Regulation of the production of the biologically active vitamin D<em>3</em> sterol 1,25-dihydroxyvitamin D<em>3</em> [1,25-(OH)2D<em>3</em>] by cultured pulmonary alveolar macrophages (PAM) obtained from 6 patients with pulmonary sarcoidosis and from 9 normal subjects was studied. The sarcoid cells, all collected from patients with normal calcium metabolism, synthesized 1,25-(OH)2-[<em>3</em>H]D<em>3</em> from the substrate 25-hydroxyvitamin [<em>3</em>H]D<em>3</em> (25OH-[<em>3</em>H]D<em>3</em>), whereas in vitro incubation with recombinant human <em>interferon</em>-gamma (IFN gamma) or lipopolysaccharide (LPS) was required for induction of synthesis of the hormone by normal PAM. Exogenous 1,25-(OH)2D<em>3</em> (10-100 nmol/L) decreased endogenous hormone production by normal PAM by approximately 45%. The relative inhibitory effect of 1,25-(OH)2D<em>3</em> was less pronounced in sarcoid PAM, in which 10-100 nmol/L 1,25-(OH)2D<em>3</em> inhibited 250HD<em>3</em>-1-hydroxylase by approximately 25%. An accompanying induction of the 250HD<em>3</em>-24-hydroxylase, which is typical for renal cells, was found at low levels in only <em>3</em> of 10 experiments; in this regard, no differences between sarcoid and normal PAM were apparent. PTH or forskolin did not influence 250HD<em>3</em> metabolism by PAM. 1,25-(OH)2D<em>3</em> production by sarcoid PAM was enhanced by lipopolysaccharide and IFN gamma. Likewise, recombinant human interleukin-2 stimulated 1,25-(OH)2D<em>3</em> production by sarcoid PAM, suggesting a possible role for both IFN gamma and interleukin-2 in the induction of 1,25-(OH)2D<em>3</em> synthesis by sarcoid PAM in vivo. Recombinant human IFN <em>alpha</em>, IFN beta, and granulocyte-macrophage colony-stimulating factor had little effect. Dexamethasone and chloroquine, which have in vivo antihypercalcemic activity in sarcoidosis, both inhibited 1,25-(OH)2D<em>3</em> synthesis by sarcoid PAM; chloroquine simultaneously stimulated the 24-hydroxylase. Our studies suggest that the 250HD<em>3</em>-metabolizing system in PAM is in some respects different from renal metabolism of 250HD<em>3</em>.

Blastic natural killer (NK) cell lymphoma corresponding to CD4+CD56+ malignancies is a novel disease entity, according to the results of clinical, morphologic, and immunologic studies. It is especially noteworthy that this disease likely arises from plasmacytoid dendritic cells (pDCs), described previously as plasmacytoid T-cells, which have an important role in innate and adaptive immunity. However, the exact relationship between the tumor cells and pDCs remains to be elucidated. We encountered a patient with typical blastic NK cell lymphoma, which later converted to leukemic manifestations, and tried to establish a cell line using the leukemic cells. We succeeded in establishment of a novel cell line, CAL-1, which originated from the primary malignant cells. The genetic and phenotypic features of CAL-1 cells bear a similarity to those of pDCs, namely, plasmacytoid morphology at light and electron microscopy; negative results for CD11c and lineage-associated markers of CD<em>3</em>, CD14, CD19, and CD16; positive results for HLA-DR, CD4, CD56, CD45RA, and CD12<em>3</em>; and negative results for TCR and IgH gene rearrangements. An interesting finding was that CAL-1 cells change morphologically into the mature DC appearance with many long dendrites after short-term culture in the presence of granulocyte-macrophage colony-stimulating factor and interleukin <em>3</em>. CAL-1 cells can secrete tumor necrosis factor <em>alpha</em> but not <em>interferon</em> <em>alpha</em>. Thus although they do not share in part phenotypic and functional features with their normal counterparts, CAL-1 cells mostly exhibit a striking pDC phenotype. We describe the first novel pDC cell line of CAL-1. This cell line should open the opportunity for study not only of CD4+CD56+ tumor cells but also of pDCs in vitro.

