Human peripheral blood mononuclear cells activated with concanavalin A (Con A) or lipopolysaccharide (LPS) produce, respectively, lymphokines (LK) of 50,000 Mr or monokines (MK) of 20,000 Mr that inhibit the growth and collagen production of cultured human dermal fibroblasts. Because antigenic typing of the antiviral activity of these LK and MK preparations revealed that LK contained mainly gamma <em>interferon</em> (IFN-gamma), and MK, primarily IFN-beta, we investigated if any of the fibroblast-inhibiting activities could be attributed to human IFN. Unlike LK and MK, which act within 24 h to inhibit the growth of subconfluent foreskin and adult dermal fibroblasts, samples of purified, natural derived IFN-<em>alpha</em>, -beta, and -gamma and recombinant DNA-derived IFN-<em>alpha</em> 2 and -gamma were ineffective inhibitors at 24 h and required 48-72 h to significantly inhibit growth. However, all IFN did mimic LK/MK action in causing concentration-dependent inhibition of collagen production by confluent fibroblast microcultures. Furthermore, the collagen production-inhibiting activity of Con A-induced LK supernatant and its 50,000 Mr fraction was completely suppressed by 10(<em>3</em>) neutralizing U/ml of either polyclonal or monoclonal antibody to IFN-gamma, while polyclonal antibodies to IFN-<em>alpha</em> and -beta had no effect. Similarly, the collagen production-inhibiting activity of LPS-induced MK supernatant and its 20,000 Mr fraction was suppressed by polyclonal anti-IFN-beta but not by anti-IFN-<em>alpha</em> or -gamma. Anti-IFN failed to reverse early-acting LK or MK growth-inhibiting activities. These data suggest collagen production-inhibiting LK and MK are IFN-gamma and IFN-beta, respectively, and that early acting, growth-inhibiting LK and MK are not IFN.

A liver fibrosis index was recently prospectively validated in a cross-sectional study where patients infected by hepatitis C virus (HCV) had only one biopsy and no longitudinal follow-up. The aim of this study was to retrospectively assess the diagnostic value of this index in patients included in a randomized trial of <em>interferon</em> (IFN) using repeated measurements, two biopsies and hyaluronic acid as a comparative reference. One-hundred and sixty-five patients who had had two interpretable liver biopsies and at least one stored serum sample before IFN treatment were selected. Seventy-eight patients received <em>3</em> MU of IFN-<em>alpha</em> thrice weekly for 24 weeks and 87 followed a reinforced regimen for 48 weeks. A fibrosis index combining five biochemical markers (<em>alpha</em>2-macroglobulin, haptoglobin, apolipoprotein A1, gamma-glutamyl transpeptidase (GGT) and total bilirubin adjusted for gender and age) as well as hyaluronic acid was assessed on 461 samples available at baseline, at the end of treatment and at the end of follow-up (72 weeks). There was a significant decrease of the fibrosis index score among the 17 sustained virologic responders, from 0.<em>3</em><em>3</em> +/- 0.06 (mean +/- SE) at baseline to 0.18 +/- 0.06 at 72 weeks in comparison with 92 nonresponders (from 0.41 +/- 0.0<em>3</em> at baseline to 0.44 +/- 0.0<em>3</em> at 72 weeks; P < 0.001) and in comparison with 56 relapsers (from 0.<em>3</em>6 +/- 0.0<em>3</em> at baseline to 0.<em>3</em>2 +/- 0.0<em>3</em> at 72 weeks; P=0.05). No significant differences were observed for hyaluronic acid.Hence, this fibrosis index could be used as a surrogate marker of the antifibrotic effect of treatments in patients with chronic hepatitis C.

