Based upon the prosthodontic literature, subjects who are at the transition stage between natural dentition and edentulism are called "terminal dentition" (TD) cases. The aim of the present cross-sectional investigation was to characterize the local and systemic inflammatory responses in 2 groups of patients with terminal dentition periodontitis. Eight severe adult periodontitis terminal dentition (AP-TD) subjects and 8 early onset periodontitis terminal dentition (EOP-TD) subjects were entered into the study. Our purpose was to measure an extended battery of cytokines in the gingival crevicular fluid (GCF) and in lipopolysaccharide (LPS)-stimulated monocytic culture supernatants as well as gingival mononuclear cell messenger RNA (mRNA) transcripts determined from biopsy samples. Within the GCF there were <em>3</em> tiers (levels) of mediators based upon approximate 10-fold differences in concentration. The highest tier included prostaglandin E2 (PGE2), interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2), the intermediate tier included tumor necrosis factor <em>alpha</em> (TNF <em>alpha</em>) and <em>interferon</em> gamma (IFN-gamma) and at the lowest concentration level were interleukin-4 (IL-4) and interleukin-6 (IL-6). Thus, the GCF analysis clearly indicated that in both AP-TD and EOP-TD groups the monocytic, i.e. IL-1 beta and PGE2 and Th1, i.e. IL-2 and IFN-gamma, inflammatory mediator levels quantitatively dominated over the Th2 mediators, i.e. IL-4 and IL-6. LPS-stimulated monocytic release of IL-1 beta, PGE2 and TNF <em>alpha</em> was significantly elevated in both AP-TD and EOP-TD groups compared to those of a control group of 21 subjects with moderate to advanced adult periodontitis. The cytokine mRNA expression of isolated gingival mononuclear cells showed that in both the AP-TD and the EOP-TD groups Th1 and Th2 cytokines were expressed, with low levels of IL-4 and IL-12. In conclusion, our data suggest that this cross-sectional TD periodontitis model may reflect progressive periodontal disease associated with tooth loss. Furthermore, although Th1 cytokine levels in the GCF dominate over the Th2 response, monocytic activation provides the main source of proinflammatory mediators. In addition, LPS-stimulated peripheral blood monocytes demonstrate an upregulated inflammatory mediator secretion in the terminal dentition.

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from <em>interferon</em> <em>alpha</em>2b-(Ser-Hyp)(20), and a 14-residue Hyp-AG isolated from (Ala-Hyp)(51)-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1->><em>3</em> beta-linked trigalactosyl blocks linked by a beta-1-->6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an <em>alpha</em>-L-Araf-(1-->5)-<em>alpha</em>-L-Araf-(1->><em>3</em>)-<em>alpha</em>-L-Araf-(1->><em>3</em>- unit and an <em>alpha</em>-L-Rhap-(1-->4)-beta-D-GlcUAp-(1-->6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the beta-1-->6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.

OBJECTIVE

The inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) are produced by activated macrophages, are key mediators of pathogenesis, and are validated therapeutic targets in rheumatoid arthritis (RA) and seronegative spondylarthritis (SpA). IL-10 is a potent antiinflammatory cytokine that suppresses macrophage TNFalpha and IL-1 production, yet is not effective in suppressing inflammatory arthritis. To gain insight into IL-10 responses in inflammatory arthritis, we used microarray analysis to determine the patterns of IL-10-inducible gene expression in freshly isolated RA and seronegative SpA synovial macrophages.

METHODS

Macrophages from the synovial fluid of 5 patients with RA and 3 with seronegative SpA (2 with psoriatic arthritis and 1 with ankylosing spondylitis) were isolated by positive selection and stimulated ex vivo with IL-10 or interferon-gamma (IFNgamma). Gene expression was analyzed using Affymetrix microarrays and protocols. Real-time polymerase chain reaction was used to confirm changes in gene expression.

RESULTS

The number of genes induced by IL-10 in arthritic macrophages was markedly smaller than that induced in control macrophages, and the strength of induction was lower in arthritic macrophages for most genes. The residual response of arthritic macrophages to IL-10 stimulation was qualitatively altered, such that IL-10 preferentially increased expression of IFNgamma-inducible genes. In contrast, arthritic macrophages expressed many IFNgamma-inducible genes prior to stimulation, and their response to IFNgamma remained mostly intact.

