<em>Interferon</em> <em>alpha</em> (IFN<em>alpha</em>) has been used in the treatment of several types of cancer for almost <em>3</em>0 years, yet the mechanism(s) responsible for its anti-tumoral action remains unknown. A variety of cellular responses, including inhibition of cell growth and induction of apoptosis are induced by IFNs, and apoptotic induction by this cytokine has been proposed to be of importance for both its anti-tumoral in addition to its anti-viral responses. The aim of the present study was to delineate the pathways activated during IFN<em>alpha</em>-induced apoptosis in malignant cell lines. We found that apoptosis induced by IFN<em>alpha</em> was associated with activation of caspases-1, -2, -<em>3</em>, -8 and -9 and that this activation was a critical event. Caspase-<em>3</em> activation was dependent on activity of caspases-8 and -9, moreover, activation of caspase-8 seems to be the upstream event in IFN<em>alpha</em>-induced caspase cascade. We also found loss of mitochondrial membrane potential as well as release of cytochrome c post IFN-treatment, clearly implicating the involvement of mitochondria in IFN-mediated apoptosis. Furthermore, IFN<em>alpha</em>-induced apoptosis was found to be independent on interactions between the Fas-receptor and its ligand. These studies form the basis for further investigations aiming to improve IFN therapy and the development of future strategies to overcome the IFN resistance observed in some malignancies.

This work aims to demonstrate that CD4(+)CD56(+) malignancies arise from transformed cells of the lymphoid-related plasmacytoid dendritic cell (pDC) subset. The analysis of malignant cells from 7 patients shows that in all cases, like pDCs, leukemic cells are negative for lineage markers CD<em>3</em>, CD19, CD1<em>3</em>, CD<em>3</em><em>3</em>, and CD11c but express high levels of interleukin-<em>3</em> receptor <em>alpha</em> chain (IL-<em>3</em>R<em>alpha</em>), HLA-DR, and CD45RA. Tumor cells produce <em>interferon</em>-<em>alpha</em> in response to influenza virus, while upon maturation with IL-<em>3</em> they become a powerful inducer of naive CD4(+) T-cell proliferation and promote their T-helper 2 polarization. As pDCs, leukemic cells also express pre-T<em>alpha</em> and lambda-like 14.1 transcripts, arguing in favor of a lymphoid origin. In addition, malignant cells express significant levels of CD56 and granzyme B. Overall, those observations suggest that CD4(+)CD56(+) leukemic cells could represent the malignant counterpart of pDCs, both of which are closely related to B, T, and NK cells.

BACKGROUND

Whether antidepressants prevent depression during interferon-alpha/ribavirin treatment for hepatitis C virus infection has yet to be established.

OBJECTIVE

To investigate the use of paroxetine in a prospective, double-blind, placebo-controlled study for this indication.

METHODS

Sixty-one hepatitis C virus-infected patients were randomly assigned to the antidepressant, paroxetine (n = 28), or placebo (n = 33), begun 2 weeks before and continued for 24 weeks during interferon-alpha/ribavirin treatment. Primary endpoints included development of major depression and severity of depressive symptoms measured by the Montgomery Asberg Depression Rating Scale (MADRS).

RESULTS

Rates of major depression during the study were low (17%) and did not differ between groups. Nevertheless, using published MADRS cut-off scores, the percent of subjects who met criteria for mild, moderate or severe depression during interferon-alpha/ribavirin therapy was significantly lower in paroxetine- vs. placebo-treated subjects (P = 0.02, Fisher's exact test). Assignment to paroxetine was also associated with significantly reduced depressive symptom severity. This effect was largely accounted for by participants with depression scores above the median (MADRS>> 3) at baseline in whom paroxetine was associated with a maximal reduction in MADRS scores of 10.3 (95% CI: 2.1-18.5) compared with placebo at 20 weeks (P < 0.01). Study limitations included a small sample size and high drop-out rate.

CONCLUSIONS

This double-blind, placebo-controlled trial provides preliminary data in support of antidepressant pre-treatment in hepatitis C virus patients with elevated depressive symptoms at baseline.