The murine coronavirus mouse hepatitis virus (MHV) induced the expression of type I <em>interferon</em> (<em>alpha</em>/beta <em>interferon</em> [IFN-<em>alpha</em>/beta]) in mouse oligodendrocytic N20.1 cells. This induction is completely dependent on virus replication, since infection with UV light-inactivated virus could no longer induce IFN-<em>alpha</em>/beta. We show that MHV infection activated both transcription factors, the IFN regulatory factor <em>3</em> (IRF-<em>3</em>) and nuclear factor kappaB (NF-kappaB), as evidenced by phosphorylation and nuclear translocation of IRF-<em>3</em> and an increased promoter binding activity for IRF-<em>3</em> and NF-kappaB. Furthermore, the cytoplasmic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) was induced by MHV infection. Knockdown of RIG-I by small interfering RNAs blocked the activation of IRF-<em>3</em> and subsequent IFN-<em>alpha</em>/beta production induced by MHV infection. Knockdown of another cytoplasmic receptor, the melanoma-differentiation-associated gene 5 (MDA5), by small interfering RNAs also blocked IFN-beta induction. These results demonstrate that MHV is recognized by both RIG-I and MDA5 and induces IFN-<em>alpha</em>/beta through the activation of the IRF-<em>3</em> signaling pathway. However, knockdown of RIG-I only partially blocked NF-kappaB activity induced by MHV infection and inhibition of NF-kappaB activity by a decoy peptide inhibitor had little effect on IFN-<em>alpha</em>/beta production. These data suggest that activation of the NF-kappaB pathway might not play a critical role in IFN-<em>alpha</em>/beta induction by MHV infection in oligodendrocytes.

Most patients with human immunodeficiency virus (HIV) who remain CD4(+) T-cell deficient on antiretroviral therapy (ART) exhibit marked immune activation. As CD4(+) T-cell activation may be mediated by microbial translocation or <em>interferon</em>-<em>alpha</em> (IFN-α), we examined these factors in HIV patients with good or poor CD4(+) T-cell recovery on long-term ART. Messenger RNA levels for <em>3</em> <em>interferon</em>-stimulated genes were increased in CD4(+) T cells of patients with poor CD4(+) T-cell recovery, whereas levels in patients with good recovery did not differ from those in healthy controls. Poor CD4(+) T-cell recovery was also associated with CD4(+) T-cell expression of markers of activation, senescence, and apoptosis, and with increased serum levels of the lipopolysaccharide receptor and soluble CD14, but these were not significantly correlated with expression of the <em>interferon</em>-stimulated genes. Therefore, CD4(+) T-cell recovery may be adversely affected by the effects of IFN-α, which may be amenable to therapeutic intervention.

BACKGROUND

The purpose of this study was to determine whether anti-tumour necrosis factor alpha (anti-TNF-α) antibody, infliximab, can inhibit T helper 17 (Th17) differentiation in uveitis patients who have Behçet's disease (BD).

METHODS

To measure inflammatory cytokines, ocular fluid samples from BD patients being treated with infliximab were collected. Cluster of differentiation 4 (CD4)+ T cells from BD patients with active uveitis were co-cultured with anti-cluster of differentiation 3/cluster of differentiation 28 (CD3/CD28) antibodies in the presence of infliximab. For the induction of Th17 cells, CD4+ T cells from BD patients were co-cultured with anti-CD3/CD28, anti-interferon-gamma (anti-IFN-γ), anti-interleukin-4 (anti-IL-4), and recombinant proteins such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-23 (IL-23), and TNF-α. The BD T cells were co-cultured with infliximab, and the production of interleukin-17 (IL-17) was evaluated by ELISA and flow cytometry, and the expression of retinoid-acid receptor-related orphan receptor gamma t (RORγt) was also evaluated by flow cytometry. In addition, intraocular cells collected from mice with experimental autoimmune uveitis (EAU) were used for the assay with anti-TNF-α blocking antibody.

RESULTS

Ocular fluids from active uveitis patients who have BD contained significant amounts of inflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-6, and IL-17, while ocular fluids from infliximab patients did not contain any inflammatory cytokines. Activated CD4+ T cells from BD patients produced large amounts of TNF-α and IL-17, whereas T cells in the presence of infliximab failed to produce these cytokines. Polarized Th17 cell lines from BD patients produced large amounts of IL-17, and Th17 cells exposed to infliximab had significantly reduced IL-17 production. Polarized BD Th17 cells expressed large amounts of transcription factor RORγt. In contrast, in vitro-treated infliximab Th17 cells expressed less RORγt. Moreover, intraocular T cells from EAU mice had a high population of IL-17+ cells, and retinal antigen-specific T cells from EAU mice produced large amounts of IL-17 in the presence of retinal peptide. However, the EAU T cells produced less IL-17 if the T cells were treated with anti-TNF-α antibody.

