The vaccinia virus double-stranded RNA binding protein E<em>3</em> has been demonstrated to inhibit the expression of cytokines, including beta <em>interferon</em> (IFN-beta) and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E<em>3</em> suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-<em>alpha</em>, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-beta induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E<em>3</em> antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p<em>3</em>8 and NF-kappaB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-<em>alpha</em> required only PKR-dependent NF-kappaB activation. In contrast, induction of IL-6 and IFN-beta was largely PKR independent. IL-6 induction is regulated by NF-kappaB, while IFN-beta induction is mediated by IFN-beta promoter stimulator 1 and IFN regulatory factor <em>3</em>/NF-kappaB. Collectively, these results indicate that E<em>3</em> suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines.

The BCR/ABL tyrosine kinase has been implicated in the pathogenesis of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL). STI571 is a novel anticancer agent that selectively inhibits the BCR/ABL tyrosine kinase. The cytotoxic effects of STI571 were studied in combination with antileukemic agents against Ph(+) leukemia cell lines, KU812, K-562, TCC-S, and TCC-Y. The cells were exposed to STI571 and to other agents simultaneously for 5 or 7 days. Cell growth inhibition was determined by MTT assay. The cytotoxic effects in combinations at the inhibitory concentration of 80% level were evaluated by the isobologram. STI571 produced synergistic effects with recombinant and natural <em>alpha</em>-<em>interferons</em> in 2 of <em>3</em> and <em>3</em> of <em>3</em> cell lines, respectively. STI571 produced additive effects with hydroxyurea, cytarabine, homoharringtonine, doxorubicin, and etoposide in all 4 cell lines. STI571 with 4-hydroperoxy-cyclophosphamide, methotrexate, or vincristine produced additive, antagonistic, and synergistic effects in <em>3</em> of 4 cell lines, respectively. These findings suggest that the simultaneous administration of STI571 with other agents except methotrexate would be advantageous for cytotoxic effects against Ph(+) leukemias. Among them, the simultaneous administration of STI571 and <em>alpha</em>-<em>interferons</em> or vincristine would be highly effective against Ph(+) leukemias and these combinations would be worthy of clinical trials. In contrast, the simultaneous administration of STI571 with methotrexate would have little therapeutic efficacy. Although there are gaps between in vitro studies and clinical trials, the present findings provide useful information for the establishment of clinical protocols involving STI571. (Blood. 2001;97:1999-2007)

<em>Interferon</em> regulatory factor (IRF)-1 and IRF-2 have been implicated for the virus-induced expression of the <em>interferon</em>-<em>alpha</em> and beta (type I IFN) genes. However, recent gene disruption studies in mice suggested the presence of other factor(s) interacting with overlapping promoter elements. In the present paper, we describe the characterization of a DNA binding factor which is strongly induced after virus infection and recognizes these promoter elements. After extensive purification, the factor was revealed to be identical to IFN-stimulated gene factor <em>3</em> (ISGF<em>3</em>), a transcription factor complex activated by IFN treatment. ISGF<em>3</em> binds to the promoter element of IFN-beta, positive regulatory domain I (PRDI), with significantly higher affinity than IRF-1, 2, and mutational analysis of PRDI showed that the gene expression and binding of ISGF<em>3</em>, but not of IRF-1, 2, are highly correlated. Furthermore, our functional analysis involving a dominant negative inhibitor for ISGF<em>3</em> activation and an anti-IFN neutralizing antibody clearly demonstrated the presence of a positive feedback path way for type I IFN genes mediated by ISGF<em>3</em>.

OBJECTIVE

On the basis of preclinical studies, we hypothesized that interleukin (IL)12 would potentiate the antitumor actions of an antihuman epidermal growth factor receptor-2 (HER2) monoclonal antibody (trastuzumab). We conducted a Phase I trial to determine the safety and optimal biological dose of IL-12 when given in combination with trastuzumab.

