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Type 1 interferon (IFN) is a major contributor to the pathogenesis of systemic lupus erythematosus (SLE). A landmark study by Caielli et al. now shows that erythrocytes from lupus patients that fail to switch from glycolysis to oxidative phosphorylation during differentiation retain their mitochondria. These mitochondria-containing erythrocytes represent a novel source of IFN when phagocytosed by macrophages., Competing Interests: Declaration of interests The author declares no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Nanoparticle-cell-nanoparticle communication by stigmergy was demonstrated using two capped nanodevices. The first community of nanoparticles (i.e.S(RA)IFN) is loaded with 9-cis-retinoic acid and capped with interferon-γ, whereas the second community of nanoparticles (i.e.S(sulf)PIC) is loaded with sulforhodamine B and capped with poly(I:C). The uptake of S(RA)IFN by SK-BR-3 breast cancer cells enhanced the expression of TLR3 receptor facilitating the subsequent uptake of S(sulf)PIC and cell killing.

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS -/- ), and STING-deficient (STING gt/gt ) mice to warm liver I/R injury and that found cGAS -/- mice had significantly increased liver injury compared with WT or STING gt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS -/- mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS -/- hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

Normally, quiescent hematopoietic stem cells (HSC) rapidly enter the cell cycle following exposure to inflammatory stimuli. The analysis of HSC cell cycle activity in murine bone marrow during inflammation is often complicated by the relative rarity of HSCs and shifts in Sca-1, a key cell surface marker used to identify HSCs. Here, we report a method to analyze HSC proliferation and cell cycle distribution under inflammatory conditions. Our approach uses EdU incorporation and Ki67 staining coupled with DNA content quantification by DAPI. We also incorporate the surface marker ESAM to help minimize the potential for contaminating events that may confound analysis in the HSC compartment.

Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC., Competing Interests: The authors have declared that no competing interests exist.

The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals.

Background: Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains., Results: Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection., Conclusions: Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken.

Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.

Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10-carboxymethyl-9-acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell-intrinsic type I IFN response, depending on the ER-resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C-terminal ligand-binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate (c-diGMP)-binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c-diGMP. Altogether, these results provide novel insight into ligand-sensing properties of STING and, furthermore, unravel unexpected species-specific differences of this innate sensor.

Objective: Myasthenia gravis (MG) is an autoimmune disease mediated mainly by anti-acetylcholine receptor (AChR) antibodies. The thymus plays a primary role in MG pathogenesis. As we recently showed an inflammatory and antiviral signature in MG thymuses, we investigated whether pathogen-sensing molecules could contribute to an anti-AChR response., Methods: We studied the effects of toll-like receptor agonists on the expression of α-AChR and various tissue-specific antigens (TSAs) in human thymic epithelial cell (TEC) cultures. As polyinosinic-polycytidylic acid (poly[I:C]), which mimics double-stranded RNA (dsRNA), stimulated specifically α-AChR expression, the signaling pathways involved were investigated. In parallel, we analyzed the expression of dsRNA-signaling components in the thymus of MG patients, and the relevance of our data was investigated in vivo in poly(I:C)-injected mice., Results: We demonstrate that dsRNA signaling induced by poly(I:C) specifically triggers the overexpression of α-AChR in TECs and not of other TSAs. A poly(I:C) effect was also observed on MG TECs. This induction is mediated through toll-like receptor 3 (TLR3) and protein kinase R (PKR), and by the release of interferon (IFN)-β. In parallel, human MG thymuses also display an overexpression of TLR3, PKR, and IFN-β. In addition, poly(I:C) injections specifically increase thymic expression of α-AChR in wild-type mice, but not in IFN-I receptor knockout mice. These injections also lead to an anti-AChR autoimmune response characterized by a significant production of serum anti-AChR antibodies and a specific proliferation of B cells., Interpretation: Because anti-AChR antibodies are highly specific for MG and are pathogenic, dsRNA-signaling activation could contribute to the etiology of MG., (Copyright © 2012 American Neurological Association.)

We have previously demonstrated that IL-27 is a novel anti-HIV cytokine, which inhibits HIV replication in CD4 T cells and macrophages as interferon (IFN)-α does. To further understand the mechanism of the antiviral effect, we performed Affymetrix DNA microarray and gene functional annotation analysis using DAVID (the Database for Annotation, Visualization, and Integrated Discovery). DAVID is a web-based bioinformatics application that systematically identifies enriched biology associated with large gene list(s) derived from high-throughput genomic experiments, such as microarray. The enriched annotation terms identified by DAVID will give important insights into understanding the biological themes under study. Having used the DAVID bioinformatics tools, we have shown that IL-27 differentially regulates the gene expression between T cells and macrophages. IL-27 significantly induces IFN-inducible genes including antiviral genes in macrophages as does IFN-α, suggesting that IL-27 inhibits HIV replication in macrophages via a mechanism similar to that of IFN-α.