CONCLUSIONS

These results indicate that anti-TNF-α therapy suppresses effector T-cell differentiation in BD patients with uveitis. Thus, suppression of effector T-cell differentiation by anti-TNF-α therapy may provide protection from severe ocular inflammation in BD.

Interferon-gamma inhibits experimental renal fibrosis.

BACKGROUND

Recent evidence has implicated myofibroblasts as a cell type responsible for the laying down of extracellular matrix components during fibrosis in a number of organs. In this study, we examined the capacity of interferon-gamma (IFN-gamma) to inhibit the activation of fibroblasts to the myofibroblastic phenotype and hence reduce the extent of renal scarring in the rat subtotal nephrectomy (SNx) model using a novel method of intrarenal delivery.

METHODS

Rats were divided into four groups: sham, SNx (group 1), SNx + drug vehicle (group 2) and SNx + IFN-gamma (400 units/day; group 3) for 30 days. Rats were sacrificed on days 15, 30, 45, and 90 following SNx.

RESULTS

Clinical data showed a marked reduction in proteinuria in the group treated with IFN-gamma (161 vs. 280 mg/24 hr by day 45, P < 0.01) and a preservation of the creatinine clearance (1.16 vs. 0. 84 ml/min by day 45, P < 0.05) when compared to the SNx or SNx + vehicle groups throughout the time course. Immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) revealed a reduction in myofibroblastic cell types (6.5 +/- 3.1% glomerular alpha-SMA in group 3 compared with 14.8 +/- 4.2% glomerular alpha-SMA in group 2, P < 0.05, 3.8 +/- 1.4% tubulointerstitial alpha-SMA in group 3 compared with 8.8 +/- 2.0% tubulointerstitial alpha-SMA in group 2 on day 45, P < 0.05). There was also a reduction in immunostaining for collagens III and IV in the IFN-gamma-treated group. Scoring for both glomerulosclerosis and tubulointerstitial fibrosis in the IFN-gamma group (group 3) was lower than the other two operated groups.

CONCLUSIONS

We conclude that IFN-gamma, administered at a dose of 400 units/day, has a strong inhibitory effect on myofibroblasts and that as a possible result of this action, renal fibrosis is reduced and renal function is preserved in the rat SNx model. The IFN-gamma renoprotective effect lasted only for the extent of its administration and subsided when discontinued.

Surface lymphotoxin (LT) is a heteromeric complex of LT-<em>alpha</em> and LT-beta chains that binds to the LT-beta receptor (LT-beta-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-beta-R. A soluble form of the surface complex was produced by coexpression of LT-<em>alpha</em> and a converted form of LT-beta wherein the normally type II LT-beta membrane protein was changed to a type I secreted form. Recombinant LT-<em>alpha</em> 1/beta 2 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-<em>3</em> when added with the synergizing agent <em>interferon</em> (IFN) gamma. When immobilized on a plastic surface, anti-LT-beta-R monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-beta-R. Anti-LT-beta-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-beta-R antibody combined with human IFN-gamma arrested tumor growth. The delineation of a biological signaling event mediated by the LT-beta-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-beta-R may have an application in tumor therapy.