African swine fever virus (ASFV) multigene family <em>3</em>60 and 5<em>3</em>0 (MGF<em>3</em>60/5<em>3</em>0) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:<em>3</em>066-<em>3</em>076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF<em>3</em>60/5<em>3</em>0 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF<em>3</em>60/5<em>3</em>0 deletion mutant (Pr4 Delta <em>3</em>5). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4 Delta <em>3</em>5 at <em>3</em> and 6 h postinfection (hpi). While at <em>3</em> hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, <em>3</em>8 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4 Delta <em>3</em>5-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr Delta <em>3</em>5 up-regulated genes were part of a type I <em>interferon</em> (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein <em>3</em>, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG4<em>3</em>, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4 Delta <em>3</em>5 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-<em>alpha</em> mRNA and secreted IFN-<em>alpha</em> levels at <em>3</em>, 8, and 24 hpi revealed undetectable IFN-<em>alpha</em> in mock- and Pr4-infected macrophages but significant IFN-<em>alpha</em> levels at 24 hpi in Pr4 Delta <em>3</em>5-infected macrophages. The absence of IFN-<em>alpha</em> in Pr4-infected macrophages suggests that MGF<em>3</em>60/5<em>3</em>0 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4 Delta <em>3</em>5 in macrophages and its attenuation in swine.

OBJECTIVE

To determine the risk factors for the development of thyroid diseases during interferon-alpha therapy, we analyzed the patients with chronic hepatitis C who were treated with interferon-alpha.

METHODS

One hundred nine patients with chronic hepatitis C (77 men and 32 women, ages 20-72 yr) were treated with interferon-alpha (alpha, 48; alpha 2a, 38; alpha 2b, 23) for 14-40 wk. Thyroid function tests and seven autoantibodies were assessed at the beginning and end of interferon-alpha therapy, and every other month. A logistic multiple regression model was used in the statistical analysis of risk factors for development of thyroid diseases.

RESULTS

Among the 106 patients with normal pretreatment thyroid function tests, nine patients (three men and six women, ages 33-62 yr) developed thyroid diseases. However, among three patients with abnormal thyroid function tests, exacerbation of thyroid disease was not observed during interferon-alpha therapy. Logistic multiple regression model revealed that positivity for microsome antibody was a significant risk factor for the development of thyroid disease (p < 0.0001, chi 2 = 20.18). Actually, compared to patients without microsome antibody at the beginning of therapy, the incidence of thyroid diseases in the patients with pretreatment microsome antibody was very high: 3.3% (3/99) versus 60% (6/10), respectively. Six patients developed hyperthyroidism and three patients developed hypothyroidism. The patients with hyperthyroidism had atypical clinical features.

CONCLUSIONS

Our study revealed that positivity for microsome antibody at the beginning of interferon-alpha therapy is a risk factor for thyroid dysfunction.

An analysis of the National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplant Registry data shows that the greater the viral load at the time of transplantation, the more rapidly clinically evident posttransplantation hepatitis C virus (HCV) disease recurs. These data suggest that aggressive pretransplantation treatment of HCV might delay recurrent posttransplantation HCV disease and enhance posttransplantation survival. We have taken an aggressive approach to treating HCV infection pretransplantation with the use of high-dose (5 MU) daily <em>interferon</em> <em>alpha</em>(2b) in an effort to clear the virus before transplantation. A total of 27 patients with HCV-induced cirrhosis were seen and underwent transplantation at Loyola University Medical Center (Maywood, IL) between February 1997 and December 2001. There were 22 men and five women, with a mean age of 56 +/- 2 years. The majority had genotype 1 disease (67%). Of the 27 patients, 7 had a baseline platelet count <50,000/mm(<em>3</em>) and were excluded from <em>interferon</em> therapy. The remaining 20 were treated for a mean of 14 +/- 2.5 (range, 0.5 to <em>3</em><em>3</em>.5) months before orthotopic liver transplantation (OLT). Twelve (60%) responded to the therapy with serologic clearance of HCV before OLT. The mean time from initiation of therapy to the first negative qualitative polymerase chain reaction was 4.5 +/- 1.5 (range, 0.5 to 12) months. Four of the 12 patients in whom the virus cleared did not have evidence of HCV recurrence after OLT, representing 20% of those treated and <em>3</em><em>3</em>% of those who had HCV clearance before OLT. The duration of post-OLT freedom from HCV infection in these individuals has been <em>3</em><em>3</em>.6 +/- 11.<em>3</em> (range, 0 to 47.4) months. These data suggest that with careful supervision, cirrhotic patients can tolerate high-dose <em>interferon</em>. In addition, a viral clearance can be achieved in a significant number of cirrhotic patients with high-dose <em>interferon</em>. One third of patients, in whom the HCV cleared before OLT, did not have evidence of disease recurrence after OLT. It is thus anticipated that with early and aggressive pre-OLT HCV therapy, possibly with the use of pegylated <em>interferon</em>, even better results may be obtained.