The purpose of this study was to characterize intestinal mucosal cytokine profiles in subjects with HIV-1 infection and their relation to mucosal viral load (MVL). Intestinal mucosal cytokine mRNA (interleukin [IL]-2, <em>interferon</em> [IFN]-gamma, IL-12, IL-10, IL-1beta, tumor necrosis factor [TNF]-<em>alpha</em>, IL-6, and regulated upon activation, normal T-cell expressed and secreted [RANTES]) and HIV-1 RNA were quantified using real-time polymerase chain reaction (PCR). On the basis of MVL quantification, the HIV-1-infected subjects were divided into <em>3</em> groups: undetectable MVL (<50 copies/microg of tissue total RNA), low MVL (>50 but <5000 copies/microg of tissue total RNA), and high MVL (>5000 copies/microg of tissue total RNA). Compared with the control group, significant reductions in RANTES, IL-2, and IFNgamma expression were seen in the undetectable MVL group (P < 0.005). IL-6 was significantly increased in all the HIV groups (P < 0.005), and RANTES, IL-10, and IFNgamma were increased in the high MVL group (P < 0.005). Subjects with high MVL have generalized immune activation with increases in T helper (Th)1, Th2, and proinflammatory cytokines, whereas subjects with undetectable MVL have reduced expression of multiple cytokines. The pathologic basis for these observations is unclear but may relate to the success or failure of antiretroviral therapy in controlling mucosal viral replication.

The manifestations of tuberculous infection reflect the immune response to infection. Most healthy tuberculin reactors develop protective immunity; tuberculous pleuritis reflects a resistant response manifest by mild disease, whereas advanced pulmonary and miliary tuberculosis reflect ineffective immunity. The role of gamma delta T cells was assessed in tuberculous infection by evaluating expansion of these cells from blood mononuclear cells after stimulation with Mycobacterium tuberculosis. After culture in vitro, the percentages of gamma delta+ cells were significantly greater in patients with protective and resistant immunity (tuberculin reactors, 25% +/- 4%; tuberculous pleuritis, <em>3</em>0% +/- 7%) than in those with ineffective immunity (advanced pulmonary tuberculosis, 9% +/- <em>3</em>%; miliary tuberculosis, 2% +/- 1%). In leprosy, expansion of gamma delta+ cells was greater in immunologically resistant tuberculoid patients (<em>3</em>2% +/- 4%) than in Mycobacterium leprae-unresponsive lepromatous patients (9% +/- 2%). M. tuberculosis-reactive gamma delta T cell lines produced <em>interferon</em>-gamma, granulocyte-macrophage colony-stimulating factor, interleukin-<em>3</em>, and tumor necrosis factor-<em>alpha</em>, cytokines that activate macrophages and may contribute to mycobacterial elimination. These findings suggest that gamma delta T cells contribute to immune resistance against M. tuberculosis.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of <em>alpha</em>/beta <em>interferon</em>, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-<em>3</em>. Studies with a BUN minireplicon system showed that active replication induced an IRF-<em>3</em>-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-<em>3</em>, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-<em>3</em>-mediated apoptosis.

Obesity-related glomerulopathy (ORG) is an important complication of obesity. The pathophysiological mechanism of glomerular injury in ORG is incompletely understood. Gene expression profiles in the glomeruli obtained from renal biopsy samples of patients with ORG were investigated, using a microdissection technique combined with Affymetrix microarray analysis. Six patients presented with obesity, proteinuria, and biopsy-proven ORG were enrolled. Two sex- and age-matched donor kidneys were applied as the controls. Glomeruli were dissected out from renal biopsy samples under microscope, and total RNA was extracted using RNeasy Micro kit. After two rounds of T7 promoter-based RNA amplification, gene expression profiles of the glomeruli samples were detected using Affymetrix U1<em>3</em><em>3</em>A gene chips. Bioinformatic tools were applied to analyze the microarray data. Results of candidate ORG-related genes were further confirmed by real-time quantitative PCR and immunohistochemistry staining using renal biopsy samples of a larger pool of 15 ORG patients. Genes related to lipid metabolism, inflammatory cytokines, and insulin resistance were the most highlighted subgroups that significantly changed in the glomerular gene expression profiles of the ORG patients, compared with the controls. The expression levels of several key genes in lipid metabolism were increased over 2-fold, including low-density lipoprotein receptor, fatty acid binding protein <em>3</em>, and sterol regulatory element binding protein 1. Moreover, some inflammatory cytokines and their downstream molecules were increased as well, including TNF-<em>alpha</em> and its receptors, IL-6 signal transducer, and <em>interferon</em>-gamma. As the indicators of insulin resistance in the local glomerular cells, levels of glucose-transporter 1, leptin receptor, peroxisome proliferator-activated receptor-gamma, and vascular endothelial growth factor increased, too. In addition to lipid dysmetabolism and insulin resistance, the activation of an inflammatory process in the glomeruli might play a unique role in the development of obesity-related glomerulopathy. Our results expand the understanding of obesity-induced glomerular injuries and shed light on new approaches in the treatment of this disease.