CONCLUSIONS

These results demonstrate that IL-10 responses are dysregulated in RA synovial macrophages. An altered biologic response to IL-10, with attenuation of its antiinflammatory function and a concomitant retention of IFNgamma-like activating functions, provides a basis for the lack of efficacy of IL-10 in suppressing inflammatory arthritis.

OBJECTIVE

Viral infections contribute to the pathogenesis of type 1 diabetes. Viruses, or viral products such as double-stranded RNA (dsRNA), affect pancreatic beta-cell survival and trigger autoimmunity by unknown mechanisms. We presently investigated the mediators and downstream effectors of dsRNA-induced beta-cell death.

METHODS

Primary rat beta-cells and islet cells from wild-type, toll-like receptor (TLR) <em>3</em>, type I <em>interferon</em> receptor (IFNAR1), or <em>interferon</em> regulatory factor (IRF)-<em>3</em> knockout mice were exposed to external dsRNA (external polyinosinic-polycytidylic acid [PICex]) or were transfected with dsRNA ([PICin]).

RESULTS

TLR<em>3</em> signaling mediated PICex-induced nuclear factor-kappaB (NF-kappaB) and IRF-<em>3</em> activation and beta-cell apoptosis. PICin activated NF-kappaB and IRF-<em>3</em> in a TLR<em>3</em>-independent manner, induced eukaryotic initiation factor 2 alpha phosphorylation, and triggered a massive production of <em>interferon</em> (IFN)-beta. This contributed to beta-cell death, as islet cells from IFNAR1(-/-) or IRF-<em>3</em>(-/-) mice were protected against PICin-induced apoptosis.

CONCLUSIONS

PICex and PICin trigger beta-cell apoptosis via the TLR<em>3</em> pathway or IRF-<em>3</em> signaling, respectively. Execution of PICin-mediated apoptosis depends on autocrine effects of type I IFNs.

OBJECTIVE

In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms.

METHODS

Rat colon was exposed to tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>) and <em>interferon</em> gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (<em>3</em>)H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM).

RESULTS

Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R(t) was decreased (101+/-10 versus 189+/-10 Omega x cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45+/-10%, 16+/-7%, 42+/-8%, and 42+/-1<em>3</em>% of controls, respectively), while claudin-2 increased to 208+/-<em>3</em>2%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.7<em>3</em>+/-0.09 to 8.55+/-2.92 pmol x h(-1) x cm(-2).

CONCLUSIONS

A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.

The chromosome 21q22.11 cytokine receptor cluster contains four genes that encode subunits of the receptors for the cytokines interleukin-10 and <em>interferon</em>-<em>alpha</em>, -beta and -gamma that may have a role in malaria pathogenesis. A total of 15 polymorphic markers located within these genes were initially genotyped in 190 controls and 190 severe malaria cases from The Gambia. Two <em>interferon</em>-<em>alpha</em> receptor-1 (IFNAR1) gene SNPs (17470 and L168 V) showed evidence for an association with severe malaria phenotypes and were typed in a larger series of samples comprising 5<em>3</em>8 severe malaria cases, <em>3</em><em>3</em>8 mild malaria cases and 562 controls. Both the 17470-G/G and L168V-G/G genotypes were associated with protection against severe malaria, in general, and cerebral malaria, in particular (P=0.004 and 0.00<em>3</em>, respectively). IFNAR1 diplotypes were then constructed for these two markers using the PHASE software package. The (17470-G L168V-G/17470-G L168V-G) diplotype was found to be associated with a reduced risk of cerebral malaria and the (17470-C L168V-C/17470-G L168V-G) diplotype with an increased risk of cerebral malaria (overall <em>3</em> x 2 chi(2)=12.8, d.f.=2, P=0.002 and <em>3</em> x 2 chi(2)=15.2, d.f.=2, P=0.0005, respectively). These data suggest a role for the type I <em>interferon</em> pathway in resistance to cerebral malaria.