METHODS

Patients with metastatic HER2-positive malignancies received trastuzumab on day 1 of each weekly cycle. Beginning in week <em>3</em>, patients also received intravenous injections of IL-12 on days 2 and 5. The IL-12 component was dose-escalated within cohorts of <em>3</em> patients (<em>3</em>0, 100, <em>3</em>00, or 500 ng/kg). Correlative assays were conducted using serum samples and peripheral blood cells obtained during the course of therapy.

RESULTS

Fifteen patients were treated, including 12 with HER2 2+ or <em>3</em>+ breast cancer. The regimen was well tolerated with IL-12-induced grade 1 nausea and grade 2 fatigue predominating. Evaluation of dose-limiting toxicity and biological end points suggested that the <em>3</em>00 ng/kg dose was both the maximally tolerated dose and the optimal biological dose of IL-12 for use in combination with trastuzumab. Two patients with HER2 <em>3</em>+ breast cancer within the 500 ng/kg dose level experienced grade 1 asymptomatic decreases in left ventricular ejection fraction of 12% and 19% after <em>3</em> and 10 months of therapy, respectively. There was one complete response in a patient with HER2 <em>3</em>+ breast cancer metastatic to the axillary, mediastinal, and supraclavicular nodes, and 2 patients with stabilization of bone disease lasting 10 months and >12 months, respectively. Correlative assays showed sustained production of interferon (IFN)gamma by natural killer cells only in those patients experiencing a clinical response or stabilization of disease. Elevated serum levels of macrophage inflammatory protein-1alpha, tumor necrosis factor-alpha, and the antiangiogenic factors IFN-gamma inducible protein-10 and monokine induced by gamma were also observed in these patients. Patient genotyping suggested that a specific IFN-gamma gene polymorphism might have been associated with increased IFN-gamma production. The ability of patient peripheral blood cells to conduct antibody-dependent cellular cytotoxicity against tumor targets in vitro did not correlate with clinical response or dose of IL-12.

CONCLUSIONS

The addition of IL-12 to trastuzumab therapy did not appear to enhance the efficacy of this antibody treatment. Sustained production of IFN-gamma and other cytokines were observed in three patients: One who exhibited a complete response and two others who had stabilization of disease lasting over 6 months. Given the small sample size and heterogeneity of the patient population, the effects of IL-12 on the innate immune response to trastuzumab therapy should be further explored in the context of a larger clinical trial.

Dendritic cells (DCs) are professional antigen-presenting cells in the immune system and can be generated in vitro from hematopoietic progenitor cells, DC precursors, and monocytes in peripheral blood. Serial analysis of gene expression (SAGE) was conducted in lipopolysaccharide (LPS)-stimulated mature and activated DCs (MADCs) derived from human blood monocytes. A total of <em>3</em>1 8<em>3</em>7 tag sequences from an MADC cDNA library represented 10 962 different genes, and these data were compared with SAGE data for monocyte-derived immature DCs (IMDCs). Many of the genes, such as germinal center kinase-related protein kinase, cystatin F, <em>interferon</em> (IFN)-<em>alpha</em>-inducible protein p27, EBI<em>3</em>, HEM45, actin-bundling protein, ELC, DC-LAMP, serine/threonine kinase 4, and several genes in expressed sequence tags, were differentially expressed in MADCs, and those encode proteins related to cell structure, antigen-processing enzymes, chemokines, and IFN-inducible proteins. The profile of MADCs was also compared with that of LPS-stimulated monocytes. The Epstein-Barr virus-induced gene <em>3</em> and IFN-<em>alpha</em>-inducible protein p27 are newly identified to be specifically and highly expressed in MADCs, but not in LPS-stimulated monocytes. The comprehensive identification of specific genes expressed in human IMDCs and MADCs should provide candidate genes to define heterogeneous subsets as well as the function and maturation stage of DCs.

OBJECTIVE

Carbon monoxide (CO) is generated from vascular smooth muscle cells via the degradation of heme by the enzyme heme oxygenase-1. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, we investigated whether CO regulates apoptosis in vascular smooth muscle.