OBJECTIVE

To determine whether innate receptor signals play an important role in the development of autoimmune nephritis in MRL/lpr mice, an experimental model of lupus nephritis.

METHODS

MyD88 is a critical adaptor that is involved in signaling pathways through all of the Toll-like receptors (TLRs) except TLR-<em>3</em>. We therefore generated MyD88-knockout (MyD88-KO) MRL/lpr mice and examined them for histopathologic changes in the kidneys, cumulative survival rates, extent of lymphadenopathy and splenomegaly, serum chemistry, and immunologic parameters. In addition, to define the role of the MyD88-independent pathway in autoimmune nephritis, we injected MyD88-KO MRL/lpr mice intraperitoneally with either poly(I-C) (50 or 100 microg per mouse) or phosphate buffered saline and examined them for survival as well as for histopathologic, serologic, and immunologic parameters.

RESULTS

In comparison with wild-type mice, MyD88-KO MRL/lpr mice exhibited a prolonged lifespan, with no apparent development of autoimmune nephritis. Their kidneys showed no glomerular cell proliferation or crescent formation, along with a drastic decrease in the mesangial matrix. Lymphadenopathy and splenomegaly were less pronounced. Serum titers of anti-double-stranded DNA (anti-dsDNA) and production of cytokines, including interferon-alpha (IFNalpha), interleukin-12 (IL-12), IL-6, and IFNgamma, in splenocytes were significantly reduced in MyD88-KO MRL/lpr mice. Interestingly, MyD88-KO MRL/lpr mice that had been treated with the MyD88-independent TLR-<em>3</em> ligand poly(I-C) showed an almost complete reversion to the features of wild-type mice, demonstrating crescentic glomerulonephritis, with significant elevation of serum anti-dsDNA titers and increased cytokine production in splenocytes.

CONCLUSIONS

The findings indicate that both MyD88-dependent and MyD88-independent innate signals play a crucial role in the development of autoimmune nephritis in MRL/lpr mice.

OBJECTIVE

To evaluate prospectively interferon alpha (IFN-alpha) associated effects on cerebral glucose metabolism and its correlation to neuropsychiatric symptoms during low-dose IFN-alpha-treatment.

METHODS

Eleven patients treated with low-dose IFN-alpha for chronic hepatitis C were prospectively evaluated by neuropsychiatric tests and cerebral [18F]deoxyglucose positron emission tomography (FDG-PET) before and in the 12th week of treatment. PET images were spatially normalized, corrected for variance in global activity and pixel-based t-statistics were calculated for each set of PET scans using SPM96 software. Pixel-cluster with P<0.001 for hypo- or hypermetabolism were displayed in parametric images. Covariance analysis with neuropsychiatric tests was calculated for each cluster.

RESULTS

In week 12 of IFN-alpha treatment, significant hypometabolism with a decrease of local activity ranging from 8 to 12% was found in all patients bilaterally in the prefrontal cortex (BA 9), which correlated in a covariate analysis with changes in depression score as measured by Beck's Depression Inventory. Additionally, hypermetabolism with a maximum increase in local activity of 6-8% was seen in all patients in putamina as well as the left occipital region (BA 18). Before IFN-alpha treatment, only 1/11 patient showed depressive symptomatology. After 3 months of treatment, 6/11 patients were classified as having mild to moderate depressive symptoms (P<0.1; Wilcoxon test).

CONCLUSIONS

Low-dose IFN-alpha therapy is associated with significant prefrontal hypometabolism. This hypometabolism covaried with depression score, but was even found in clinically non-depressed patients. These findings may reflect a possible predisposing factor for IFN-alpha associated neuropsychiatric syndromes and might contribute to a pathophysiological model of affective disorders, as endogenous IFN-alpha levels are elevated in a subset of psychotic patients during acute disease.