Statins are <em>3</em>-hydroxyl-<em>3</em>-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti-HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) or HCV nonstructural (NS)5B polymerase or NS<em>3</em> protease inhibitors results in an additive antiviral activity in short-term (<em>3</em> days) antiviral assays. Neither statins, at a concentration of five-fold their median effective concentration (EC(50)) value, nor polymerase, protease inhibitors, or IFN-<em>alpha</em>, at concentrations 10- or 20-fold their EC(50) value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV-796.

CONCLUSIONS

A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors.

A new experimental drug, pirfenidone (5-methyl-1-phenyl-1H-pyridine-one; S-7701), has been reported to have beneficial effects for the treatment of certain fibrotic diseases. We investigated the anti-inflammatory properties in murine endotoxic shock to determine the pharmacological characteristics. The present study describes the prophylactic effect, cytokine regulatory profiles and therapeutic effect of pirfenidone in murine endotoxic shock, which was induced in mice using an intraperitoneal (i.p.) injection of lipopolysaccharide and D-galactosamine. First, we examined the prophylactic effect and cytokine regulatory profiles. A single oral administration of pirfenidone prior to lipopolysaccharide/D-galactosamine challenge inhibited the production of circulating tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), interleukin-12 and <em>interferon</em>-gamma, markedly enhanced that of interleukin-10, and offered protection from subsequent lethal symptoms in a dose-dependent manner. Second, we examined the therapeutic effect. A single oral administration of pirfenidone 1, 2, <em>3</em>, 4 and 5 h post lipopolysaccharide/D-galactosamine challenge provided protection against lethal shock in a time-dependent manner. At the histopathological level, apoptotic positive cells were found to be suppressed in the liver. The transforming growth factor (TGF)-beta1 level was markedly elevated in the liver of lipopolysaccharide/D-galactosamine-challenged mice, suppressed in pirfenidone-treated mice. These findings may offer an alternative for both protective and therapeutical treatment of several human acute or chronic inflammatory diseases by pirfenidone.

CPG 10101, a synthetic oligodeoxynucleotide (ODN), is a toll-like receptor 9 (TLR9) agonist with antiviral and immunomodulatory properties that could potentially influence chronic infection with HCV. In this multicenter Phase 1b trial, 60 HCV-positive patients (50 genotype 1 HCV) were randomized and received either placebo or CPG 10101 at 0.25, 1, 4, 10, or 20 mg subcutaneously (SC) twice weekly for 4 weeks or at 0.5 or 0.75 mg/kg SC once weekly for 4 weeks. Dose-dependent cytokine induction was observed after administration of CPG 10101. At 24 hours after administering the highest dose of 0.75 mg/kg CPG 10101, <em>interferon</em> (IFN)-gamma-inducible protein 10 (IP-10) had a mean increase over baseline levels (+/-SD) of 15,057 (+/-9769) pg/ml (P < 0.01, compared to placebo); IFN-<em>alpha</em> had a 106 (+/-6<em>3</em>.<em>3</em>) pg/ml increase (P < 0.01); and 2'5'-oligoadenylate synthetase (OAS) had a 16<em>3</em> (+/-120.6) pmol/dl increase (P < 0.01). Decreases in HCV RNA also were dose-dependent, with the greatest group geometric mean maximum reduction of 1.69 +/- 0.618 log(10) (P < 0.05) observed in the 0.75 mg/kg dose group. Decreases>>/=1 log(10) were seen in 22 of 40 patients who received>>/=1 mg CPG 10101, with <em>3</em> patients exceeding a 2.5-log(10) reduction. CPG 10101 was well tolerated, and adverse events were consistent with CPG 10101's mechanism of action.

CONCLUSIONS

In this Phase 1 study, CPG 10101 was associated with dose-dependent increases in markers of immune activation and decreases in HCV RNA levels. The data support further clinical studies of CPG 10101 for treating chronic HCV infection.