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D<em>3</em> [1,25(OH)<em>3</em>D<em>3</em>] and express the vitamin D receptor (VDR). However, 1 <em>alpha</em>-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D<em>3</em> and 24,25-dihydroxyvitamin D<em>3</em> [24,25(OH)2D<em>3</em>] synthesis, specific [<em>3</em>H]-1,25(OH)2D<em>3</em> binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D<em>3</em> into 1,25(OH)2D<em>3</em> (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.<em>3</em> +/- 4.<em>3</em> pmol/10(6) cells/6 h, n = 5). <em>Interferon</em>-gamma (IFN-gamma) increased baseline 1,25(OH)2D<em>3</em>-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D<em>3</em>-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D<em>3</em> did not influence constitutive 1,25(OH)2D<em>3</em> synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D<em>3</em> also stimulated 24,25(OH)2D<em>3</em> synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D<em>3</em> synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D<em>3</em> occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D<em>3</em>. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D<em>3</em>, (2) a loss of 24,25(OH)2D<em>3</em>-synthesizing activity, and (<em>3</em>) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D<em>3</em> was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D<em>3</em>.

Early studies showed that the administration of the anti-inflammatory cytokine interleukin-10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO-related molecular mechanisms. IL10 was over-expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase <em>3</em> compared with wild type mice. Under basal conditions, anti-inflammatory factors such as nerve growth factor and GSH were up-regulated and the pro-inflammatory cytokine IL1beta was down-regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro-inflammatory cytokines, including tumor necrosis factor-<em>alpha</em>, <em>interferon</em>-gamma, and IL1beta. Our data indicate that constitutive IL10 over-expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.

Dengue is a pantropic public health problem. In children, dengue shock syndrome (DSS) is the most common life-threatening complication. The ability to predict which patients may develop DSS may improve triage and treatment. To this end, we conducted a nested case-control comparison of the early host transcriptional features in 24 DSS patients and 56 sex-, age-, and virus serotype-matched uncomplicated (UC) dengue patients. In the first instance, we defined the "early dengue" profile. The transcriptional signature in acute rather than convalescent samples (≤72 h post-illness onset) was defined by an overabundance of <em>interferon</em>-inducible transcripts (<em>3</em>1% of the 551 overabundant transcripts) and canonical gene ontology terms that included the following: response to virus, immune response, innate immune response, and inflammatory response. Pathway and network analyses identified STAT1, STAT2, STAT<em>3</em>, IRF7, IRF9, IRF1, CEBPB, and SP1 as key transcriptional factors mediating the early response. Strikingly, the only difference in the transcriptional signatures of early DSS and UC dengue cases was the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS, a finding supported by higher plasma concentrations of several canonical proteins associated with neutrophil degranulation (bactericidal/permeability-increasing protein [BPI], elastase 2 [ELA2], and defensin 1 <em>alpha</em> [DEF1A]). Elevated levels of neutrophil-associated transcripts were independent of the neutrophil count and also of the genotype of the infecting virus, as genome-length sequences of dengue virus serotype 1 (DENV-1) (n = 15) and DENV-2 (n = <em>3</em>) sampled from DSS patients were phylogenetically indistinguishable from those sampled from uncomplicated dengue patients (<em>3</em>2 DENV-1 and 9 DENV-2 sequences). Collectively, these data suggest a hitherto unrecognized association between neutrophil activation, pathogenesis, and the development of DSS and point to future strategies for guiding prognosis.

Dendritic cells, macrophages, and granulocytes are derived from hematopoietic stem cells and provide a first line of defense against infectious pathogens. Toll-like receptors (TLRs) expressed on these cells recognize molecular stuctures present in the pathogens. Upon binding of a pathogen ligand, TLRs trigger a cascade of signaling pathways that is conserved from insect to plants to humans, which ultimately activates NFkappaB. In mammalian cells, this leads to the induction of cytokine genes and the establishment of innate immunity. For example, TLR signals induce type I <em>interferons</em> (IFN <em>alpha</em>/beta) in dendritic cells conferring an antiviral state upon host cells. Moreover, TLR signals stimulate not only pro-inflammatory cytokines such as IFNs, IL-1, TNF<em>alpha</em>, and IL-12 but also anti-inflammatory cytokines such as IL-10 and IL-6 IL-12 and IL-10 are cytokines that bridge early innate responses and the ensuing specific immune responses. TLR signals also enhance an antigen presentation capacity in dendritic cells and macrophages. Recent studies with mouse and human cells indicate that TLRs activate multiple signaling cascades that involve chromatin structure alterations as well as activation of many transcription factors (e.g., IRF-<em>3</em>, IRF-8/ICSBP, and PU.1). Together, although the basic backbone is conserved throughout evolution, the TLR signaling system in mammalian species has an added complexity to accommodate a mechanism that links innate and adaptive immunity.