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in <em>interferon</em> response factors <em>3</em> and 7 (IRF<em>3</em>/7(-/-)) is lethal. Mortality was associated with undetectable levels of <em>alpha</em>/beta <em>interferon</em> (IFN-α/β) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/β receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF<em>3</em>/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF<em>3</em>/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/β induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/β responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

Immunotherapies, although promising in preclinical studies, have not yet enhanced the survival of patients with glioblastomas. To further understand the immunobiology of glioblastomas in clinical settings, we examined 5<em>3</em> cytokine or cytokine receptor transcripts in 12 human glioblastomas and 6 human glioblastoma cell lines and correlated the findings with the degree of inflammation. Multi-probe RNase protection assays were used to examine Th1, Th2, and Th<em>3</em> cytokine and cytokine receptor expression. Th2 [interleukin (IL)-6, leukemia inhibitory factor and oncostatin M] and Th<em>3</em> (transforming growth factor-beta1, 2, <em>3</em>) cytokine and their receptor transcripts were strongly expressed in almost all glioblastomas and glioma cell lines. Two other Th2 cytokine receptor subunit transcripts (IL-4R<em>alpha</em> and IL-1<em>3</em>R<em>alpha</em>) were also commonly detected. In contrast, although Th1 cytokine receptors tumor necrosis factor (TNF) RI, <em>interferon</em> (IFN)-gammaR<em>alpha</em>, IFN-gammaRbeta, were detected, their cytokines (IFN-gamma, TNF-<em>alpha</em>, lymphotoxin-<em>alpha</em>) were not. Transcripts for IL-2 family cytokine (IL-2, IL-7, IL-9, IL-15) and receptors (IL-2R<em>alpha</em>, IL-2Rbeta, gammac, IL-7R<em>alpha</em>, IL-9R<em>alpha</em>, IL15R<em>alpha</em>) and IL-12 family cytokine (IL-12p40) and receptors (IL-12Rbeta1 and IL-12beta2) were essentially absent in both tumors and cell lines. Immunohistochemical methods showed sparse T lymphocyte infiltrates and numerous microglia in the glioblastomas. This pattern indicates an 'immunosuppressive status' in glioblastomas and could account for the failure of immunotherapy in such tumors.

A breakdown in intestinal barrier function and increased bacterial translocation are key events in the pathogenesis of sepsis and liver disease. Altering gut microflora with noninvasive and immunomodulatory probiotic organisms has been proposed as an adjunctive therapy to reduce the level of bacterial translocation and prevent the onset of sepsis. The purpose of this study was to determine the efficacy of a probiotic compound in attenuating hepatic and intestinal injury in a mouse model of sepsis. Wild-type and interleukin-10 (IL-10) gene-deficient 129 Sv/Ev mice were fed the probiotic compound VSL#<em>3</em> for 7 days. To induce sepsis, the mice were injected with lipopolysaccharide (LPS) and D-galactosamine (GalN) in the presence and absence of the peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662. The mice were killed after 6 hours, and their colons were removed for the measurement of the cytokine production and epithelial function. The functional permeability was assessed by the mannitol movement and cyclic adenosine monophosphate-dependent chloride secretion in tissue mounted in Ussing chambers. The livers were analyzed for bacterial translocation, cytokine production, histological injury, and PPARgamma levels. The tissue levels of tumor necrosis factor <em>alpha</em>, <em>interferon</em> gamma, IL-6, and IL-12p<em>3</em>5 ribonucleic acid were measured by semiquantitative reverse transcription polymerase chain reaction. Mice injected with LPS/GalN demonstrated a breakdown in colonic barrier function, which correlated with enhanced proinflammatory cytokine secretion, bacterial translocation, and significant hepatic injury. A pretreatment with oral probiotics prevented the breakdown in intestinal barrier function, reduced bacterial translocation, and significantly attenuated liver injury. The inhibition of PPARgamma with GW9662 abrogated the protection induced by probiotics.

CONCLUSIONS

Orally administered probiotics prevented liver and intestinal damage in a mouse model of sepsis through a PPARgamma-dependent mechanism.

OBJECTIVE

In celiac disease (CD), gluten induces both adaptive and innate immune responses. Non-celiac gluten sensitivity (NCGS) is another form of gluten intolerance where the immune response is less characterized. The aim of our study was to explore and compare the early mucosal immunological events in CD and NCGS.

METHODS

We challenged <em>3</em>0 HLA-DQ2(+) NCGS and 15 CD patients, all on a gluten-free diet, with four slices of gluten-containing bread daily for <em>3</em> days. Duodenal biopsy specimens were collected before and after challenge. The specimens were examined for cytokine mRNA by quantitative reverse transcriptase-PCR and for MxA-expression and CD<em>3</em>(+) intraepithelial lymphocytes (IELs) by immunohistochemistry and compared with specimens from untreated CD patients and disease controls.