Differential expression of the human <em>interferon</em> A (IFN-A) gene cluster is modulated following paramyxovirus infection by the relative amounts of active <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and IRF-7. IRF-<em>3</em> expression activates predominantly IFN-A1 and IFN-B, while IRF-7 expression induces multiple IFN-A genes. IFN-A1 gene expression is dependent on three promoter proximal IRF elements (B, C, and D modules, located at positions -98 to -45 relative to the mRNA start site). IRF-<em>3</em> binds the C module of IFN-A1, while other IFN-A gene promoters are responsive to the binding of IRF-7 to the B and D modules. Maximal expression of IFN-A1 is observed with complete occupancy of the three modules in the presence of IRF-7. Nucleotide substitutions in the C modules of other IFN-A genes disrupt IRF-<em>3</em>-mediated transcription, whereas a G/A substitution in the D modules enhances IRF7-mediated expression. IRF-<em>3</em> exerts dual effects on IFN-A gene expression, as follows: a synergistic effect with IRF-7 on IFN-A1 expression and an inhibitory effect on other IFN-A gene promoters. Chromatin immunoprecipitation experiments reveal that transient binding of both IRF-<em>3</em> and IRF-7, accompanied by CBP/p<em>3</em>00 recruitment to the endogenous IFN-A gene promoters, is associated with transcriptional activation, whereas a biphasic recruitment of IRF-<em>3</em> and CBP/p<em>3</em>00 represses IFN-A gene expression. This regulatory mechanism contributes to differential expression of IFN-A genes and may be critical for <em>alpha</em> <em>interferon</em> production in different cell types by RIG-I-dependent signals, leading to innate antiviral immune responses.

BACKGROUND

Hypertrophic scars (HSc) are a dermal fibroproliferative disorder that leads to considerable morbidity. Preliminary evidence suggests that <em>interferon</em> (IFN) may improve HSc clinically. The aims of this study were (1) to compare the cell density in HSc and in wounds that heal without the development of HSc (normotrophic scars), (2) to examine the presence of myofibroblasts and apoptosis in normotrophic and HSc scars over time, and (<em>3</em>) to determine if the systemic administration of IFN-alpha2b can induce apoptosis.

METHODS

Two groups of patients underwent serial tissue biopsies. Six burn patients were studied prospectively by obtaining biopsy specimens from wound granulation tissue, normal skin, post-burn HSc, and normotrophic scars (healed donor sites). A second patient group with HSc was treated with systemic IFN-alpha2b and had biopsy material taken before, during, and after IFN therapy. The tissue was analyzed by immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) and in situ DNA fragmentation terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis.

RESULTS

The total numbers of fibroblasts in HSc were found to be similar to granulation tissue and twice that of normal skin and normotrophic scar. Over time the numbers of cells in HSc tissue decreased toward normal skin levels. There was a significantly higher percentage of fibroblasts staining for alpha-SMA in HSc as compared with normotrophic scar or normal skin obtained from the same patient (P>>.05). Serial biopsy specimens of resolving HSc tissue obtained from the patients who received systemic IFN-alpha2b showed a general reduction in total number of fibroblasts and myofibroblasts associated with a significant increase in the percentage of apoptotic cells compared with normal dermis from the same patient.

CONCLUSIONS

HSc tissues have greater numbers of fibroblasts and myofibroblasts than normal skin and normotrophic scars. As HSc remodels, the numbers of fibroblasts and myofibroblasts reduces, possibly by the induction of apoptosis. Systemic IFN-alpha2b may contribute to the resolution of HSc in part by the enhanced induction of apoptosis.

OBJECTIVE

To compare the response rate, survival, and toxicity of treatment with high-dose intravenous (IV) bolus interleukin-2 (IL-2) plus interferon alfa-2a (IFN-alpha) with high-dose IL-2 alone in patients with advanced melanoma in a randomized phase III trial design.

METHODS

Eighty-five patients with advanced melanoma were randomly assigned to receive IL-2 6 X 10(6) U/m2 per dose every 8 hours as tolerated for a maximum of 14 doses on days 1 through 5 and 15 through 19, or IL-2 4.5 X 10(6) U/m2 per dose, plus IFN-alpha 3 X 10(6) U/m2 using an identical schedule. A planned interim analysis was performed after 85 patients were entered, which forms the basis for this report.