Therapy of chronic hepatitis delta with standard <em>interferon</em> therapy has met with limited efficacy. This study was designed to examine the efficacy and safety of peg<em>interferon</em> with or without ribavirin. Thirty-eight serum hepatitis B surface antigen- and HDV RNA-positive patients with alanine aminotransferase (ALT) more than 1.5 times the upper normal limit received peg<em>interferon</em> <em>alpha</em>-2b (1.5 microg/kg) alone as monotherapy (n=16) or in combination with ribavirin (n=22), for 48 weeks. Thereafter, all the patients were maintained on peg<em>interferon</em> for 24 weeks and followed for 24 weeks off therapy. The primary end point studied was the virological and biochemical response at the end of follow-up. HDV RNA was determined by single or nested polymerase chain reaction assays. Twenty-seven patients (71%), 11 receiving monotherapy and 16 receiving the combination treatment, completed the follow-up. At the end of treatment, a virological response was observed in <em>3</em> of the patients treated with peg<em>interferon</em> (19%) and in 2 of the patients treated with combination therapy (9%), and a biochemical response was observed in 6 (<em>3</em>7.5%) and 9 patients (41%), respectively. In nonresponders, ALT diminished from a mean of 174+/-5<em>3</em> to 86+/-41 IU/L. At the end of follow-up, serum HDV RNA was negative in 8 patients (21%), and a biochemical response was detected in 10 patients (26%). Treatment was discontinued in 25% of the patients, and dosing was modified in 58%. In conclusion, a prolonged course of peg<em>interferon</em> <em>alpha</em>-2b resulted in clearance of serum HDV RNA and ALT normalization in a fifth of patients with chronic hepatitis D, while ribavirin had no effect on the viral clearance rate. Overall tolerance of therapy was poor.

BACKGROUND

Owing to the limited efficacy of therapy on melanoma at the stage of distant metastases, a well-tolerated adjuvant therapy is needed for patients with high-risk primary melanoma. Our hypothesis was that an adjuvant treatment with low doses of interferon alpha could be effective in patients with localised melanoma.

METHODS

After resection of a primary cutaneous melanoma thicker than 1.5 mm, patients without clinically detectable node metastases were randomly assigned to receive either 3x10(6) IU interferon alpha-2a, three-times weekly for 18 months, or no treatment. The primary endpoint was the relapse-free interval.

RESULTS

499 patients were enrolled, of whom 489 were eligible. When used as part of a sequential procedure, interferon alpha-2a was of significant benefit for relapse-free interval (p=0.038). A long-term analysis, after a median follow-up of 5 years, showed a significant extension of relapse-free interval (p=0.035) and a clear trend towards an increase in overall survival (p=0.059) in interferon alpha-2a-treated patients compared with controls. There were 100 relapses and 59 deaths among the 244 interferon alpha-2a-treated patients compared with 119 relapses and 76 deaths among the 245 controls. The estimated 3-year-relapse rates were 32% in the interferon alpha-2a group and 44% in controls; the 3-year death rates were 15% and 21%, respectively. Only 10% of patients experienced WHO grade 3 or 4 adverse events. Treatment was compatible with normal daily life.

CONCLUSIONS

Adjuvant therapy of high-risk melanoma with low doses of interferon alpha-2a for 18 months is safe and is beneficial when started before clinically detectable node metastases develop.