RESULTS

In CD patients, tumor necrosis factor alpha (P=0.02) and interleukin 8 (P=0.002) mRNA increased after in vivo gluten challenge. The interferon gamma (IFN-γ) level of treated CD patients was high both before and after challenge and did not increase significantly (P=0.06). Four IFN-γ-related genes increased significantly. Treated and untreated CD patients had comparable levels of IFN-γ. Increased expression of MxA in treated CD patients after challenge suggested that IFN-α was activated on gluten challenge. In NCGS patients only IFN-γ increased significantly (P=0.0<em>3</em>). mRNA for heat shock protein (Hsp) 27 or Hsp70 did not change in any of the groups. Importantly, we found that the density of IELs was higher in NCGS patients compared with disease controls, independent of challenge, although lower than the level for treated CD patients.

CONCLUSIONS

CD patients mounted a concomitant innate and adaptive immune response to gluten challenge. NCGS patients had increased density of intraepithelial CD<em>3</em>(+) T cells before challenge compared with disease controls and increased IFN-γ mRNA after challenge. Our results warrant further search for the pathogenic mechanisms for NCGS.

Recent studies have documented evidence for the death of smooth muscle cells (SMCs) within advanced human atheroma. These lesions contain macrophages and T lymphocytes in addition to SMCs. We therefore investigated whether <em>interferon</em>-gamma (IFN-gamma), a cytokine secreted by T lymphocytes, or interleukin-1 beta (IL-1 beta) and tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), two cytokines characteristically produced by activated macrophages, can trigger apoptosis of vascular SMCs. Simultaneous treatment with IFN-gamma and TNF-<em>alpha</em> and/or IL-1 beta but not with each cytokine alone promoted death of human and rat SMCs. Exposure for 48 hours to a combination of IFN-gamma (400 U/mL), TNF-<em>alpha</em> (400 U/mL), and IL-1 beta (100 U/mL) significantly (P < .001) increased the accumulation of oligonucleosomes comprising DNA fragments and histones in human SMCs. Electrophoresis of genomic DNA showed internucleosomal fragments of genomic DNA isolated from the cytokine-cotreated SMCs of both humans and rats. These cells exhibited morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. In situ <em>3</em>' end labeling of DNA fragments with terminal transferase confirmed the fragmentation of genomic DNA in these cells. Simultaneous treatment with IFN-gamma and TNF-<em>alpha</em> or IL-1 beta induced elaboration of nitrite, an end product of nitric oxide, in rat but not human SMCs. NG-monomethyl-L-arginine inhibited nitrite accumulation and also partly blocked cytokine-induced apoptosis of rat SMCs but had little effect on human SMCs, suggesting operation of both nitric oxide-dependent and -independent mechanisms for cytokine-induced apoptosis in vascular SMCs. Production of immune cytokines by vascular cells and/or infiltrating leukocytes may regulate apoptotic death of SMCs during atherogenesis.

Primary biliary cirrhosis (PBC) is characterized by chronic nonsuppurative destructive cholangitis (CNSDC) associated with destruction of small bile ducts. Although there have been significant advances in the dissection of the adaptive immune response against the mitochondrial autoantigens, there are increasing data that suggest a contribution of innate immune mechanisms in inducing chronic biliary pathology. We have taken advantage of our ability to isolate subpopulations of liver mononuclear cells (LMC) and examined herein the role of Toll-like receptors (TLRs), their ligands, and natural killer (NK) cells in modulating cytotoxic activity against biliary epithelial cells (BECs). In particular, we demonstrate that Toll-like receptor 4 ligand (TLR4-L)-stimulated NK cells destroy autologous BECs in the presence of <em>interferon</em> <em>alpha</em> (IFN-α) synthesized by TLR <em>3</em> ligand (TLR<em>3</em>-L)-stimulated monocytes (Mo). Indeed, IFN-α production by hepatic Mo is significantly increased in patients with PBC compared to disease controls. There were also marked increases in the cytotoxic activity of hepatic NK cells from PBC patients compared to NK cells from controls but only when the NK cells were prepared following ligation of both TLR<em>3</em>-L- and TLR4-L-stimulated LMC. These functional data are supported by the immunohistochemical observation of an increased presence of CD56-positive NK cells scattered around destroyed small bile ducts more frequently in liver tissues from PBC patients than controls.

CONCLUSIONS

These data highlight critical differences in the varied roles of Mo and NK cells following TLR<em>3</em>-L and TLR4-L stimulation.

Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-<em>interferon</em> (1,000 U/ml) plus tumor necrosis factor-<em>alpha</em> (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a>><em>3</em>-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-<em>3</em>0% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (<em>3</em>9 +/- <em>3</em>%) and iNOS4-/- (<em>3</em><em>3</em> +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- <em>3</em>%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and <em>3</em>) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.