RESULTS

Partial response (PR) occurred in two of 44 patients (5%; 95% confidence interval, 1% to 15%) receiving IL-2 alone, compared with four of 41 patients (10%; 95% confidence interval, 3% to 23%) receiving IL-2/IFN-alpha (P = .30). There were no complete responses (CRs). The median duration of response was 11.5 months (range, 2.0 to 15.7+). There was no significant difference in the median survival duration for patients receiving IL-2 alone (10.2 months) compared with patients receiving IL-2/IFN-alpha (9.7 months). The median and mean number of doses of IL-2 were equivalent in both groups, as was toxicity. There were three treatment-related deaths, two in the IL-2-alone arm and one in the IL-2/IFN-alpha arm. The trial was terminated after the first interim analysis based on predefined early-stopping rules, which included termination if the response rate in the IL-2/IFN-alpha arm was less than 25%.

CONCLUSIONS

Using the preparation, dose, and schedule of IL-2 in our trial, IFN-alpha failed to enhance significantly the response rate to high-dose IL-2 in the treatment of patients with advanced melanoma.

BACKGROUND

Less than half of patients with melanoma that has spread to local draining regional lymph nodes (stage III melanoma) live with no disease for 5 years or longer after surgery. We aimed to see whether interferon alpha-2a increased survival prospects in these patients.

METHODS

444 patients from 23 centres in the WHO Melanoma Programme had complete lymphadenectomy for pathologically proven regional nodal spread of melanoma and were randomly assigned to receive either 3 MU subcutaneously of recombinant interferon alpha-2a three times a week for 3 years, or to observation alone after surgery. Patients were stratified by centre, nodes with macroscopic or microscopic melanoma, number of affected nodes, and nodal metastatic spread. Treatment was continued for 3 years or until first sign of relapse.

RESULTS

424 patients entered the study. 5-year disease-free survival of those who had surgery plus interferon alpha-2a was 27.5% (95% CI 21.7-33.6); for those who received surgery alone, survival was 28.4% (22.5-34.6) (p=0.50). Neither Kaplan-Meier cumulative survival rates, nor multivariate analysis of survival, showed a difference between those who had surgery and interferon alpha-2a (35%, 95% CI 29-42) and those who had surgery alone (37%, 31-44).

CONCLUSIONS

Patients with melanoma that has spread to the local draining regional lymph nodes tolerate well 3 MU of interferon alpha-2a given subcutaneously three times a week for 3 years, but this treatment does not improve either disease-free or overall survival.

We examined purified human large granular lymphocytes, peripheral monocytes, and T cells for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) with murine monoclonal antibodies. We also evaluated the effects of pretreatment of cells with interleukin 2 and <em>interferon</em> to augment ADCC activity. MB<em>3</em>.6, a murine monoclonal antibody directed against the GD<em>3</em> ganglioside, induced high levels of ADCC. This ADCC was mediated predominantly, if not completely, by human killer cells (large granular lymphocytes) whereas other effector cell populations demonstrated no significant cytotoxic activity in 6- or 18-hr assays. The IgG2a an anti-melanoma antibody 9.2.27 generated low or no ADCC with most normal donors or melanoma patients. IL 2 was a very potent booster of ADCC activity. <em>Interferon</em> <em>alpha</em> also was effective, whereas <em>interferon</em> gamma did not augment but rather inhibited reactivity. We tested a large panel of antibodies of various isotype against colon carcinoma cells and found that gamma-<em>3</em> isotype antibodies more frequently generated ADCC and produced higher levels of cytotoxic activity than did IgG1 or IgG2 antibodies. It appears that a variety of parameters can affect ADCC reactions, including the type of effector cell and its level of activation, the isotype of the antibody, and properties of the target cell line such as its susceptibility to lysis.