RESULTS

Treatment of cultured rat aortic smooth muscle cells with a combination of cytokines (interleukin-1beta, 5 ng/ml; tumor necrosis factor-<em>alpha</em>, 20 ng/ml; <em>interferon</em>-gamma, 200 U/ml) for 48 h stimulated apoptosis, as demonstrated by DNA laddering, annexin V binding, and caspase-<em>3</em> activation. However, the exogenous administration of CO inhibited cytokine-mediated apoptosis. The antiapoptotic action of CO was partially dependent on the activation of soluble guanylate cyclase and was associated with the inhibition of mitochondrial cytochrome c release and with the suppression of p5<em>3</em> expression. Incubation of smooth muscle cells with the cytokines also resulted in a pronounced increase in heme oxygenase-1 protein after 24 h of stimulation. The addition of the heme oxygenase inhibitor, zinc protoporphyrin-IX, or the CO scavenger, hemoglobin, stimulated apoptosis following 24 h of cytokine exposure.

CONCLUSIONS

These results demonstrate that CO, either administered exogenously or endogenously derived from heme oxygenase-1 activity, inhibits vascular smooth muscle cell apoptosis. The ability of CO to block smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.

Host cells activate innate immune signaling pathways to defend against invading pathogens. To survive within an infected host, viruses have evolved intricate strategies to counteract host immune responses. Herpesviruses, including herpes simplex virus type 1 (HSV-1), have large genomes and therefore have the capacity to encode numerous proteins that modulate host innate immune responses. Here we define the contribution of HSV-1 tegument protein VP16 in the inhibition of beta <em>interferon</em> (IFN-β) production. VP16 was demonstrated to significantly inhibit Sendai virus (SeV)-induced IFN-β production, and its transcriptional activation domain was not responsible for this inhibition activity. Additionally, VP16 blocked the activation of the NF-κB promoter induced by SeV or tumor necrosis factor <em>alpha</em> treatment and expression of NF-κB-dependent genes through interaction with p65. Coexpression analysis revealed that VP16 selectively blocked IFN regulatory factor <em>3</em> (IRF-<em>3</em>)-mediated but not IRF-7-mediated transactivation. VP16 was able to bind to IRF-<em>3</em> but not IRF-7 in vivo, based on coimmunoprecipitation analysis, but it did not affect IRF-<em>3</em> dimerization, nuclear translocation, or DNA binding activity. Rather, VP16 interacted with the CREB binding protein (CBP) coactivator and efficiently inhibited the formation of the transcriptional complexes IRF-<em>3</em>-CBP in the context of HSV-1 infection. These results illustrate that VP16 is able to block the production of IFN-β by inhibiting NF-κB activation and interfering with IRF-<em>3</em> to recruit its coactivator CBP, which may be important to the early events leading to HSV-1 infection.

OBJECTIVE

Hepatitis B surface antigen (HBsAg) loss is the hallmark of a complete response to antiviral therapy for chronic hepatitis B. In this study, we investigated the frequency of HBsAg loss after treatment with pegylated (Peg)-interferon alpha-2b.

METHODS

In a multicenter randomized controlled trial, 266 HBeAg-positive patients were treated for 52 wks with Peg-interferon alpha-2b (100 microg/wk) in combination with either lamivudine (100 mg/day) or placebo. Posttreatment follow-up was 26 wks.

RESULTS

At the end of follow-up, 95 (36%) of the 266 patients exhibited HBeAg loss, 18 (7%) HBsAg loss, and 16 (6%) HBsAg seroconversion. Addition of lamivudine did not enhance HBeAg loss, HBsAg loss, or development of anti-HBs. All 18 patients who showed HBsAg loss had normal ALT; 11 (61%) of these patients were also hepatitis B virus (HBV) DNA negative (<400 copies/mL) at the end of follow-up. Loss of HBsAg differed according to HBV genotype: 14% for genotype A, 9% for genotype B, 3% for genotype C, and 2% for genotype D (A vs D: p = 0.006).

CONCLUSIONS

One year of Peg-interferon alpha-2b for HBeAg-positive patients led to HBsAg loss in 7%. Our study indicates that treatment with Peg-interferon alpha-2b is the best therapy to achieve HBsAg clearance in patients with genotype A.