We examined the production and utilization of interleukin 6 (IL-6), a multifunctional cytokine with diverse biological effects, by both ovarian cancer cell lines and primary ovarian tumor cultures. We have found that epithelial ovarian cancer cell lines (CAOV-<em>3</em>, OVCAR-<em>3</em>, and SKOV-<em>3</em>) constitutively produce varying amounts of IL-6. This molecule is biologically active as determined by the proliferation of an IL-6-dependent hybridoma cell line, MH60.BSF-2, and is detectable by an IL-6 enzyme-linked immunosorbent assay. By cytoplasmic immunoperoxidase staining, greater than 98% of the cells produce at least some IL-6, with variation in the staining intensity between individual cells. The ovarian cancer cell-produced protein has a molecular weight of approximately 24,000, and exhibits some molecular weight heterogeneity, with Mr 27,000 and 28,000 minor forms of IL-6. The levels of IL-6 produced by ovarian cancer cells can be modulated by other inflammatory cytokines, such as tumor necrosis factor-<em>alpha</em>, interleukin-1 beta, and <em>interferon</em>-gamma. Our results suggest that IL-6 is not an autocrine growth factor for these established ovarian tumor cell lines, because the addition of either exogenous IL-6 or antibodies to IL-6 did not affect the cellular proliferation of the cell lines. We also found significant levels (greater than <em>3</em> ng/ml) of IL-6 in ascitic fluids of ovarian cancer patients and in the supernants of primary cultures from freshly excised ovarian tumors. The production of IL-6 by epithelial ovarian cancer cells may prove to be a useful diagnostic tool and aid in investigation of the host immune response to ovarian cancer.

The observation that HIV-1 replication is concentrated in lymphoid follicles (F) compared to extrafollicular (EF) lymphoid tissue is not fully understood. The purpose of this study was to quantify HIV-1 replication in these compartments and evaluate the hypothesis that heightened replication in F occurs because of diminished antiretroviral mechanisms. In situ hybridization for HIV-1 RNA and immunohistochemical staining for CD4, CD8, CD20, and multiple antiretroviral proteins was performed in lymph nodes from 15 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 70% of virus-producing cells were detected in F, defined morphologically by CD20 staining, although only a minority of tissue (median, 19%) consisted of F. Frequencies of virus-producing cells were higher in F (median, 1.<em>3</em>5 cells/mm2) compared to EF (median, 0.<em>3</em>5 cells/mm2; p < 0.0001). A CD4+ cell in F had a median <em>3</em>1-fold greater likelihood of harboring HIV-1 RNA than a CD4+ cell in EF (p = 0.0006). The most highly expressed antiretroviral proteins were <em>alpha</em>-defensins 1, 2, and <em>3</em> (median, 4.6% tissue staining), RANTES (median, 728.4 cells/mm2), and granzyme A (median, 412.6 cells/mm2). Less <em>alpha</em>-defensins (p = 0.0127), RANTES (p = 0.0007), granzyme A (p = 0.0018), MIP-1<em>alpha</em> (p = 0.0054), <em>interferon</em> (IFN)-<em>alpha</em> (p = 0.0186), and CD8 (p < 0.0001) was expressed in F compared to EF; amounts in F ranged from 0.15 to 0.50 of those in EF. Expression of IFN-gamma (median, <em>3</em>.1 cells/mm2) and perforin (median, 4.0 cells/mm2) was low and not significantly different in F and EF. Deficiencies of multiple antiretroviral proteins within F may contribute to heightened replication at that site.

Infection with wild-type adenovirus 5, but not with a mutant lacking the E1A gene, prevented the induction by <em>interferon</em> (IFN) <em>alpha</em> of chloramphenicol acetyltransferase (CAT) activity in HeLaM cell lines that had been permanently transfected with chimeric CAT reporter genes driven by the transcriptional regulatory regions of the IFN-responsive genes 561 and 6-16. Similar inhibition of IFN-inducible CAT activity was observed in cells that were cotransfected with the same reporter genes and plasmids expressing either the E1A 289- or 24<em>3</em>-amino acid protein. These proteins also prevented the induction of CAT activity by IFN-gamma from a cotransfected HLA-DR <em>alpha</em>-CAT gene. Experiments with E1A mutants mapped the inhibitory activity to amino acid residues <em>3</em>8-65 of these proteins. In a HeLa cell line permanently expressing the E1A 289-amino acid protein, the replication of vesicular stomatitis virus and encephalomyocarditis virus was not inhibited by IFN-<em>alpha</em>, suggesting a global blockade of IFN responses. In accord with this theory, induction of 561, 1-8, and (2'-5')oligoadenylate synthetase mRNAs by IFN was blocked in these cells at the transcriptional level. The observed transcriptional inhibition could be attributed to the lack of formation of the crucial IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>) transcriptional complex. As shown by mobility shift assays, this complex was not formed in the nuclear extracts of IFN-treated adenovirus-infected cells or IFN-treated E1A-producing cells. These nuclear extracts were deficient in both ISGF<em>3</em> <em>alpha</em> and ISGF<em>3</em> gamma subunits. However, they did not block the formation of ISGF<em>3</em> complex from exogenously added components.