Thyroid autoimmunity and dysfunction have been widely reported as side effects of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) treatment, but the literature lacks data regarding the long-term course of these complications, clinical observation being limited to 6-12 months off therapy. Our study is the first that has aimed to evaluate the natural history of IFN-related thyroid autoimmunity during a 6.2-yr follow-up after the IFN-<em>alpha</em> withdrawal as well as to investigate the potential role of the autoantibody pattern at the end of treatment to predict the long-term outcome. Our study group included 114 patients (79 males, <em>3</em>5 females), mean age 48 yr (range 2<em>3</em>-67 yr) with no preexisting thyroid disease, undergoing a 12-month treatment with recombinant IFN-<em>alpha</em> for C virus-related chronic active hepatitis. Thyroid autoimmunity (serum TgAb and TPOAb) and function (serum FT(4), FT(<em>3</em>), TSH) were retrospectively evaluated at the end of IFN therapy, 6 months after IFN withdrawal and after a median period of 6.2 yr (range 5.5-8.4 yr). At the end of treatment, 78 patients were negative for thyroid autoantibodies (Abs-) and all but one of them remained so for the following evaluations. The remaining <em>3</em>6 patients had thyroid autoantibodies (Abs+) at the end of treatment, and they subsequently showed a heterogeneous behavior: 16 patients remained Abs+ for the whole length of the study (persistent thyroiditis); 10 patients became Abs- 6 months off therapy but were again Abs+ 6.2 yr later (remitting/relapsing thyroiditis); 10 patients reverted to autoantibody negativity at different observation times (transient thyroiditis). The absence of thyroid autoantibodies at the end of treatment was a protective factor for the successive development of thyroiditis (odds ratio: 0.02, confidence interval (CI) 95%: 0-0.1). On the contrary, the positivity for TgAb and/or TPOAb at high titers at the end of IFN treatment was significantly related to the highest risk of having chronic thyroiditis (odds ratio: 17.<em>3</em>, CI 95%: <em>3</em>.2-91.7 for TgAb levels>> 50 degree percentile; odds ratio: 7.<em>3</em>, CI 95%: 1.5-<em>3</em>5.2 for TPOAb levels>> 50 degree percentile). None of the patients showed overt thyroid dysfunction throughout the study, whereas a subclinical hypothyroidism was found in 12 patients. In all 12 cases, the functional abnormality was accompanied by the presence of thyroid autoantibodies. Eight of these 12 patients belonged to the group with persistent thyroiditis (P < 0.05). The absence of thyroid autoantibodies at the end of treatment was a protective factor for the successive development of thyroid dysfunction (odds ratio: 0.06, CI 95%: 0.01-0.56). On the contrary, the positivity for both TgAb and TPOAb at the end of IFN therapy was significantly correlated with the highest risk of having subclinical hypothyroidism 6.2 yr. later (odds ratio: <em>3</em>8.7; CI 95%: 6.2-242). Our study demonstrates that in patients undergoing an IFN-<em>alpha</em> therapy for chronic hepatitis C and with no evidence of preexisting thyroid disease: 1) the negativity for thyroid autoantibodies after IFN treatment is a protective factor for the developing thyroid autoimmunity and/or dysfunction in following years; 2) the IFN-<em>alpha</em>-related thyroid autoimmunity is not a complete reversible phenomenon because some patients can develop chronic thyroiditis; <em>3</em>) high autoantibody levels at the end of IFN therapy are related to the risk of having chronic thyroid autoimmunity; and 4) the coexistence of TgAb and TPOAb at the end of treatment is a predictive factor for the presence of thyroid dysfunction, even if subclinical, many years after IFN withdrawal.

Phosphoinositide <em>3</em>-kinases (PI<em>3</em>K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI<em>3</em>K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR<em>3</em> and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR<em>3</em> or TLR4 engagement. In the same models of DC activation, PI<em>3</em>K inhibition increased DNA-binding activity of NF-kappaB, but not <em>interferon</em> response factor (IRF)-<em>3</em>, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-<em>alpha</em>/beta phosphorylation and IkappaB-<em>alpha</em> degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 29<em>3</em>T cells stably expressing TLR<em>3</em> or TLR4 confirmed that inhibition of PI<em>3</em>K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 29<em>3</em>T cells, while overexpression of catalytically active PI<em>3</em>K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI<em>3</em>K physically interacted with TRIF. We conclude that inhibition of PI<em>3</em>K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR<em>3</em> or TLR4 ligands.

