alpha</em>2, and <em>3</em>-fold tighter compared with IFNbeta. Binding of YNS to IFNAR2 was comparable with wild-type IFN<em>alpha</em>2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN<em>alpha</em>2, whereas its antiviral activity was increased by only <em>3</em>.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA2<em>3</em>1 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN<em>alpha</em>2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFN<em>alpha</em>2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an <em>interferon</em> variant with highly increased biological potency.

BACKGROUND

Peginterferon alpha-2b plus ribavirin therapy in previously untreated patients with chronic hepatitis C yields the highest sustained virological response rates of any treatment strategy but is expensive.

OBJECTIVE

To estimate the cost effectiveness of treatment with peginterferon alpha-2b plus ribavirin compared with interferon alpha-2b plus ribavirin for initial treatment of patients with chronic hepatitis C.

METHODS

Individual patient level data from a randomised clinical trial with peginterferon plus ribavirin were applied to a previously published and validated Markov model to project lifelong clinical outcomes. Quality of life and economic estimates were based on German patient data. We used a societal perspective and applied a 3% annual discount rate.

RESULTS

Compared with no antiviral therapy, peginterferon plus fixed or weight based dosing of ribavirin increased life expectancy by 4.2 and 4.7 years, respectively. Compared with standard interferon alpha-2b plus ribavirin, peginterferon plus fixed or weight based dosing of ribavirin increased life expectancy by 0.5 and by 1.0 years with incremental cost effectiveness ratios of 11,800 euros and 6600 euros per quality adjusted life year (QALY), respectively. Subgroup analyses by genotype, viral load, sex, and histology showed that peginterferon plus weight based ribavirin remained cost effective compared with other well accepted medical treatments.

CONCLUSIONS

Peginterferon alpha-2b plus ribavirin should reduce the incidence of liver complications, prolong life, improve quality of life, and be cost effective for the initial treatment of chronic hepatitis C.

BACKGROUND

Recent reports suggest that the metabolic activity of the gut microbiota may contribute to the pathogenesis of obesity and hepatic steatosis.

OBJECTIVE

The objective was to determine whether the fat composition of host tissues might be influenced by oral administration of commensal bifidobacteria previously shown by us to produce bioactive isomers of conjugated linoleic acid (CLA).

METHODS

Murine trials were conducted in which linoleic acid-supplemented diets were fed with or without Bifidobacterium breve NCIMB 702258 (daily dose of 10(9) microorganisms) to healthy BALB/c mice and to severe combined immunodeficient mice for 8-10 wk. To ensure that the observations were not peculiar to mice, a similar trial was conducted in weanling pigs over 21 d. Tissue fatty acid composition was assessed by gas-liquid chromatography.

RESULTS

In comparison with controls, there was an increase in cis-9, trans-11 CLA in the livers of the mice and pigs after feeding with linoleic acid in combination with B. breve NCIMB 702258 (P < 0.05). In addition, an altered profile of polyunsaturated fatty acid composition was observed, including higher concentrations of the omega-<em>3</em> (n-<em>3</em>) fatty acids eicosapentaenoic acid and docosahexaenoic acid in adipose tissue (P < 0.05). These changes were associated with reductions in the proinflammatory cytokines tumor necrosis factor-<em>alpha</em> and <em>interferon</em>-gamma (P < 0.05).

CONCLUSIONS

These results are consistent with the concept that the metabolome is a composite of host and microbe metabolic activity and that the influence of the microbiota on host fatty acid composition can be manipulated by oral administration of CLA-producing microorganisms.

OBJECTIVE

We reported here a series of 49 patients with unresectable hepatocellular carcinoma (HCC) who underwent nonsurgical treatment to downstage the disease followed by salvage surgery, their long-term outcome, and pattern of recurrence.

BACKGROUND

Most HCC patients present with unresectable disease and are treated with chemotherapy or intra-arterial therapy with a palliative intent. Occasionally, there are good responses to treatment so that salvage surgery becomes feasible afterward. However, long-term outcomes of these patients are seldom reported.

METHODS

Patients with unresectable hepatocellular carcinoma, from September 1993 to June 2002, who received salvage surgery after downstaging by systemic chemotherapy, intra-arterial yttrium-90 microspheres, or sequential treatment were included in this study. Systemic chemotherapy consisted of combination doxorubicin, cisplatin, interferon-alpha and 5-fluorouracil (5-FU), or single-agent doxorubicin. The choice of treatment was according to stage of disease and contemporary clinical trial protocol. Survival, recurrence pattern, and surgical outcome were studied.