The effects of <em>interferons</em> (IFNs) on lymphocyte adhesion to cultured human vascular endothelial cells (EC) were investigated using an in vitro assay. Endothelial cells obtained from umbilical vein were first cultured at a low density with a conditioned medium (CM) from 12-O-tetra decanoylphorbol 1<em>3</em>-acetate-concanavalin A (TPA-Con A) stimulated human peripheral blood lymphocytes (PBL), or with recombinant (r) gamma <em>interferon</em> (IFN-gamma) or r <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>), and then were incubated with freshly isolated PBL. Natural IFN-gamma in the TPA-Con A CM and rIFN-gamma (12.5-500 U/ml) induced major histocompatibility complex-class II antigens (HLA-DR, HLA-DP, and HLA-DQ) and significant lymphocyte adhesion to the EC, whereas rIFN-<em>alpha</em> did not. The lymphocyte adhesion to the EC and the expression of DR antigens on the EC were well correlated in terms of both kinetics and the dose-response pattern of rIFN-gamma. When EC expressing I region associated (Ia) antigen were preincubated with monoclonal anti-DR antibody before the addition of lymphocytes, the lymphocyte adhesion was significantly inhibited in both allogeneic and syngeneic combinations, whereas anti-HLA-DP, anti-HLA-DQ, and anti-HLA-ABC antibodies did not inhibit the binding at all. Cell fractionation experiments indicated that the majority of lymphocytes adhering to Ia-expressed EC were Leu-<em>3</em>+ T cells, whose binding was again almost completely inhibited by anti-DR antibody. Moreover, anti-Leu-<em>3</em>a, but not anti-Leu-2a, antibody effectively inhibited the T cell adhesion to the EC. These results strongly suggest that the interaction of the Leu-<em>3</em>(T4) receptor of T cells with IFN-gamma-induced DR antigens on EC plays a central role in the selective adhesion of Leu-<em>3</em>+ T cell to EC.

We have previously shown that swine inoculated with recombinant, replication-defective human adenovirus type 5 containing the porcine <em>interferon</em> <em>alpha</em> gene (Ad5-pIFN<em>alpha</em>) are completely protected when challenged 1 day later with virulent foot-and-mouth disease virus (FMDV). In the current study, we examined the duration of protection afforded swine by Ad5-pIFN<em>alpha</em> and the ability of a combination of Ad5-pIFN<em>alpha</em> and a FMDV subunit vaccine delivered by Ad5-A24 (an Ad5 vector containing the capsid coding region of FMDV serotype A24 Cruzeiro and the <em>3</em>C proteinase coding region of FMDV serotype A12) to induce immediate as well as long-lasting protection against homologous FMDV challenge. Groups of swine were inoculated with Ad5-pIFN<em>alpha</em> and challenged with virulent FMDV A24 1, <em>3</em>, 5, and 7 days postinoculation (dpi) or 1 day preinoculation. All animals challenged 1 and <em>3</em>dpi were completely protected from disease. The animals in the remaining groups had either no clinical signs of disease or clinical signs were delayed and less severe compared to the control group. Swine inoculated with a combination of Ad5-pIFN<em>alpha</em> and Ad5-A24 and challenged 5dpi were all completely protected from disease and developed a significant FMDV-specific neutralizing antibody response.

Distinct NF-kappa B subunit combinations contribute to the specificity of NF-kappa B-mediated transcriptional activation and to the induction of multiple cytokine genes including <em>interferon</em>-beta (IFN-beta). To evaluate the regulatory influence of different homo- and heterodimers, NF-kappa B subunits were analyzed for transcriptional activity in vitro using test templates containing two types of NF-kappa B recognition elements (the human immunodeficiency virus type 1 enhancer and the IFN-beta-positive regulatory domain-II (PRDII) as well as IFN-beta PRDIII-PRDI-PRDII linked to the -56 minimal promoter of rabbit beta-globin. Recombinant NF-kappa B subunits (p50, p65, c-Rel, p52, and I kappa B <em>alpha</em>) and <em>interferon</em> regulatory factor 1 were produced from either Escherichia coli or baculovirus expression systems. Transcriptional analysis in vitro demonstrated that 1) various dimeric complexes of NF-kappa B differentially stimulated transcription through the human immunodeficiency virus enhancer or PRDII up to 20-fold; 2) recombinant I kappa B <em>alpha</em> specifically inhibited NF-kappa B-dependent transcription in vitro; and <em>3</em>) different NF-kappa B complexes and <em>interferon</em> regulatory factor 1 cooperated to stimulate transcription in vitro through the PRDIII-PRDI-PRDII virus-inducible regulatory domains of the IFN-beta promoter. These results demonstrate the role of NF-kappa B protein dimerization in differential transcriptional activation in vitro and emphasize the role of cooperativity between transcription factor families as an additional regulatory level to maintain transcriptional specificity.