Natural <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR(+) CD11c(-) lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-<em>3</em> and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-<em>alpha</em> production and their differentiation into DCs. After incubation with either NL4-<em>3</em> or JR-CSF, IPCs produced a large amount of IFN-<em>alpha</em> and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-<em>alpha</em> production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-<em>alpha</em> production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.

Cells of the mononuclear phagocyte system arise from circulating blood monocytes. Upon emigration from the vasculature, monocytes differentiate into macrophages, a process that monocytes similarly undergo in vitro. We have established primary cultures from elutriated or adherence-purified blood monocytes and analyzed the antigenic modulation during monocyte to macrophage transformation, which could be followed by the expression of specific antigens and which required as yet unknown inducer signals present in the serum. It is shown that in the absence of serum monocytes only survive in vitro when cultured adherent to plastic but rapidly die in suspension culture. Starting at 0.5%, serum induced maturation dose-dependently, with the optimal concentration being 2 to 5%. Of those antigens not present on monocyte, the low-affinity Fc receptor (CD16), the <em>alpha</em>-chain of the vitronectin receptor (CD51), gp65-MAX.1, and gp68-MAX.<em>3</em> were expressed only upon serum-induced macrophage differentiation, whereas the transferrin receptor (CD71), MAX.26, and to some degree also gp65-MAX.11 appeared to be independent of maturation and were also found on primary cultures of adherent monocytes under serum-free conditions. In addition, the rapid induction of HLA class II antigens (within 24 hr) was similar with and without serum, as was the continued high-density expression in long-term culture. The monocyte-specific CD14 antigen was down-regulated in the absence of serum but kept its level of expression on differentiated macrophages. In comparison, alveolar and peritoneal macrophages, respectively, differed in their antigenic phenotype: Alveolar macrophages expressed high HLA class II antigens but low CD14, whereas for peritoneal macrophages the opposite was found. Both <em>interferon</em>-gamma and -<em>alpha</em> suppressed macrophage maturation in vitro but had contrary effects on HLA class II and CD16 expression: <em>Interferon</em>-gamma up-regulated the two types of antigens, which, in contrast, were down-regulated by <em>interferon</em>-<em>alpha</em>.

We examined the effect of <em>alpha</em>-galactosylceramide (<em>alpha</em>-GalCer) on the synthesis of gamma <em>interferon</em> (IFN-gamma) and local resistance in mice infected intravenously with Cryptococcus neoformans. The level of IFN-gamma in serum increased on day <em>3</em>, reached a peak level on day 7, and decreased to the basal level on day 14 postinfection in mice treated with <em>alpha</em>-GalCer, while in vehicle-treated mice, no increase was detected at any time points except for a small increase on day 7. Such effects were not observed in NKT-KO mice. In CD4KO mice, minor synthesis of IFN-gamma was detected on day <em>3</em> in sera but was completely abolished by day 7. The <em>alpha</em>-GalCer-induced IFN-gamma production on day <em>3</em> was partially reduced in mice depleted of NK cells by treatment with anti-asialo-GM(1) antibody (Ab). Spleen cells obtained from infected and <em>alpha</em>-GalCer-treated mice on day 7 produced a large amount of IFN-gamma upon restimulation with live organisms, while only a marginal level of production was detected in splenocytes from infected and vehicle-treated mice. Such effects were abolished in CD4KO and NKT-KO mice. Finally, the fungal loads in the lungs and spleen on days 7 and 14 were significantly reduced in <em>alpha</em>-GalCer-treated mice compared to those in control mice. In NKT-KO mice, local resistance elicited by <em>alpha</em>-GalCer was completely abolished, although no obvious exacerbation of infection was detected. Furthermore, treatment with anti-IFN-gamma monoclonal Ab mostly abrogated the protective effect of this agent. Thus, our results indicated that activation of V<em>alpha</em>14(+) NKT cells resulted in an increased Th1 response and local resistance to C. neoformans through production of IFN-gamma.