RESULTS

There were 49 patients in this study with 40 males and 9 females, age ranged from 12 to 69 years. Forty patients (81.6%) were hepatitis B positive. Thirty-two patients had combination chemotherapy alone (65.3%), 8 patients had single agent chemotherapy alone (16.3%), 4 patients received intra-arterial yttrium-90 microspheres alone (8.2%), and 5 patients received sequential therapy (10.2%). Twenty-eight (57.1%) patients received major hepatic resection. Thirteen patients (26.5%) had complete necrosis of the tumor after treatment. Twenty-one patients (42.9%) had recurrence after surgery, and 14 of them were intrahepatic recurrence. The median survival was 85.9 months. The 1-year, 3-year, and 5-year survival rates were 98%, 64%, and 57%, respectively.

CONCLUSIONS

Salvage surgery after successful downstaging can provide long-term control of disease in a small proportion of patients with unresectable hepatocellular carcinoma.

Oxidized low density lipoprotein (LDL) (Ox-LDL) plays an important role in the pathogenesis of atherosclerosis. Oxidized LDL is taken up by macrophages via scavenger receptors. CD<em>3</em>6 is an 88 kDa glycoprotein expressed on platelets, monocyte-macrophages, microvascular endothelial cells, adipose tissue, skeletal muscles and heart. We found patients with CD<em>3</em>6 deficiency and identified several mutations in the CD<em>3</em>6 gene. We also reported that CD<em>3</em>6-deficient macrophages showed a 50% reduction in the binding of Ox-LDL, suggesting that CD<em>3</em>6 is one of the major receptors for Ox-LDL. CD<em>3</em>6 was expressed on macrophages in the atherosclerotic lesions of human aorta and coronary arteries especially on foamed macrophages. The distribution of CD<em>3</em>6 expression was slightly different from that of scavenger receptor class A types I and II. The expression of CD<em>3</em>6 on macrophages was up-regulated by Ox-LDL and down-regulated by <em>interferon</em> gamma. Since CD<em>3</em>6 is a transporter of long-chain fatty acids (LCFA), CD<em>3</em>6-deficient patients showed a defect in the uptake of an LCFA analog, BMIPP, by the heart. Furthermore, the secretion of IL-1beta and TNF-<em>alpha</em> from monocyte-derived macrophages induced by Ox-LDL was markedly reduced and the activation of NF-kappaB was attenuated in CD<em>3</em>6-deficient subjects compared with controls, suggesting that CD<em>3</em>6-mediated signaling is also impaired in CD<em>3</em>6 deficiency. To elucidate the roles of CD<em>3</em>6 in vivo, we characterized the clinical profile of CD<em>3</em>6-deficient patients. Most of them were accompanied by hyperlipidemia (mainly hypertriglyceridemia), increased remnant lipoproteins and mild elevation of fasting plasma glucose level and blood pressure. Glucose clamp technique revealed mean whole body glucose uptake was reduced in CD<em>3</em>6-deficient patients, indicating the presence of insulin resistance. The frequency of CD<em>3</em>6 deficiency was higher in patients with coronary heart disease (CHD) than in control subjects. Taken together, CD<em>3</em>6 deficiency is accompanied by (1) hyperlipidemia and increased remnant lipoproteins, (2) impaired glucose metabolism based upon insulin resistance, and (<em>3</em>) mild hypertension, and comprises one of the genetic backgrounds of the metabolic syndrome, leading to the development of CHD.

BACKGROUND

There is debate about whether interferon-alpha treatment lowers the risk of progression to hepatocellular carcinoma in patients with chronic viral hepatitis and cirrhosis and whether any effect is limited to certain subgroups. We investigated these issues by retrospective analysis of data for 913 patients from Italy and Argentina.

METHODS

21 centres reported patients from their records who had chronic viral hepatitis and Child's A cirrhosis, were positive for HBsAg or hepatitis-C-virus antibodies (anti-HCV), and had been screened yearly for at least 3 years by ultrasonography and alpha-1-fetoprotein testing. Prognostic risk factors for hepatocellular carcinoma defined by multivariate Cox regression analysis and individual observation time were used for group matching and conditional logistic regression analysis of the independent interferon-alpha treatment effect.