The ability of endocrine organs to express human immune response-associated antigens (Ia), such as HLA-DR, is a subject of intense current interest. In this study, the effects of various potential modulators of thyroid follicular cell HLA-DR expression were examined using in vitro cultures. A culture supernatant containing T-cell-derived lymphokines caused DR antigen expression on 1<em>3</em>-18% of thyroid cells; more consistent effects were produced by recombinant gamma-<em>interferon</em>, which led to 46-100% of the thyroid cells becoming HLA-DR positive after <em>3</em> days in culture. This effect was both time and concentration dependent and occurred in thyroid cells derived from patients with Graves' disease (n = 7) and Hashimoto's thyroiditis (n = 2) as well as from three subjects with no autoimmune thyroid disease. Thyroid cells stained with the monoclonal antibodies 4F2 and 5E9, which recognize cell activation antigens, regardless of whether they were treated with gamma-<em>interferon</em>. The lectin phytohemagglutinin also induced HLA-DR antigen expression (21-91% of cells positive). This response was dependent on T cell contamination of thyroid cell suspensions, since the effect was inhibited by cyclosporin A. HLA-DQ antigen expression, identified by the Leu-10 monoclonal antibody, was also induced on thyroid cells by gamma-<em>interferon</em> and phytohemagglutinin. In contrast, neither recombinant <em>alpha</em>-<em>interferon</em> nor interleukin-2 induced HLA-DR antigens. Irradiation reduced the response of thyroid cells to gamma-<em>interferon</em>, but two of the known inhibitors of macrophage Ia expression, prostaglandin E2 and (Bu)2cAMP, did not affect gamma-<em>interferon</em>-induced thyroid cell HLA-DR expression. We were unable to detect interleukin-1 production by thyroid cells. These results suggest that 1) under normal circumstances, thyroid cells are 4F2 and 5E9 positive, but are incapable of expressing Ia antigens and, thus, of activating T cells to initiate autoimmune thyroiditis; and 2) once activated, for example by a virus, T cells could release gamma-<em>interferon</em> and induce thyroid cell HLA-DR and -DQ antigen expression; these Ia-positive thyroid cells could then have a role in maintaining or enhancing the autoimmune response.

Pretreatment hepatitis C virus (HCV)-specific lymphoproliferative (LP) responses, neutralizing antibody (NA) responses, intrahepatic cytotoxic T lymphocyte (CTL) responses, and HCV quasi-species (QS) diversity and complexity were examined in patients with advanced hepatic fibrosis (Ishak fibrosis score of>> or = <em>3</em>) and prior nonresponse to <em>interferon</em> (IFN)- <em>alpha</em> therapy who were enrolled in the initial phase of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis Trial. Positive baseline HCV E1- and/or E2-specific NA responses (P = .01) and higher baseline HCV QS diversity (P = .01) were more commonly found in patients who did not become sustained virologic responders (SVRs) at week 72 (W72) than they were in those who did. No patients with positive results for both the LP and NA assays achieved a sustained virologic response. Multiple logistic regression analysis revealed that, when the presence of cirrhosis, prior ribavirin therapy, genotype 1 infection, log serum HCV RNA level, and receipt of >80% of the prescribed medication were controlled for, a sustained virologic response (W72) was negatively correlated with positive baseline LP assay results (P = .02) and with 1 or more positive assays (LP, NA, or CTL) (P = .02). No differences were noted in baseline intrahepatic CTL activity between SVRs and non-SVRs. Thus, in patients with advanced hepatic fibrosis due to HCV infection, pretreatment HCV-specific immune responses and increased QS variability appear to hinder viral clearance by pegylated IFN- <em>alpha</em> 2a and ribavirin combination therapy.