Background: Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response., Objectives: We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro., Methods: 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy., Results: PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, β-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction., Conclusions: PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5(+) IgM(+) WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-kappaB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed., (2009 Elsevier Inc. All rights reserved.)

The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

RNA virus replication results in expression of double-stranded RNA (ds-RNA) molecules that trigger innate immune responses through interactions with both intracellular and extracellular receptors. We investigated the contributions of the extracellular and intracellular pathways to innate immunity in murine astrocyte primary cultures using polyinosinic-polycytidylic acid (poly I:C), a synthetic ds-RNA molecule designed to mimic RNA virus infection. Whereas extracellular poly I:C (naked poly I:C) mainly induced the expression of regulated on activation normal T-cell expressed and secreted (RANTES), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-alpha), intracellular delivery of poly I:C (complexed poly I:C) chiefly induced expression of IFN-beta and IL-6. Experiments with astrocytes from Toll-like receptor 3 (TLR-3) knockout mice indicated that naked poly I:C signals via a TLR-3-dependent NF-kappaB pathway. Complexed poly I:C induced the expression of the intracellular ds-RNA sensor proteins, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA-5). However, transfection of astrocytes with dominant negative forms of the helicases implicated MDA-5, but not RIG-I, as the intracellular sensor of poly I:C. Complexed poly I:C-mediated MDA-5 stimulation transmitted "downstream" signals, resulting in activation of the transcription factors NF-kappaB and IRF-3. Our results illustrate the intricacy of extracellular and intracellular ds-RNA recognition in viral infections of the central nervous system and indicate the importance of MDA-5 helicase as an intracellular ds-RNA sensor in astrocytes.

The induction of type I interferon (IFN) and the development of the innate antiviral response are mediated by the activation of interferon regulatory factor (IRF)-3 and IRF-7 under the control of the non-canonical kinases TBK-1 and IKKepsilon. The initial sensing of infection by RNA viruses is mediated by the cytoplasmic, retinoic acid inducible gene I (RIG-I), via a Toll-like receptor (TLR) independent signaling pathway. In the present study, we identify key residues involved in IRF-3 and IRF-7 phosphorylation using TAP-tag purification of TBK-1 and IKKepsilon proteins. Based on the identification of an extended sequence motif--SxSxxxS--common to both IRF-3 and IRF-7, an IRF-7 pSer477/479 phosphospecific antibody was generated. Virus infection, TBK-1/IKKepsilon expression or co-expression of different signaling adaptors such as RIG-I, MAVS and TRIF, all stimulated pSer477/479 phosphorylation. Furthermore, the newly identified adaptor of the RIG-I pathway (MAVS/IPS-1/VISA/Cardif) was able to induce IRF and NF-kappaB dependent promoter activity as efficiently as the constitutively active form of RIG-I (DeltaRIG-I). Co-expression of the NS3/4A protease activity of hepatitis C virus however blocked MAVS-mediated gene activation in a dose dependent manner. These studies link RIG-I sensing of viral RNA to downstream kinase signaling and phosphorylation of IRF-3 and IRF-7 via the MAVS/IPS/VISA/Cardif adaptor.

TLR3 recognizes viral dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). TLR3 expression is commonly considered to be restricted to dendritic cells, NK cells, and fibroblasts. In this study we report that human gammadelta and alphabeta T lymphocytes also express TLR3, as shown by quantitative real-time PCR, flow cytometry, and confocal microscopy. Although T cells did not respond directly to poly(I:C), we observed a dramatic increase in IFN-gamma secretion and an up-regulation of CD69 when freshly isolated gammadelta T cells were stimulated via TCR in the presence of poly(I:C) without APC. IFN-gamma secretion was partially inhibited by anti-TLR3 Abs. In contrast, poly(I:C) did not costimulate IFN-gamma secretion by alphabeta T cells. These results indicate that TLR3 signaling is differentially regulated in TCR-stimulated gammadelta and alphabeta T cells, suggesting an early activation of gammadelta T cells in antiviral immunity.