The gene for the RNA-dependent eIF-2 <em>alpha</em> protein kinase (PKR) was isolated from mouse genomic DNA and characterized. The mouse PKR gene contains 16 exons and spans about 28 kilobase pairs. Exon 1 is untranslated; the AUG translation initiation site is located early in the second exon. Exon 16 includes the UAG translation termination site. ATTAAA polyadenylylation signal, and a putative TA rather than CA <em>3</em>' cleavage site. Primer extension analysis determined one major as well as multiple minor transcription initiation sites; the major site was 159 bp upstream of the translation initiation site. The complete cDNA of mouse PKR is, therefore, 2<em>3</em><em>3</em>4 bp in length excluding the <em>3</em>' poly(A)+ tail. The PKR gene 5' flanking region was a functional promoter in <em>interferon</em>-treated, transfected cells as measured with chloramphenicol acetyltransferase as the reporter gene. Sequence analysis of the 5' flanking region disclosed numerous potential binding sites for transcription factors including both an ISRE element and a GAS element involved in <em>interferon</em> inducibility; Ets, Myb, MyoD, and E2F sites commonly associated with growth control regulation and differentiation; and NF-kappa B-like sites as well as sites for two types of interleukin 6-activated factors, NF-IL6 and APRF, often associated with acute-phase, immune, and inflammatory response genes.

BACKGROUND

Exploratory subgroup analyses from the phase <em>3</em> global advanced renal cell carcinoma (ARCC) trial were conducted to determine if baseline levels of the tumor molecular markers PTEN and HIF1 <em>alpha</em> correlated with efficacy in patients treated with temsirolimus (Torisel) versus <em>interferon</em>-<em>alpha</em> (IFN).

METHODS

Patients in the IFN group received <em>3</em> million U (MU) subcutaneously <em>3</em>x weekly, escalating to 18 MU. Patients in the temsirolimus group received 25 mg intravenously weekly. PTEN and HIF1 <em>alpha</em> baseline levels were measured in archived tumor specimens by immunohistochemistry.

RESULTS

There was no correlation between baseline PTEN and HIF1 alpha levels and treatment effect with respect to overall survival (OS), progression-free survival, or objective response rate (ORR) in patients with advanced renal cell carcinoma with poor-risk prognostic factors.

CONCLUSIONS

The baseline status of the molecular markers PTEN and HIF1 <em>alpha</em> did not correlate with efficacy in renal cell carcinoma patients treated with temsirolimus versus IFN. Patients demonstrated OS and progression-free survival benefit when treated with temsirolimus regardless of PTEN and HIF1 <em>alpha</em> status. Thus, baseline PTEN and HIF-1 levels may not predict response to temsirolimus. Alternatively, the lack of correlation may be due to the variability in tumor specimens that occurred because of the global nature of the clinical trial. Other markers in the phosphoinositide <em>3</em>-kinase (PI<em>3</em>K)/Akt pathway may be of utility as predictors of response to temsirolimus in patients with advanced renal cell carcinoma.

Symptoms of autoimmune disease were evaluated in 125 patients with chronic myelogenous leukemia (CML) and in 12 patients with essential thrombocythemia undergoing treatment with recombinant <em>interferon</em> (IFN)-<em>alpha</em>-2b plus/minus low-dose recombinant IFN-gamma. Twenty-seven of 1<em>3</em>7 patients (20%) developed rheumatoid symptoms. Furthermore, the incidence of antinuclear antibody (ANA) formation was studied. Elevated ANA titers were found in 5/19 (26%) of CML patients at the time of diagnosis and in <em>3</em>/18 (17%) of patients treated with hydroxyurea or busulfan. During IFN treatment, 18 of 25 tested patients (72%) had elevated ANA titers. In 15 of these ANA-positive patients, clinical signs of autoimmune disease appeared. All these patients were under long-term IFN treatment and were in remission of disease. In three patients criteria for systemic lupus erythematosus were fulfilled. Severity of side effects had led to the discontinuation of IFN treatment in these patients. The data indicate that IFN-<em>alpha</em> and IFN-gamma can induce ANA associated with autoimmune disease in patients with myeloproliferative disorders.

OBJECTIVE

To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions.

METHODS

Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-<em>3</em> cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days.

RESULTS

Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC.

CONCLUSIONS

These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT<em>3</em>. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the <em>alpha</em> and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p<em>3</em>8 mitogen-activated protein kinase family members. In addition, we have observed the activation of <em>interferon</em> regulatory factor <em>3</em>, leading to the synthesis of beta <em>interferon</em> mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.