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a <em>3</em>-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.<em>3</em> to <em>3</em>.<em>3</em> log10 HCV RNA IU/ml across dosing cohorts (1, <em>3</em>, 10, <em>3</em>0, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, <em>3</em>0, <em>3</em>1, or 9<em>3</em> in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y9<em>3</em>H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for <em>3</em>/10 subjects at the 1-mg dose and for all subjects dosed at ≥<em>3</em> mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the <em>3</em>0- and 90-mg doses. Subject-derived M28T, Q<em>3</em>0R, L<em>3</em>1M, and Y9<em>3</em>C mutations all conferred>><em>3</em>0-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), <em>interferon</em> <em>alpha</em>, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT0119<em>3</em>478.).

CONCLUSIONS

Pegylated <em>interferon</em> with ribavirin (Peg/R) is the most effective therapy for chronic hepatitis C virus (HCV) but its utility and effectiveness after liver transplantation has been difficult to assess. We evaluated efficacy, tolerability, and safety of Peg/R in liver transplant candidates and recipients with HCV cirrhosis. We searched medical databases and conference proceedings between January 1999 and January 2008 selecting randomized and nonrandomized studies. Primary end points meta-analytically were: (1) sustained viral response (SVR) and (2) histological response. Secondary end points were: (1) treatment discontinuation, (2) mortality, and (<em>3</em>) rejection episodes. Pegylated <em>interferons</em> using either 1-1.5 mcg/kg of pegylated <em>interferon</em> <em>alpha</em>-2b or 180 microg (pegylated <em>interferon</em> <em>alpha</em>-2a combined with ribavirin 800-1200 mg/day were the most effective compared to any other regimen or no therapy. In three pretransplant studies the median SVR was 19.6% (19.6-50%). In six postransplant studies where a meta-analysis was done the cumulative risk difference in SVR was 0.<em>3</em>1% (95% CI, 0.18-0.44, p < 0.001). However histological response was not significantly better compared to no therapy or other antiviral regimens. There were no significant differences in discontinuation of therapy, acute or chronic rejection or mortality between optimal Peg/R vs no treatment or other regimens. Hence pegylated <em>interferon</em> plus ribavirin in full doses is effective pre and post transplant but has a low SVR rate. To date no significant histological improvement has been reported.

<em>Interferon</em> alfa (IFN-<em>alpha</em>) is an approved therapeutic agent for chronic hepatitis C. To directly characterize the effects of IFN-<em>alpha</em> in humans, we used microarrays to profile gene expression in peripheral blood mononuclear cells (PBMCs) from hepatitis C patients treated with IFN-<em>alpha</em>. Seven patients were studied using two strategies: (1) in vivo: PBMCs were collected immediately before the first dose of IFN-<em>alpha</em>, and <em>3</em> and 6 hours after the dose; (2) ex vivo: PBMCs that were collected before the first IFN-<em>alpha</em> dose were incubated with IFN-<em>alpha</em> for <em>3</em> and 6 hours. The microarray datasets were analyzed with significance analysis of microarrays (SAM) to identify genes regulated by IFN-<em>alpha</em>. We identified 516 named genes up-regulated at least 2-fold, at a false discovery rate (FDR) of less than 1%. In vivo and ex vivo studies generated similar results. No genes were identified as regulated differently between these 2 experimental conditions. The up-regulated genes belonged to a broad range of functional pathways and included multiple genes thought to be involved in the direct antiviral effect of IFN-<em>alpha</em>. Of particular interest, 88 genes directly relating to functions of immune cells were up-regulated, including genes involved in antigen processing and presentation, T-cell activation, lymphocyte trafficking, and effector functions, suggesting that IFN-<em>alpha</em> up-regulates multiple genes involving different aspects of immune responses to enhance immunity against hepatitis C virus. In conclusion, IFN-<em>alpha</em>-inducible genes can be identified in human PBMCs in vivo as well as ex vivo. Signature changes associated with different treatment outcomes may be found among these genes.

To determine the naturally occurring immunological responses to the Schistosoma mansoni antigens paramyosin, IrV-5, Sm-2<em>3</em> (MAP-<em>3</em>), and triose phosphate isomerase (MAP-4), a total of 119 subjects from an area of endemicity for schistosomiasis, including "resistant" subjects (n = 17) were evaluated. Specific immunoglobulin G1 (IgG1), IgG2, IgG<em>3</em>, IgG4, and IgA levels for each of the antigens and the cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC) were determined. Although all the subjects had a high degree of contaminated water exposure, their infection levels were variable (0 to 1,128 eggs/g of stool). There were direct correlations between infection levels and levels of SWAP- and paramyosin-specific IgG1 and IgG4 (P < 0.05). However, an inverse correlation between infection levels and specific IgG2 to IrV-5 (P < 0.01) was observed. The evaluation of the cytokine profile (interleukin 5 [IL-5], IL-10, gamma <em>interferon</em> [IFN-gamma], and tumor necrosis factor <em>alpha</em>) in response to these antigens showed inverse correlations between the degree of infection and IFN-gamma levels in PBMC supernatants stimulated with paramyosin (P < 0.05) and IrV-5 (P < 0.01). Additionally, inverse correlations between the degree of infection and IL-5 levels in MAP-<em>3</em>- and MAP-4-stimulated PBMC supernatants (P < 0.01) were found. Logistic regression analysis was performed to adjust the results of cytokine profile by age. IL-5 production in MAP-<em>3</em>-stimulated PBMC supernatants was associated with lower infection levels (odds ratio = 11.2 [95% confidence interval, 2.7 to 45.8]).