In order to identify novel proteins produced by activated macrophages, a cDNA library was made from cultures of the mouse macrophage-like cell line RAW 264.7 that had been treated with conditioned medium from mitogen-stimulated spleen cells, and the library was screened by differential plaque hybridization. A cDNA clone was isolated that detected a 1.4-kilobase mRNA that accumulated dramatically in response to the spleen cell conditioned medium. The 1.4-kilobase mRNA encodes a predicted protein of 98 amino acids, designated CRG-2, molecular weight (Mr) 10,781, with a 21-residue signal peptide. The amino acid sequence indicates that CRG-2 is a member of the platelet factor 4 family (PF4) of cytokines. The CRG-2 mRNA was induced by <em>alpha</em>-, beta-, and gamma-<em>interferons</em> (IFNs) and by lipopolysaccharide. In response to IFN-gamma, the CRG-2 mRNA level reached a peak between <em>3</em> and 6 h. The accumulation of CRG-2 mRNA was not blocked by cycloheximide. Among the known members of the PF4 family, CRG-2 is most closely related to the <em>interferon</em>-inducible human protein IP-10. The 5'-flanking region of the crg-2 gene was isolated, and comparisons between crg-2 and IP-10 genes, mRNAs, and proteins reveal conserved features of possible functional importance. CRG-2 may play a role in host defense, particularly in the response to viral infection.

The response rate to <em>interferon</em> alfa (IFN-<em>alpha</em>) in patients infected with hepatitis C virus (HCV) genotype 1 isolates is poor. A region associated with sensitivity to IFN has been identified in subtype HCV-1b isolates from Japanese patients in the carboxyterminal half of the nonstructural protein NS5A (between codon 2209 and 2248). HCV-1b isolates with at least four amino acid changes in this region compared with the HCV-1b prototype sequence were sensitive, whereas isolates identical to the prototype sequence were resistant to IFN-<em>alpha</em>. Patients infected with HCV-1b isolates carrying 1 to <em>3</em> mutations in NS5A(2209-2248) showed an intermediate response pattern. Because of the large geographical differences observed for HCV it is unknown whether this putative IFN-<em>alpha</em> sensitivity determining region is also predictive for European isolates. We analyzed <em>3</em>2 patients chronically infected with HCV-1a or HCV-1b isolates who were treated with <em>3</em> million units of recombinant IFN-<em>alpha</em> three times per week for 1 year. Before initiation, during, and after treatment serum HCV-RNA levels were assessed by a quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. The amino acid sequence of NS5A(2209-2248)was determined by direct sequencing of the PCR-amplified HCV genome and was compared with the reference sequence HCV-J. In patients chronically infected with subtype HCV-1a or HCV-1b the initial or sustained response to IFN-<em>alpha</em> was not related to the number of amino acid substitutions in the NS5A(2209-2248) region. In addition, the number of amino acid changes in NS5A(2209-2248) was not related to pretreatment HCV-RNA serum levels. In three patients with a pronounced initial decline of HCV-RNA levels >><em>3</em> log) sequence analyses of NS5A(2209-2248) were performed before and after therapy. Compared with the pretreatment amino acid sequence the HCV isolates of these patients revealed more mutations in the NS5A(2209-2248) region after therapy. These findings from European patients indicate that the NS5A(2209-2248) region of HCV does not represent a common <em>interferon</em> sensitivity determining region.

The promoter region from the cloned glyceraldehyde-<em>3</em>-phosphate dehydrogenase (GPD) gene of Saccharomyces cerevisiae (Musti et al., 198<em>3</em>) has been characterized. A 65<em>3</em>-bp TaqI restriction fragment with a <em>3</em>' border 24 bp upstream from the ATG initiation codon was isolated and demonstrated to contain all sequences necessary for promoter function in vivo. This DNA segment was converted to a portable promoter by cloning it into M1<em>3</em>mp9, and the entire nucleotide sequence of the portable promoter was determined. Two generalized yeast expression vectors have been constructed utilizing the GPD portable promoter. The expression vectors include the yeast 2 mu origin of replication and amplification functions, such that the plasmids are maintained at high copy number in ciro yeast hosts. These vectors direct synthesis of a consensus <em>alpha</em>-<em>interferon</em> (IFN-<em>alpha</em> Con1) as 1% of total cell protein. Hepatitis B surface antigen (HBsAg) was also expressed from these vectors. The 5' end of the HBsAg gene was replaced with a synthetic DNA segment which restored the deleted GPD untranslated leader and utilized optimal yeast codons for the first <em>3</em>0 amino acids. The partially synthetic gene resulted in a 10- to 15-fold increased expression level from GPD vectors yielding HBsAg polypeptide as 2-4% of total cell protein.