OBJECTIVE

Obestatin is a newly discovered peptide encoded by the ghrelin gene whose biological functions are poorly understood. We investigated obestatin effect on survival of beta-cells and human pancreatic islets and the underlying signaling pathways.

METHODS

beta-Cells and human islets were used to assess obestatin effect on cell proliferation, survival, apoptosis, intracellular signaling, and gene expression.

RESULTS

Obestatin showed specific binding on HIT-T15 and INS-1E beta-cells, bound to glucagon-like peptide-1 receptor (GLP-1R), and recognized ghrelin binding sites. Obestatin exerted proliferative, survival, and antiapoptotic effects under serum-deprived conditions and <em>interferon</em>-gamma/tumor necrosis factor-<em>alpha</em>/interleukin-1 beta treatment, particularly at pharmacological concentrations. Ghrelin receptor antagonist [D-Lys(<em>3</em>)]-growth hormone releasing peptide-6 and anti-ghrelin antibody prevented obestatin-induced survival in beta-cells and human islets. beta-Cells and islet cells released obestatin, and addition of anti-obestatin antibody reduced their viability. Obestatin increased beta-cell cAMP and activated extracellular signal-related kinase 1/2 (ERK1/2) and phosphatidylinositol <em>3</em>-kinase (PI <em>3</em>-kinase)/Akt; its antiapoptotic effect was blocked by inhibition of adenylyl cyclase/cAMP/protein kinase A (PKA), PI <em>3</em>-kinase/Akt, and ERK1/2 signaling. Moreover, obestatin upregulated GLP-1R mRNA and insulin receptor substrate-2 (IRS-2) expression and phosphorylation. The GLP-1R antagonist exendin-(9-<em>3</em>9) reduced obestatin effect on beta-cell survival. In human islets, obestatin, whose immunoreactivity colocalized with that of ghrelin, promoted cell survival and blocked cytokine-induced apoptosis through cAMP increase and involvement of adenylyl cyclase/cAMP/PKA signaling. Moreover, obestatin 1) induced PI <em>3</em>-kinase/Akt, ERK1/2, and also cAMP response element-binding protein phosphorylation; 2) stimulated insulin secretion and gene expression; and <em>3</em>) upregulated GLP-1R, IRS-2, pancreatic and duodenal homeobox-1, and glucokinase mRNA.

CONCLUSIONS

These results indicate that obestatin promotes beta-cell and human islet cell survival and stimulates the expression of main regulatory beta-cell genes, identifying a new role for this peptide within the endocrine pancreas.

BACKGROUND

Thyroid gland dysfunction has been reported to occur with variable frequency during interferon alfa (IFN-alpha) therapy in patients with the hepatitis C virus (HCV). We prospectively evaluated if the prevalence of autoimmune thyroid disease in patients with HCV differs from that in patients with the hepatitis B virus (HBV) before, at the end of, and 6 months after stopping treatment with IFN-alpha.

METHODS

One hundred thirty-four patients with HCV and 41 patients with HBV were studied. Measurements of serum free thyroxine, free triiodothyronine, thyrotropin, thyroid peroxidase antibodies (TPOAbs), thyroglobulin antibodies (TgAbs), and thyrotropin-binding inhibitory immunoglobulin were performed.

RESULTS

Positive levels of TPOAb and TgAb were found in 20% and 11% of patients with HCV compared with 5% and 3% of patients with HBV, respectively. At the end of IFN-alpha therapy, thyroid gland dysfunction was more prevalent in patients with HCV (12%) compared with those with HBV (3%), with thyrotropin levels significantly higher in the HCV group (P = .03). Titers of TPOAb, TgAb, and thyrotropin-binding inhibitory immunoglobulin increased significantly (P = .02, P = .04, and P = .02, respectively) at the end of IFN-alpha therapy in patients with HCV but not in those with HBV. Patients who developed thyroid gland dysfunction were predominantly female (P = .03), had decreased levels of free triiodothyronine (P<.001), and had a higher prevalence of TPOAb (P = .03) before treatment with IFN-alpha. Thyroid gland dysfunction was reversed in 60% of those with HCV 6 months after discontinuing treatment with IFN-alpha.

CONCLUSIONS

Patients with HCV are more susceptible than patients with HBV to autoimmune thyroid disease. Systematic screening of thyroid gland function and TPOAb titers in all patients with HCV before, during, and after IFN-alpha therapy appears warranted. This precaution is not necessary for patients with HBV.