RESULTS

After group matching, the number of patients was reduced to 637. Age, male sex, and portal hypertension were significant risk factors for hepatocellular carcinoma (each p < 0.001); hepatic inflammation (p = 0.21) and iron storage (p = 0.18) were also included in the model 66 (19%) of 356 untreated patients and 29 (10%) of 281 treated patients developed hepatocellular carcinoma (relative risk 1.99 [95% CI 1.09-3.64]); the corresponding proportions for anti-HCV-positive patients were 48 (18.5%) of 259 versus 21 (9.1%) of 232 (3.14 [1.46-6.80]), and those for hepatitis-B-virus-infected (HBV) patients were 18 (10%) of 97 and eight (16%) of 49 (0.98 [0.33-2.92]). Among anti-HCV patients without HBV markers, 29 (20%) of 129 untreated and six (5%) of 116 treated patients developed hepatocellular carcinoma (6.28 [1.65-23.8]).

CONCLUSIONS

Interferon treatment lowered the rate of progression to hepatocellular carcinoma two fold. The risk reduction was apparently greater for patients with chronic hepatitis C and no evidence of HBV infection. Future studies should stratify HCV-infected patients by HBV status.

The effect of recombinant human (rh) cytokines, interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>), interleukin-2 (IL-2), interleukin-<em>3</em> (IL-<em>3</em>), interleukin-4 (IL-4), granulocyte/macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte/macrophage colony stimulating factor (M-CSF), <em>interferon</em>-<em>alpha</em> (IF-<em>alpha</em>), <em>interferon</em>-gamma (IF-gamma), and the tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) on differentiation and function of metachromatic cells (MCS) was studied. Among all cytokines tested, rh interleukin-<em>3</em> (rhIL-<em>3</em>) selectively induced a significant formation of MCS (IL-<em>3</em>: 1.1 +/- 0.6 x 10(5) v control: 0.02 +/- 0.15 x 10(5) MCS/mL suspension) and dose dependent increase in formation of intracellular histamine (IL-<em>3</em>, 100 U/mL: 95 +/- 2<em>3</em> ng/mL v control: 1.8 +/- 0.8 ng/mL) in a bone marrow suspension culture system (analyzed on day 14 of culture). Besides MCS, formation of eosinophils was observed in this culture system in the continuous presence of rhIL-<em>3</em>, whereas IL-<em>3</em> pulse-stimulation for three hours and subsequent exposure to control medium induced growth of MCS but not of eosinophils. By combined immunofluorescence/toluidine blue staining, MCS were found to express a cell surface marker profile that corresponds to the immunological phenotype of peripheral blood basophils (MY-7(CD1<em>3</em>)+, VIM12(CD11b)+, VIM2+, MAX1-, MAX24- and YB5B8-). Furthermore, cultured MCS expressed surface membrane receptors for IgE and could be triggered for nontoxic histamine release by a monoclonal anti-IgE antibody. To evaluate a possible influence of IL-<em>3</em> on basophil function, studies were extended to freshly obtained blood basophils (healthy volunteers, n = <em>3</em>). However, like all other cytokines tested, rhIL-<em>3</em> failed to induce basophil histamine release. Taken together, our studies demonstrate that IL-<em>3</em> is a differentiation factor for human basophils.

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of gamma delta thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5% and <em>3</em>0% of total gamma delta thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1dull gamma delta thymocytes from DBA/2 mice with anti-gamma delta monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including <em>interferon</em>-gamma (IFN-gamma), IL-4, IL-10, and IL-<em>3</em>. In contrast, only IFN-gamma was detected in parallel cultures of Thy-1bright gamma delta thymocytes. Virtually all Thy-1dull gamma delta thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1dull gamma delta thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1dull gamma delta thymocytes from DBA/2 mice express TCR encoded by the V gamma 1 gene and a novel V delta 6 gene named V delta 6.4. Sequence analysis of these functionally rearranged gamma and delta genes revealed highly restricted V delta-D delta-J delta junctions, and somewhat more diverse V gamma-J gamma junctions. We conclude that Thy-1dull gamma delta thymocytes exhibit properties that are equivalent to those of natural killer TCR <em>alpha</em> beta T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1beta (IL-1beta) alone or a combination of IL-1beta, tumor necrosis factor-<em>alpha</em>, and gamma-<em>interferon</em> resulted in a time- and concentration-dependent decrease of cell viability in the <em>3</em>-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1beta alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1beta alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

Hepatitis C virus (HCV) infection induces the endogenous <em>interferon</em> (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-<em>alpha</em>. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS<em>3</em>-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and <em>3</em> than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.

Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp<em>3</em> expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-<em>3</em>. Addition of IL-1 beta, IL-6, IL-10, <em>interferon</em> (IFN)-<em>alpha</em>, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.

OBJECTIVE

To evaluate the direct impact of n-<em>3</em> polyunsaturated fatty acids (n-<em>3</em> PUFAs) on the functions and viability of pancreatic beta-cells.

METHODS

We developed an mfat-1 transgenic mouse model in which endogenous production of n-<em>3</em> PUFAs was achieved through overexpressing a C. elegans n-<em>3</em> fatty acid desaturase gene, mfat-1. The islets and INS-1 cells expressing mfat-1 were analyzed for insulin secretion and viability in response to cytokine treatment.

RESULTS

The transgenic islets contained much higher levels of n-<em>3</em> PUFAs and lower levels of n-6 PUFAs than the wild type. Insulin secretion stimulated by glucose, amino acids, and glucagon-like peptide-1 (GLP-1) was significantly elevated in the transgenic islets. When challenged with tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and gamma-interferon (IFN-gamma), the transgenic islets completely resisted cytokine-induced cell death. Adenoviral transduction of mfat-1 gene in wild-type islets and in INS-1 cells led to acute changes in the cellular levels of n-<em>3</em>- and n-6 PUFAs and recapitulated the results in the transgenic islets. The expression of mfat-1 led to decreased production of prostaglandin E(2) (PGE(2)), which in turn contributed to the elevation of insulin secretion. We further found that cytokine-induced activation of NF-kappaB and extracellular signal-related kinase 1/2 (ERK(1/2)) was significantly attenuated and that the expression of pancreatic duodenal hemeobox-1 (PDX-1), glucokinase, and insulin-1 was increased as a result of n-<em>3</em> PUFA production.

CONCLUSIONS

Stable cellular production of n-<em>3</em> PUFAs via mfat-1 can enhance insulin secretion and confers strong resistance to cytokine-induced beta-cell destruction. The utility of mfat-1 gene in deterring type 1 diabetes should be further explored in vivo.

The present study aimed to determine plasma lipid levels in 95 HIV-infected patients divided into four groups according to the CD4 lymphocyte counts comparatively to a control group of 20 HIV-negative normolipidaemic subjects. A relationship between lipidic abnormalities and immune or nutritional status was also investigated. The patients below 200 CD4 lymphocyte mm-<em>3</em> (groups 1 and 2) had significantly lower total cholesterol than the controls. The patients below 400 CD4 lymphocytes mm-<em>3</em> (groups 1, 2, <em>3</em>) had significantly higher triglycerides and Lp(a) but lower LDL-cholesterol than the controls. In all HIV-positive patients, whatever their CD4 lymphocyte count, HDL-C and apoA1 were lower than in the controls. By multivariate analysis triglycerides were positively correlated to acute opportunistic infections and to <em>interferon</em>-<em>alpha</em> levels, while cholesterol was negatively correlated to TNF-<em>alpha</em>, and LDL-C was positively correlated to albuminaemia. The latter parameter was the only lipidic value to correlate with nutritional markers. The contamination route, or the presence of wasting, was not correlated to any lipidic disorder.