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma <em>interferon</em> (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th<em>3</em>) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-<em>alpha</em> levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID <em>3</em> in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.

Eight chemokines were tested for ability to elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes. The C-C chemokines monocyte chemotactic protein (MCP)-2, MCP-<em>3</em>, RANTES, macrophage inflammatory protein (MIP)-1 <em>alpha</em>, MIP-1 beta, and, as previously described, MCP-1 induced significant, dose-dependent transendothelial chemotaxis of CD<em>3</em>+ T lymphocytes. In contrast, the C-X-C chemokines interleukin-8 (IL-8) and <em>interferon</em>-gamma inducible protein-10 (IP-10) failed to induce transendothelial chemotaxis of CD<em>3</em>+ T lymphocytes or T lymphocyte subsets. RANTES, MIP-1 <em>alpha</em>, and MIP-1 beta induced significant transendothelial chemotaxis of CD4+, CD8+, and CD45R0+ T lymphocyte subsets. Phenotyping of mononuclear cells that underwent transendothelial migration to MCP-2, MCP-<em>3</em>, RANTES, or MIP-1 <em>alpha</em> showed both monocytes and activated (CD26 high), memory-type (CD45R0+) T cells. Both CD4+ and CD8+ T lymphocytes were recruited, but not natural killer cells or significant numbers of B cells. MCP-2 was the only C-C chemokine tested that attracted a significant number of naive-type (CD45RA+) T lymphocytes. In the absence of endothelium, IL-8 but not IP-10 promoted modest but significant chemotoxis of CD<em>3</em>+ T lymphocytes. Our data support the hypothesis that C-C, not the C-X-C chemokines IL-8 or IP-10, promote transendothelial chemotaxis of T lymphocytes.

OBJECTIVE

The therapeutic mechanisms of ribavirin for hepatitis C are unclear. Microarray analyses have shown that ribavirin increases induction of interferon-stimulated genes. We evaluated viral kinetics, serum cytokine expression, and viral mutagenesis during early stages of peginterferon therapy with and without ribavirin.

METHODS

Fifty patients with chronic hepatitis C virus (HCV) infection genotype 1 were randomly assigned to groups that were given peginterferon alpha-2a, with or without ribavirin, for 4 weeks; all patients then received an additional 44 weeks of combination therapy. First- and second-phase viral kinetics were evaluated. Serum levels of interferon-gamma-inducible protein-10 (IP10), monokine induced by interferon-gamma, and monocyte chemoattractant protein 1 were quantified as measures of the interferon-stimulated genes response. NS5A and NS5B were partially sequenced, and mutation rates were calculated.

RESULTS

The first-phase decrease in HCV RNA was similar between groups. Patients who received ribavirin had a more rapid second-phase decrease, compared with patients who did not receive ribavirin-particularly those with an adequate first-phase decrease (0.61 vs 0.35 log10 IU/mL/week; P = .018). At 12 hours, fold induction of serum IP10 was higher in patients given the combination therapy than those given peginterferon only (7.6- vs 3.8-fold; P = .01); however, the difference was greatest in patients with an adequate first-phase decrease in HCV RNA. IP10-induction correlated with first- and second-phase kinetics and with ribavirin serum concentrations on day 3. HCV mutation rates were similar between groups.

CONCLUSIONS

Ribavirin improves the kinetics of the early response to therapy in patients with an adequate initial response to peginterferon. Induction of interferon-stimulated cytokines correlates with viral kinetics following ribavirin therapy, suggesting that ribavirin promotes interferon signaling.

OBJECTIVE

The therapeutic effect of pegylated interferon (peg-IFN)-alpha-2a combination with ribavirin on patients with chronic hepatitis C virus (HCV) infection is dependent on the rapidity of the virological response. The aim of this study was to investigate the predictive value of rapid virological response (RVR) and early virological response (EVR) on sustained virological response (SVR) in HCV patients treated with peg-IFN-alpha-2a and ribavirin.