Fluorouracil (FUra) is the most active agent in advanced colorectal carcinoma, and this activity can be enhanced by various modulating agents both in vitro and in vivo. To determine whether <em>interferon</em> (IFN) is capable of augmenting the cytotoxic and cytokinetic effects of FUra, combinations of FUra and IFN <em>alpha</em>, -beta, and -gamma were tested against 2 human colon cancer cell lines in vitro. In a clonogenic assay, IFN <em>alpha</em> and -beta, at concentrations that produced less than 1 log cell kill, significantly increased the cytotoxic effects of FUra in both cell lines. IFN gamma also enhanced the cytotoxic effects of FUra, but unlike IFN <em>alpha</em> and -beta, only at the highest concentrations tested. Median effects analysis demonstrated that all <em>3</em> IFNs exhibited synergy with FUra. Combinations of IFNs were no more effective at modulating FUra activity than single agent IFN. Flow cytometric studies indicated that these effects did not correlate with cytokinetic alterations. Only the combination of FUra and IFN beta produced cytokinetic effects different from those of FUra alone. Incubation with IFN <em>alpha</em> or IFN gamma for 24 h resulted in only modest cytokinetic alterations, and they did not modify the effects of FUra. These results indicate that IFN is capable of increasing the cytotoxic actions of FUra and that this is separable from any cytokinetic effects produced by the <em>interferons</em>.

OBJECTIVE

Treatment of chronic hepatitis C with interferon (IFN)-alpha often has hematotoxic effects. We evaluated the effects of acute vs. chronic and standard vs. pegylated IFN-alpha on hematopoiesis.

METHODS

We studied hematopoiesis in 46 patients with chronic hepatitis C receiving single high-dose IN-F<em>alpha</em>2b followed by daily dose standard or weekly pegylated IFN before combination antiviral therapy.

RESULTS

Single high-dose therapy resulted in a significant drop in hemoglobin (HB), leukocytes, and platelet count. Although platelets, stimulated by a significant increase in thrombopoietin (TPO), and leukocytes recovered quickly, HB remained below baseline for 7 days. Daily standard or weekly pegylated IFN-alpha leads to a more pronounced drop in all 3 lineages with concomitant increases in TPO and erythropoietin (EPO). No difference was observed between standard and pegylated IFN, except for HB, which fell more during pegylated IFN therapy. Consecutive combination antiviral therapy aggravated the anemia but not the drop in leukocytes or thrombocytes.

CONCLUSIONS

The drop in all 3 hematopoietic lineages through IFN-alpha treatment, high-dose standard, standard daily dose, or pegylated, is caused by a combination of bone marrow inhibition and probably some other rapid acting mechanisms. Hematopoietic growth factors are increased as a consequence but cannot overcome the bone marrow suppression.

Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>) and <em>interferon</em> gamma (IFNgamma) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a <em>3</em>-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement <em>3</em> (C<em>3</em>) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

We reanalyzed the results of a pilot study of recombinant <em>alpha</em>-<em>interferon</em> therapy for chronic non-A, non-B hepatitis in light of the recent discovery of the hepatitis C virus and the development of diagnostic assays for this agent. Stored serum samples from 10 patients treated between 1984 and 1986 were tested for antibody to hepatitis C virus and hepatitis C virus RNA before, during and after therapy. In addition, the current clinical, serum biochemical and virological statuses of these patients were evaluated to determine the long-term effects of <em>interferon</em> therapy. All patients had evidence of hepatitis C virus infection, with hepatitis C viral RNA, antibody to hepatitis C virus or both markers detectable in serum. Serum hepatitis C virus RNA was found to disappear in seven of eight patients whose aminotransferase levels became normal with <em>interferon</em> therapy but remained present in two patients who did not respond to therapy. Levels of hepatitis C virus RNA decreased and disappeared when serum aminotransferases fell to normal levels but rose with subsequent elevation of aminotransferase levels in two patients who had relapses in disease when <em>interferon</em> was stopped. During a follow-up of <em>3</em> to 6 yr, hepatitis C virus RNA remained undetectable in the six patients whose serum aminotransferase levels remained normal after <em>interferon</em> therapy. However, neither initial titers of hepatitis C virus RNA nor disappearance of viral RNA from serum during treatment predicted a sustained response. Thus long-term beneficial responses to <em>alpha</em>-<em>interferon</em> can occur in patients with chronic hepatitis C and are associated with sustained loss of hepatitis C virus RNA from serum.