The E <em>alpha</em> MHC class II gene with 1.4 kb of 5'-flanking and 0.5 kb of <em>3</em>'-flanking sequences was introduced into (H-2b X s)F2 mice, which do not express their endogenous E <em>alpha</em> gene. The transgene was expressed in thymic tissue and in adherent spleen cells and was induced in peritoneal exudate cells by gamma-<em>interferon</em>. In contrast to the normal E <em>alpha</em> gene, there was no expression in B lymphocytes. Since transgenic animals made with constructs containing <em>3</em>.2 kb and 2 kb of 5'-flanking sequences show normal expression pattern of the E <em>alpha</em> gene, it appears that deletion of 5'-flanking sequences between -1.4 kb and -2 kb inactivated or eliminated regulatory sequences required for expression of E <em>alpha</em> specifically in B cells. The presence of pBR<em>3</em>27 DNA linked to the -1.4 kb E <em>alpha</em> transgene suppresses expression in peripheral adherent cells, yielding mice expressing E <em>alpha</em> only in the thymus. These mice appear to be tolerant to I-E, as measured in mixed leukocyte response experiments.

Toll-like receptors and the beta-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88-/-) mice. Early (<48 h) after infection, MyD88-/- mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88-/- mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88-/- mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88-/- mouse lung tissues, MyD88-/- cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af29<em>3</em>); histologically, MyD88-/- mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88-/- lung homogenates contained comparatively decreased amounts of interleukin-1beta (IL-1beta), IL-6, KC, and gamma <em>interferon</em> and paradoxically increased amounts of tumor necrosis factor <em>alpha</em> and macrophage inflammatory protein 1<em>alpha</em>. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a <em>3</em>-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.

C 57 BL/6 mice developed resistance to lethal intravenous challenge with virulent (Moscow strain) ectromelia virus between 2 and <em>3</em> weeks of age. The fraction of C57 BL/6 mice in which virus was detected in spleen was significantly lower than for DBA/2 mice by day <em>3</em>. Thereafter, C 57 BL/6 mice had significantly reduced virus titers in spleen compared with those of DBA/2 mice. Resistance was abrogated by treatment with anti-asialo GM1 gammaglobulin, which blocks NK cell activity, or with anti-<em>interferon</em> (IFN) <em>alpha</em>, beta. C 57 BL/6 mice carrying the bg/bg mutation, associated with a deficiency of NK cells, were highly susceptible to lethal infection as were athymic mice derived from a resistant genetic background. Virus titers in spleens of C 57 BL/6 mice treated with anti-asialo GM1 or anti-IFN <em>alpha</em>, beta were significantly higher 4 days after virus challenge than were titers in C 57 BL/6 mice treated with normal rabbit serum. The results strongly suggest that genetic resistance to lethal ectromelia virus infection requires non-specific host defenses such as NK cells and IFN <em>alpha</em>, beta that are activated during the first <em>3</em> to 4 days of infection.

Cytokine gene transcription has been analyzed by direct analysis of RNA obtained from mouse heterotopic cardiac transplants. The level of expression of the cytokine genes was assessed using semiquantitative polymerase chain reaction (PCR). Expression of the cytokines investigated fell into three groups. The first group included interleukin 1 beta (IL-1 beta), IL-5, IL-6, and <em>interferon</em> gamma (IFN gamma). These genes were expressed in normal heart tissue at low level and were upregulated following both syngeneic and allogeneic transplantation. Genes in the second group (IL-1 <em>alpha</em>, IL-<em>3</em>) were not expressed at detectable levels in normal heart but were induced following either syngeneic or allogeneic heart grafting. IL-2, IL-4, and tumor necrosis factor beta (IFN beta) comprised the third group and these cytokines were expressed only in allogeneic grafts after transplantation.