METHODS

The HCV genotypes of 105 patients with chronic hepatitis C were detected by enzyme-immunoassay. Patients received subcutaneous 180 microg peg-IFN-alpha-2a once weekly plus daily ribavirin. Patients with genotype 1 were treated for 48 weeks and patients with genotype 2 or 3 were treated for 24 weeks. HCV RNA was assessed by qualitative PCR at pretreatment, at weeks 4 and 12 during treatment, and at week 24 of follow-up. Virological response rates at different weeks were investigated, with RVR defined as serum HCV RNA undetectable after 4 weeks and EVR defined as HCV RNA either undetectable or decrease by>>or=2 log(10) after 12 weeks. The effects of virological response rates at different weeks on SVR were analyzed.

RESULTS

Of the 105 patients, 44 (41.9%) were genotype 1, 46 (43.8%) were genotype 2, and 15 (14.3%) were genotype 3. RVR rates (19.5%) of patients with genotype 1 were significantly lower than those (60.7%) of genotype 2 or 3 (chi(2) = 16.836, P = 0.000); and EVR rates (73.2%) of patients with genotype 1 were significantly lower than those (96.7%) of genotype 2 or 3 (chi(2) = 12.220, P = 0.000). The SVR rates (86.7%) of patients who had achieved RVR were significantly higher than those (43.9%) of patients who had not achieved RVR (chi(2) = 19.713, P = 0.000). The positive predictive value of RVR in all patients was higher than that of EVR, but there was no significant difference between RVR and EVR. The negative predictive value of RVR in all patients or with genotype 1 was significantly lower than that of EVR. In univariate analysis, HCV RNA level (P = 0.014), genotype (P = 0.001), RVR (P = 0.000) and EVR (P = 0.000) were associated with effect of treatment. However, in stepwise regression analysis, the independent factors associated with effect of antiviral therapy were RVR (OR = 6.501, P = 0.001), EVR (OR = 2.776, P = 0.003) and genotype (OR = 3.061, P = 0.024).

CONCLUSIONS

The RVR and EVR rates of patients with genotype 1 were significantly lower than those of patients with genotype 2 or 3. RVR had a similar predictive value as EVR on SVR. Genotype, HCV RNA level, RVR and EVR were associated with SVR. Genotype, RVR and EVR were independent factors for predicting the effect of antiviral therapy.

We have determined the three-dimensional structure of recombinant human granulocyte-colony-stimulating factor by x-ray crystallography. Phases were initially obtained at <em>3</em>.0-A resolution by multiple isomorphous replacement and were refined by solvent flattening and by averaging of the electron density of the three molecules in the asymmetric unit. The current R factor is 21.5% for all data between 6.0- and 2.2-A resolution. The structure is predominantly helical, with 104 of the 175 residues forming a four-<em>alpha</em>-helix bundle. The only other secondary structure is also helical. In the loop between the first two long helices a four-residue <em>3</em>(10)-helix is immediately followed by a 6-residue <em>alpha</em>-helix. Three residues in the short connection between the second and third bundle helices form almost one turn of left-handed helix. The up-up-down-down connectivity with two long crossover connections has been reported previously for five other proteins, which like granulocyte-colony-stimulating factor are all signaling ligands: growth hormone, granulocyte/macrophage-colony-stimulating factor, <em>interferon</em> beta, interleukin 2, and interleukin 4. Structural similarity among these growth factors occurs despite the absence of similarity in their amino acid sequences. Conservation of this tertiary structure suggests that these different growth factors might all bind to their respective sequence-related receptors in an equivalent manner.