BACKGROUND

We postulate that hypercytokinemia plays a role in immunopathogenesis of severe human influenza.

METHODS

We prospectively studied 39 consecutive patients who were hospitalized with severe influenza A virus infection. On laboratory confirmation of the diagnosis, paired acute-phase (obtained at hospital admission) and convalescent-phase (obtained >10 days after hospital admission) plasma samples were collected for assay of 11 cytokines and chemokines (interleukin [IL] 1 beta; IL-6; IL-10; IL-12p70; tumor necrosis factor alpha; IL-8; monokine induced by interferon [IFN]-gamma; IFN-inducible protein 10; monocyte chemoattractant protein 1; regulated upon activation, normal T cell-expressed and secreted; and IFN-gamma) using cytometric bead-array analysis and enzyme-linked immunosorbent assay. Simultaneously, virus concentration in the acute-phase nasopharyngeal aspirate was determined using real-time quantitative reverse-transcriptase polymerase chain reaction. Intracellular signaling molecules regulating lymphocyte activation, phospho-p38 mitogen-activated protein kinase and phospho-extracellular signal-regulated protein kinase in CD4+ and CD8+ T lymphocytes were studied in the acute-phase samples using flow cytometric analysis and were compared with results for samples from healthy control subjects.

RESULTS

Statistically significant increases in plasma IL-6 (3.7-fold increase), IL-8 (2.6-fold increase), IFN-induced protein 10 (4.9-fold increase), and monokine induced by IFN-gamma (2.3-fold increase) concentrations were detected during acute illness (P < .01 for all, by Wilcoxon signed-rank test); the highest concentrations were observed on symptom days 3 and 4. Corresponding plasma cytokine and chemokine concentrations and nasopharyngeal viral loads showed statistically significant correlations (rho = 0.41, 0.49, 0.54, and 0.46, respectively; P < or = .01). Phospho-p38 mitogen-activated protein kinase expression in CD4+ lymphocytes was increased, correlating with cytokine concentrations (e.g., for IFN-induced protein 10, rho = 0.78; P < .01); phospho-extracellular signal-regulated protein kinase was suppressed. Advanced age and comorbidity were associated with aberrant IL-6, IL-8, and monokine induced by IFN-gamma responses (P < .05, by Mann-Whitney U test). An elevated IL-6 concentration was independently associated with prolonged hospitalization (hospitalization for >5 days; P = .02), adjusted for age, comorbidity, and virus load.

CONCLUSIONS

Hypercytokinemia (of proinflammatory and T helper 1 cytokines) is detected in severe influenza, correlating with clinical illness and virus concentration. Hyperactivation of phospho-p38 mitogen-activated protein kinase (in T helper cells) is possibly involved. Early viral suppression may attenuate these potentially deleterious cytokine responses.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin <em>3</em>, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma <em>interferon</em> or tumor necrosis factor <em>alpha</em> to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin <em>3</em>, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.

OBJECTIVE

To determine Th1 and Th2 cytokine production in patients with reactive arthritis (ReA) in relation to disease outcome and in comparison with rheumatoid arthritis (RA).

METHODS

Secretion of tumor necrosis factor <em>alpha</em> (TNF<em>alpha</em>), <em>interferon</em>-gamma, interleukin-10 (IL-10), and IL-4 by peripheral blood mononuclear cells (PBMC) from 5<em>3</em> patients with early ReA (disease duration <8 weeks, 64% HLA-B27 positive) and <em>3</em>0 patients with early, untreated RA (disease duration <6 months) was determined by enzyme-linked immunosorbent assay (ELISA) after ex vivo stimulation. Intracellular cytokine staining with quantification of positive T cells by fluorescence-activated cell sorting (FACS) was performed in 12 ReA patients and 12 RA patients. In 27 ReA patients, cytokine secretion was measured again after <em>3</em> months. Patients were followed up for 1 year, and cytokine patterns were correlated with disease duration.