The production and roles of endogenous gamma <em>interferon</em> (IFN-gamma), tumor necrosis factor (TNF), and interleukin-6 (IL-6) in both lethal and nonlethal infections of Staphylococcus aureus were investigated in mice. In the case of nonlethal infection, although no bacteria were detected in the bloodstreams, bacteria that colonized and proliferated persistently for <em>3</em> weeks were found in the kidneys. All mice given lethal injections died within 7 days, and large numbers of bacteria were detected in the bloodstreams, spleens, and kidneys. The first peaks of IFN-gamma, TNF, and IL-6 were observed in the bloodstreams and spleens of the mice with nonlethal and lethal infections within 24 h. Thereafter, in the nonlethal cases, IFN-gamma, TNF, and IL-6 peaked again in the spleens and kidneys during the period of maximum growth of bacteria in the kidneys, although only IL-6 was detected in the sera. In contrast, in the case of lethal infection, the titers of IFN-gamma and IL-6 in the sera and TNF in the kidneys peaked before death. Effects of in vivo administration of monoclonal antibodies (MAbs) against IFN-gamma and TNF on the fates of S. aureus-infected mice were studied. In the nonlethal infections, anti-TNF <em>alpha</em> (anti-TNF-<em>alpha</em>) MAb-treated mice, but not anti-IFN-gamma MAb-treated mice, died as a result of worsening infection, suggesting that endogenous TNF plays a protective role in host resistance to S. aureus infection. In the mice that received lethal doses, injection of anti-TNF-<em>alpha</em> MAb accelerated death. However, although injection of anti-IFN-gamma MAb inhibited host resistance of the infected mice early in infection, most of the animals survived the lethal infection by injection of anti-IFN-gamma MAb, suggesting that endogenous IFN-gamma plays a detrimental role in S. aureus infection. Thus, this study demonstrated that IFN-gamma and TNF play different roles in S. aureus infection.

Fifty-one patients with previously untreated or minimally treated chronic myelogenous leukemia in chronic phase received human <em>alpha</em> <em>interferon</em> <em>3</em> to 9 X 10(6) units intramuscularly (IM) daily until complete hematologic remission, then at doses ranging from <em>3</em> X 10(6) units every other day to 9 X 10(6) units daily. Forty-one (80%) patients achieved a hematologic response, <em>3</em>6 (71%) of them attaining a complete hematologic remission with normal peripheral WBC and differential counts. Responding patients showed continuous but slow normalization of several other blood and marrow parameters including platelet counts, serum lactic dehydrogenase and B12 levels, and marrow cellularity and maturation index. Suppression of the Philadelphia chromosome on serial cytogenetic studies of marrow metaphases was documented in 20 of the <em>3</em>6 patients who achieved complete hematologic remission (56%; <em>3</em>9% of total group), eight of whom (22%) had a decrease of the Philadelphia chromosome-positive metaphases to less than <em>3</em>5%. These changes were persistent for 6 months or longer in 18 patients, seven of whom had continuous suppression of the Philadelphia chromosome to less than 90% for a median of <em>3</em>0+ months (range 21+ to <em>3</em>9+ months). After a median follow-up period of <em>3</em>7 months, 25 patients remain in continued disease control with <em>interferon</em> therapy. The projected <em>3</em>-year survival rate is 76%, with a yearly death rate of 6%, 9%, and 9% in the first <em>3</em> years. Response, Philadelphia chromosome suppression, and survival were significantly better among patients in the low-risk category compared to intermediate- and high-risk categories, as defined by a multivariate analysis-derived prognostic model. The projected <em>3</em>-year survival rate was 94% for patients who achieved a complete hematologic remission on <em>interferon</em> therapy and 45% for those who did not. Thirteen patients have developed blastic crisis, six with lymphoid and three with undifferentiated morphology. We conclude that human leukocyte <em>alpha</em> <em>interferon</em> effectively controls chronic myeloid leukemia and allows reappearance of diploid hemopoietic cells in some patients.

Cytokines and growth factors elicit responses in target cells through induction of gene expression. Signaling mechanisms leading to gene transcription from cell surface receptors often require tyrosine phosphorylation. A family of transcription factors comprising the <em>interferon</em> (IFN)-stimulated gene factor <em>3</em> (ISGF<em>3</em>) multimeric complex are phosphorylated and activated in response to <em>interferon</em>. We describe a protein 50% identical to the 91-kDa subunit of ISGF<em>3</em> that constitutes the acute phase response factor (APRF). This protein was rapidly activated by interleukin-6 to bind an enhancer element common to genes activated in liver cells during the acute phase response to inflammation. Remarkably, APRF was also activated by IFN <em>alpha</em>, IFN gamma, epidermal growth factor, platelet-derived growth factor, colony stimulating factor-1, and the cytokines leukemia inhibitory factor and oncostatin M. The growth factors also activated a third, distinct but related, DNA-binding protein in addition to APRF and p91. This novel factor or a closely related one, but neither APRF nor p91, was also activated in lymphoid cells by interleukin-2, erythropoietin, and interleukin-<em>3</em>. Activation of APRF, p91, and additional members of the ISGF<em>3</em> family is thus a general feature of a wide variety of signaling pathways, integrating diverse signals through common transcriptional regulators.