Renal cell carcinoma (RCC) evokes an immune response, which occasionally has resulted in spontaneous and dramatic remissions. In an attempt to reproduce or accentuate this response, various immunotherapeutic strategies have been studied. The most consistent antitumor activity has been reported with <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) and interleukin-2 (IL-2). In recent years, randomized trials have suggested that high-dose, intravenous bolus IL-2 is superior in terms of response rate and possibly in terms of response quality to regimens that involve either low-dose IL-2 and IFN-<em>alpha</em>, intermediate- or low-dose IL-2 alone, or low-dose IFN-<em>alpha</em> alone. More significantly, investigations associated with those trials suggested that the potential exists for identifying predictors of response (or resistance) and limiting IL-2 therapy to those patients who hare most likely to benefit. Although the role of low-dose, single-agent cytokines is limited, combinations of cytokines with targeted therapy may have merit. Several studies, including 2 completed, large, phase <em>3</em> trials of <em>interferon</em> plus bevacizumab versus <em>interferon</em> alone, have demonstrated superior efficacy with the combination regimen compared with cytokine monotherapy and suggested the potential of an additive effect that requires further exploration. For patients who are unlikely to benefit from IL-2 or who are unable to receive it, the emergence "targeted immunotherapy" offers hope for improved clinical outcome. Improvements in patient selection, novel agents, and combination therapy will be required to optimize the benefits of immunotherapy in metastatic RCC as the list of effective therapies grows.

Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-<em>3</em>, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor <em>alpha</em> (TNF <em>alpha</em>) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with <em>interferon</em> gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.

BACKGROUND

Ovarian surface epithelial cells undergo several rounds of division to repair the wound created by follicular rupture at the time of ovulation. This cyclical requirement for cell division, when not interrupted by the long anovulatory rest periods that occur during pregnancy and lactation, may contribute to the development of ovarian cancer.

OBJECTIVE

To test this hypothesis, we isolated rat ovarian surface epithelial cells from 10 adult female Fisher rats, initiated two mixed-population and seven clonal cell lines, and repeatedly subcultured these cells in vitro for more than 20 passages. We then tested them for the acquisition of the following four features associated with transformation: 1) the loss of contact inhibition, 2) the capacity for substrate-independent growth, <em>3</em>) the ability to form tumors when injected subcutaneously and/or intraperitoneally into athymic mice, and 4) cytogenetic abnormalities.

RESULTS

Loss of contact inhibition was observed in all nine late-passage cell lines. Six of the nine late-passage, but none of the early-passage, cell lines tested exhibited a capacity for substrate-independent growth that was augmented in a dose-dependent manner by epidermal growth factor. Two late-passage cell lines (clone 2 and mixed-population 2) generated tumors in athymic BALB/c mice within <em>3</em> weeks following subcutaneous injection of 5 x 10(6) cells, whereas similar numbers of early-passage cells from the same cell lines failed to generate palpable tumors. Late-passage clone 7 cells were tumorigenic when 5 x 10(7) cells were injected intraperitoneally. Two of the cell lines analyzed exhibited alterations involving losses of part or all of one member of the chromosome 5 pair. Clone 2 possessed an interstitial deletion, del(5)(q21.<em>3</em>q24), consistent with the loss of an uncloned putative tumor suppressor gene at 5q22q2<em>3</em> previously reported to reside near the loci for the interferon alpha, interferon beta, and c-jun genes. Early-passage clone 7 cells exhibited chromosome 5 monosomy, while late-passage cells contained one normal chromosome 5 and a derivative (5q12q). Southern analysis of the three cell lines revealed no consistent loss of loci for the interferon and c-jun genes, although early-passage clone 7 cells had one half the gene copy number for the interferon beta and c-jun genes and both early- and late-passage clone 7 cells lacked DNA sequences hybridizing with the probe for interferon alpha.

CONCLUSIONS

This pattern of passage-dependent spontaneous transformation of rat ovarian surface epithelial cells in vitro supports the hypothesis that repetitious ovulation contributes to the etiology of human ovarian cancer.