RESULTS

TNF<em>alpha</em> secreted by whole PBMC and by T cells was significantly lower, by ELISA and by FACS, in ReA patients than in RA patients, while no significant differences were detected for the other cytokines. ReA patients with a disease duration of>> or =6 months showed significantly lower TNF<em>alpha</em> secretion than patients with a disease duration of <6 months (mean +/- SD <em>3</em>85 +/- 207 pg/ml versus 684 +/- 277 pg/ml; P = 0.00<em>3</em>). Furthermore, low TNF<em>alpha</em> secretion after <em>3</em> months also correlated significantly with a more chronic course of disease. HLA-B27 positive patients secreted less TNF<em>alpha</em> than did those who were B27 negative (<em>3</em><em>3</em>8 +/- 214 pg/ml versus 512 +/- 207 pg/ml; P = 0.05), and patients with a more chronic course had a higher frequency of B27 positivity (47% versus 80%; P = 0.01). Among the 27 HLA-B27 positive patients, TNF<em>alpha</em> secretion in those with a disease duration of>> or = 6 months was lower than that in the 7 with a disease duration of <6 months (<em>3</em>08 +/- 167 pg/ml versus 562 +/- <em>3</em>08 pg/ml; P = 0.04).

CONCLUSIONS

Low TNFalpha secretion and HLA-B27 status correlate with longer disease duration in ReA patients, possibly with an additive effect. The diminished TNFalpha production might reflect a state of relative immunodeficiency contributing to bacterial persistence in ReA.

The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via <em>interferon</em> (IFN) regulatory factor <em>3</em> (IRF-<em>3</em>)-dependent pathways. The absence of IRF-<em>3</em> would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-<em>3</em>(-/-) mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 4<em>3</em>4:772-777, 2005). In contrast, the present study demonstrated that IRF-<em>3</em>(-/-) mice are significantly more susceptible to HSV infection via the corneal and intracranial routes. Following corneal infection with 2 x 10(6) PFU of HSV-1 strain McKrae, 50% of wild-type mice survived, compared to 10% of IRF-<em>3</em>-deficient mice. Significantly increased viral replication and inflammatory cytokine production were observed in brain tissues of IRF-<em>3</em>(-/-) mice compared to control mice, with a concomitant deficit in production of both IFN-beta and IFN-<em>alpha</em>. These data demonstrate a critical role for IRF-<em>3</em> in control of central nervous system infection following HSV-1 challenge. Furthermore, this work underscores the necessity to evaluate multiple routes of infection and animal models in order to fully determine the role of host resistance factors in pathogenesis.

Mice with a fat-specific insulin receptor knock-out (FIRKO) have reduced adipose tissue mass, are protected against obesity, and have an extended life span. White adipose tissue of FIRKO mice is also characterized by a polarization into two major populations of adipocytes, one small (<50 microm) and one large (>100 microm), which differ with regard to basal triglyceride synthesis and lipolysis, as well as in the expression of fatty acid synthase, sterol regulatory element-binding protein 1c, and CCAAT/enhancer-binding protein <em>alpha</em> (C/EBP-<em>alpha</em>). Gene expression analysis using RNA isolated from large and small adipocytes of FIRKO and control (IR lox/lox) mice was performed on oligonucleotide microarrays. Of the 12,488 genes/expressed sequence tags represented, 111 genes were expressed differentially in the four populations of adipocytes at the p < 0.001 level. These alterations exhibited 10 defined patterns and occurred in response to two distinct regulatory effects. 6<em>3</em> genes were identified as changed in expression depending primarily upon adipocyte size, including C/EBP-<em>alpha</em>, C/EBP-delta, superoxide dismutase <em>3</em>, and the platelet-derived growth factor receptor. 48 genes were regulated primarily by impairment of insulin signaling, including transforming growth factor beta, <em>interferon</em> gamma, insulin-like growth factor I receptor, activating transcription factor <em>3</em>, aldehyde dehydrogenase 2, and protein kinase Cdelta. These data suggest an intrinsic heterogeneity of adipocytes with differences in gene expression related to adipocyte size and insulin signaling.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the <em>interferon</em> <em>alpha</em>/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor <em>interferon</em> <em>alpha</em> signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-<em>3</em>) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in <em>interferon</em> <em>alpha</em>/beta receptor signaling.