We have investigated the effects of the two prominent inflammatory cytokines, <em>interferon</em>-gamma (IFN-gamma) and tumour necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum-free medium and exposed to IFN-gamma (1-100 U/ml, TNF-<em>alpha</em> (25-100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day <em>3</em> or from day <em>3</em> to day 6. While cell survival was not affected in any of the conditions tested, IFN-gamma dose-dependently inhibited [<em>3</em>H]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt <em>3</em>-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; by up to <em>3</em><em>3</em>%). TNF-<em>alpha</em> synergized with IFN-gamma, but was ineffective by itself. Moreover, IFN-gamma totally antagonized the induction by basic fibroblast growth factor and platelet-derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN-gamma also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN-gamma was potentiated by TNF-<em>alpha</em>, which was ineffective when tested alone. The inhibitory activity of IFN-gamma was rapidly reversible: <em>3</em> days after removal of the cytokine, administered from day 1 to day <em>3</em>, complete recovery of cll proliferation and differentiation could be documented. The cytokine-induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription-polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days <em>3</em> to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN-gamma, and further depressed in cultures treated with IFN-gamma and TNF-<em>alpha</em>, while TNF-<em>alpha</em> alone was ineffective. The above observations may help explain the role of IFN-gamma and TNF-<em>alpha</em> in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing-remitting course of the disease.

A tumor-specific, bcr-abl-derived fusion peptide vaccine can be safely administered to patients with chronic myelogenous leukemia (CML) and can elicit a bcr-abl peptide-specific T-cell immune response. In the present phase 2 trial, 14 patients with CML in chronic phase were vaccinated with 6 fusion peptides mixed with Quillaja saponaria (QS-21). No significant toxic effects were observed. In 14 of 14 patients, delayed-type hypersensitivity (DTH) and/or CD4 proliferative responses developed after beginning vaccinations, and 11 of 14 patients showed <em>interferon</em>-gamma (IFN-gamma) release by CD4 enzyme-linked immunospot (ELISPOT) at one or more time points. These responses were CD4(+)CD45RO(+). A peptide-specific CD8(+) <em>interferon</em>-gamma ELISPOT was found in 4 patients. Four patients in hematologic remission had a decrease in Philadelphia chromosome (Ph) percentage (<em>3</em> concurrently receiving <em>interferon</em>-<em>alpha</em> and 1 on imatinib mesylate), and <em>3</em> patients in molecular relapse after allogenic transplantation became transiently polymerase chain reaction (PCR) negative after vaccination; 2 of these patients received concurrent donor lymphocyte infusion (DLI). All 5 patients on IFN-<em>alpha</em> ultimately reached a complete cytogenetic remission. In conclusion, a tumor-specific bcr-abl breakpoint peptide-derived vaccine can be safely administered and can reliably elicit measurable peptide-specific CD4 immune responses, including in patients after bone marrow transplantation, on <em>interferon</em>, or on imatinib mesylate. A relationship between the clinical responses and vaccination cannot be determined from this trial.

Partially purified human leukocyte (<em>alpha</em>) <em>interferon</em> was administered i.m. at a dose of <em>3</em> x 10(6) units/day to 19 patients with metastatic renal cell carcinoma. Five patients (26%) showed partial responses; two patients (10.5%), objective minor responses; three patients (16%), mixed effects (evidence of biological effect with regression of some lesions but concomitant progression); two patients (10.5%), disease stabilization; and seven patients (<em>3</em>7%), progressive disease. All tumor responses were seen in lung or mediastinal metastases. Tumor response significantly correlated with <em>interferon</em>-induced leukopenia and granulocytopenia and with pretreatment performance status. Antibodies to <em>interferon</em> were found in one patient prior to treatment. We concluded that <em>interferon</em> is a potential active antitumor agent in patients with renal cell carcinoma.

Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, <em>3</em>'-azido-<em>3</em>'-deoxythymidine), didanosine (ddI, 2',<em>3</em>'-dideoxyinosine), and recombinant <em>interferon</em>-<em>alpha</em> A (rIFN-<em>alpha</em> A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-<em>alpha</em> A in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.