<em>Interferon</em> (IFN)-gamma-induced indoleamine 2,<em>3</em>-dioxygenase (IDO) activity is enhanced synergistically by interleukin (IL-)1, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and LPS in IFN-treated macrophages by increasing IDO mRNA concentration. These studies demonstrate that IFN-treated HeLa cells also exhibit dose-dependent enhancement of IDO induction by TNF-<em>alpha</em> and IL-1, with maximal effects at concentrations of 5 ng/ml and <em>3</em> ng/ml, respectively. Furthermore, with sub-optimal IFN concentrations, cells treated with maximally effective concentrations of TNF-<em>alpha</em> or IL-1<em>alpha</em> required <em>3</em>-5 times less IFN to induce the same level of IDO activity as that observed with IFN alone. To detect changes in transcriptional activation of the IDO gene, HeLa cells were transfected with a plasmid containing the IDO 5' regulatory region upstream of a green fluorescent protein (GFP) reporter gene. In transfected cells, IFN induced both IDO activity and GFP that was detected by flow cytometry. When cell-sorted, transfected cells were stimulated with IFN in combination with TNF-<em>alpha</em> or IL-1 but not LPS, increased GFP was detected in comparison to transfected cells treated with IFN alone. Furthermore, increases in GFP expression correlated with IDO enzymatic activity, indicating that combinations of IFN with IL-1 or TNF-<em>alpha</em> increase the transcriptional activity of the IDO promoter region.

This study was conducted to investigate the possible involvement of Fas in beta-cell death in insulitis of Type 1 (insulin-dependent) diabetes mellitus. Although primary cultured Balb/c mouse islet cells did not express Fas mRNA, 4-12 hours of treatment with 10(2)-10(<em>3</em>) U/l of mouse interleukin-1 <em>alpha</em> (IL-1 <em>alpha</em>) induced the expression of Fas mRNA. Surface Fas expression was detected by immunofluorescence flow cytometry using a non-cytolytic anti-Fas monoclonal antibody after 6 or 12 h of incubation with 10(<em>3</em>) U/l of IL-1 <em>alpha</em>. Primary islet cells were resistant to an agonistic anti-Fas monoclonal antibody. However, 12 h pretreatment with IL-1 <em>alpha</em> sensitized islet cells to its cytolytic effect. Significant cell death was observed 24 h after the addition of anti-Fas, and progressively increased until 72 h, when specific 51Cr release was 72 +/- 6%. Agarose gel electrophoresis of DNA extracted from cells exposed to IL-1 <em>alpha</em> and agonistic anti-Fas showed internucleosomal DNA fragmentation, a hallmark of apoptotic cell death. Since the Fas antibody showed no cross-reactive activity of tumour necrosis factor (TNF), the cytotoxic effect was not mediated by TNF receptors. A protein synthesis inhibitor cycloheximide augmented Fas-mediated islet cell death. The Fas-mediated killing of islet cells was not L-arginine-dependent, or blocked by N(G)-monomethyl-L-arginine. beta-TC1 cells also expressed Fas mRNA when exposed to IL-1 <em>alpha</em> or IL-1 <em>alpha</em> plus <em>interferon</em>-gamma. These observations suggest that Fas-mediated apoptosis may be a mechanism of islet cell death in autoimmune insulitis.

We have studied the activity of recombinant human tumor necrosis factor (rHuTNF) on six different human tumor xenografts derived from primary breast and bowel tumors and maintained by passage in nude mice. When 5 micrograms rHuTNF was given daily intratumorally to mice with established (approximately, 0.5 cm) tumors, total tumor regression was observed by <em>3</em>-4 weeks in three of six xenograft lines. In a further two lines tumor stasis or significant slowing of growth was seen. This antitumor action was not accompanied by any consistent macroscopic change in the tumor such as necrosis, but histological examination revealed tumor cell degeneration and a large peritumoral infiltration of host inflammatory cells after 4-7 days therapy. In contrast to these data, little effect was seen when the same dose of rHuTNF was administered i.p. to nude mice bearing these tumors. In only two of six lines was any significant slowing of tumor growth seen. A 5-fold increase in the i.p. dose resulted in improved activity on only one of two xenograft lines tested. Efficacy of the i.p. rHuTNF dose could, however, be enhanced by simultaneous administration of human <em>interferon</em>, <em>alpha</em> or gamma. No obvious signs of toxicity were observed at all rHuTNF doses administered and weights of control and treated mice at the end of the experiments were comparable.

DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase <em>3</em> (Flt<em>3</em>)-ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8(+) <em>interferon</em> (IFN) gamma(+) T cells and polyfunctional CD8(+) T cells producing IFN-gamma, tumor necrosis factor <em>alpha</em>, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac(251) and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL.

Treatment regimens based on the use of interferon-alpha (IFN-alpha) remain the cornerstone of therapy for chronic hepatitis C virus infection, which affects nearly 170 million people worldwide. Treatment options include unmodified IFN-alpha given three times weekly or pegylated IFNs given once weekly. The albumin-fusion platform takes advantage of the long half-life of human albumin to provide a new treatment approach that allows the dosing frequency of IFN-alpha to be reduced in individuals with chronic hepatitis C. Albinterferon alpha-2b (alb-IFN), a recombinant polypeptide composed of IFN-alpha registration trials are in progress. The albumin-fusion platform is currently being applied to other important bioactive peptides with short half-lives. These fusion proteins, which are at present in different phases of clinical development, might lead to improved therapies across a broad range of diseases.

Immune responses to pathogens need to be maintained within appropriate levels to minimize tissue damage, whereas such controlled immunity may allow persistent infection of certain types of pathogens. Interleukin 10 (IL-10) plays an important role in such immune regulation. We previously showed that HSV-stimulated human plasmacytoid dendritic cells (pDCs) induced naive CD4+ T cells to differentiate into <em>interferon</em> gamma (IFN-gamma)/IL-10-producing T cells. Here we show that HSV-stimulated pDCs induce allogeneic naive CD4+ T cells to differentiate into cytotoxic regulatory T cells that poorly proliferate on restimulation and inhibit proliferation of coexisting naive CD4+ T cells. IL-<em>3</em>-stimulated pDCs or myeloid DCs did not induce such regulatory T cells. Both IFN-<em>alpha</em> and IL-10 were responsible for the induction of anergic and regulatory properties. High percentages of CD4+ T cells cocultured with HSV-stimulated pDCs, and to a lesser extent those cocultured with IL-<em>3</em>-stimulated pDCs, expressed granzyme B and perforin in an IL-10-dependent manner. CD4+ T cells cocultured with HSV-stimulated pDCs accordingly exhibited cytotoxic activity. The finding that virus-stimulated pDCs are capable of inducing CD4+ cytotoxic regulatory T cells suggests that this DC subset may play an important role in suppressing excessive inflammatory responses and also in inducing persistent viral infection.

Imatinib at 400 mg daily is effective in chronic-phase chronic myeloid leukemia (CML) after <em>interferon</em> failure, although only a few patients achieve a molecular remission. We investigated whether higher doses of imatinib may be more effective. Thirty-six patients with chronic-phase CML after failure on <em>interferon</em>-<em>alpha</em> were treated with 400 mg imatinib twice daily. Median time from diagnosis was 25 months (range, 10-1<em>3</em>5 months); 4 patients (11%) had clonal evolution. All 11 patients with active disease achieved complete hematologic response. Excluding patients with fewer than <em>3</em>5% Ph-positive metaphases before the start of therapy, 19 (90%) of 21 evaluable patients achieved a major cytogenetic response. Of 27 evaluable patients, 24 (89%) achieved a complete cytogenetic response. Quantitative polymerase chain reaction was performed in bone marrow every <em>3</em> months. Of <em>3</em>2 evaluable patients, 18 (56%) showed BCR-ABL/ABL percentage ratios lower than 0.045%, including 1<em>3</em> (41%) with undetectable levels. With a median follow-up of 15 months, all patients were alive in chronic phase. Toxicities were similar to those reported with standard dose; 71% of patients continue to receive 600 mg or more of imatinib daily. In conclusion, high-dose imatinib induces complete cytogenetic responses in most patients with chronic-phase CML after <em>interferon</em> failure. This is accompanied by a high rate of molecular remission.

Recent genome-wide association studies suggest distinct roles for 12 human <em>interferon</em>-<em>alpha</em> (IFN-α) and <em>3</em> IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-β and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ<em>3</em>. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.