<em>Interferon</em>-gamma1b (IFN-gamma1b) is a pleiotropic cytokine that displays antifibrotic, antiviral, and antiproliferative activity. A total of 502 patients with compensated liver disease and an Ishak fibrosis score of 4-6 were randomized in a double-blind, placebo-controlled study, and 488 of these patients received subcutaneous injections of IFN-gamma1b 100 microg (group 1, n=169), IFN-gamma1b 200 microg (group 2, n=157), or placebo (group <em>3</em>, n=162) <em>3</em> times a week for 48 weeks. Most patients (8<em>3</em>.6%) had cirrhosis at baseline (Ishak score=5 or 6). Posttreatment liver biopsies were assessed in a blinded fashion for a reduction of 1 or more Ishak points (primary endpoint). Four hundred twenty patients with pretreatment and posttreatment liver biopsies were evaluable and showed no improvement in Ishak score between the <em>3</em> treatment groups (12.1%, 12.4%, and 16% of patients in groups 1, 2, and <em>3</em>, respectively; P>0.05). Analysis of IFN-gamma-inducible biomarkers revealed that <em>interferon</em>-inducible T cell-<em>alpha</em> chemoattractant (ITAC), an IFN-gamma-inducible CXCR<em>3</em> chemokine was an independent predictor of stable or improving Ishak score. IFN-gamma1b was well tolerated. There were similar numbers of deaths in all <em>3</em> arms (5, 5, and 4, respectively), and most were related to complications of cirrhosis.

CONCLUSIONS

IFN-gamma1b therapy was not able to reverse fibrosis in patients with advanced liver disease for 1 year. Subgroups of patients with elevated ITAC levels and perhaps less advanced disease may be considered for future studies with IFN-gamma1b.

<em>Interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) is known to participate in the transcriptional induction of <em>interferon</em> (IFN) <em>alpha</em> and IFNbeta genes, as well as of a number of <em>interferon</em>-stimulated genes (ISGs), as a result of viral infection. In the present study we demonstrate the activation of IRF<em>3</em> followed by ISG induction after exposure of cells to the bacterial cell wall component lipopolysaccharide. Engagement of Toll-like receptors by lipopolysaccharide triggered the nuclear translocation of IRF<em>3</em>, followed by its DNA binding and the subsequent induction of several <em>interferon</em>-regulated genes. Transcriptional activation of ISGs occurred in a protein synthesis independent manner, but was sensitive to inhibition of the stress-activated protein kinase, p<em>3</em>8. The activation of IRF<em>3</em> by viral particles or bacterial membrane components suggests that this signaling pathway might contribute to the evolutionary conserved innate immune response.

Monocyte or macrophage polykaryons (MP) are seen in different tissues in various inflammatory states and in normal bone (osteoclasts). The factors controlling the formation and the function of MP are not completely understood. This study was designed to evaluate the effects of the lymphokine gamma-<em>interferon</em> (IFN-gamma) on human monocyte function in vitro. Purified recombinant IFN-gamma [20-200 units/ml (0.1-1.0 nM)] caused the appearance of MP in cultures of normal human monocytes cultured in 10% unheated autologous serum. The MP were noted by as early as <em>3</em>6 hr of culture with fusion indices of 40%-60% and up to 160 nuclei per cell. The effect was seen with both recombinant IFN-gamma and natural IFN-gamma produced by Staphylococcal enterotoxin A-stimulated lymphocytes, but IFN-<em>alpha</em> (leukocyte-derived and recombinant) and IFN-beta did not induce MP formation. The activity of the IFN-gamma was destroyed by heating at 56 degrees C for 4 hr, incubating at pH 2 for <em>3</em> hr, or incubating with antibody against IFN-gamma. Populations of monocytes incubated <em>3</em> days with 100 units of IFN-gamma per ml (0.5 nM) had enhanced capacity to produce H2O2 in response to phorbol 12-myristate 1<em>3</em>-acetate and increased content of acid phosphatase and plasminogen activator. As determined by autoradiography, the MP did not incorporate [<em>3</em>H]dThd into their nuclei. Thus, the IFN-gamma appears to induce MP formation by a process of monocyte fusion, and to "activate" monocytes, as judged by various parameters.