Type I interferons are central mediators for antiviral responses. Using high-throughput functional screening of interferon inducers, we have identified here a molecule we call interferon-beta promoter stimulator 1 (IPS-1). Overexpression of IPS-1 induced type I interferon and interferon-inducible genes through activation of IRF3, IRF7 and NF-kappaB transcription factors. TBK1 and IKKi protein kinases were required for the IPS-1-mediated interferon induction. IPS-1 contained an N-terminal CARD-like structure that mediated interaction with the CARD of RIG-I and Mda5, which are cytoplasmic RNA helicases that sense viral infection. 'Knockdown' of IPS-1 by small interfering RNA blocked interferon induction by virus infection. Thus, IPS-1 is an adaptor involved in RIG-I- and Mda5-mediated antiviral immune responses.

Germinal center kinase (GCK), a member of the Ste20 family, selectively activates the Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases. Here, we show that endogenous GCK is activated by polyinosine-polycytidine [poly(IC)] and lipopolysaccharides (LPS), lipid A, interleukin-1 (IL-1), and engagement of CD40, all agonists that require TRAF6 for JNK activation. RNA interference experiments indicate that GCK is required for the maximal activation of JNK by LPS, lipid A, poly(IC), and, to a lesser extent, IL-1 and engagement of CD40. GCK is ubiquitinated in situ and stabilized by inhibitors of the proteasome, indicating that GCK is subject to proteasomal turnover. GCK is constitutively active, and the kinase activity of GCK is required for GCK ubiquitination. Agonist activation of GCK involves the TRAF6-dependent transient stabilization of the GCK polypeptide rather than an increase in intrinsic kinase activity. Our results identify a physiologic function and unexpected mode of regulation for GCK.

Double-stranded RNA (dsRNA) is a viral product recognized by Toll-like receptor 3 (TLR3), and it is a potent activator of dendritic cells (DC). We compared Langerhans cells (LC) and splenic CD11c(+) DC and investigated the responsiveness to dsRNA. We prepared highly purified LC (> 95%) using the panning method. TLR3 mRNA was expressed in LC, splenic DC, and keratinocytes (KC). The expression of IFN-beta mRNA was enhanced in LC and splenic DC by Poly(I:C) stimulation. However, cytokine/chemokine production in response to Poly(I:C) by LC was much lower than that by splenic DC. In addition, Poly(I:C) induced further maturation in splenic DC, but not in LC. Finally, we found that the mouse KC cell line, PAM212, produced a great amount of IL-1alpha by Poly(I:C) stimulation, and that IL-1alpha promoted the maturation of LC. These data altogether indicate that LC exhibit low responsiveness to dsRNA. It is possible that KC may primarily trigger anti-viral immune responses in the skin via cytokine production such as IL-1alpha.

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-beta in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use.

Cytokine mRNA expression was studied in human long-term cell cultures of different origin: J-96 and J-41 (monocytic leukemia), SW-13 (paradrenal adenocarcinoma), and MT-4 (T-cell leukemia), in response to IFN-alpha and IFN inducers (kagocel and cycloferon). Cytokine mRNA level in the cell cultures was measured by the RT-PCR method using 11 primer pairs for the following cytokines: IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNF-alpha. It was shown that IFN-alpha and IFN inducers possess an ability to regulate different cytokine mRNAs. Treatment of the cells with IFN-alpha resulted in expression of mRNAs for IL-2, IL-4, and IL-8. Kagocel induced production of IFN-alpha, IFN-gamma, and IL-2 mRNAs, and cycloferon--IFN-gamma, IL-2, IL-4, and IL-8 mRNAs. It is suggested that antiviral effects of these inducers, in general, can be attributed to imitation of cytokine responses observed in viral infection and, as a result, can lead to starting-up of cellular defense antiviral mechanisms even before action of viruses. Conclusion is made that IFN and IFN inducers may act as regulators of cytokine activity.

Antiviral and interferon inducing activity of the amixine and its some derivatives, as well as their influence on the proteolytic enzymes activity, monooxygenase activity of the microsomal fraction, level of the lipids peroxidation were studied. Lack of correlation between antiviral and interferon inducing activity in the investigated series of compounds was found. Vice versa, the good correlation between interferon inducing activity and the elastase-like activities inhibition ability of the compounds was observed. It allows to state the assumption, that only one ability of compounds to induce of an interferon doesn't suffice for obtaining of high titres of interferon, and while their rather high antiproteolitic activity is necessary. It's shown, that except for one compound the influence of amixine and its derivatives on the red-ox enzymes activity well correlates with their ability to the interferon-inducing. All presented above allows to attribute amixine and its derivatives to polymodal antiviral agents.

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M(r) was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Both noncytopathic (ncp) and cytopathic (cp) biotypes of BVDV can be isolated from persistently infected cattle suffering from the lethal mucosal disease. The cp biotype correlates with the production of the NS3 nonstructural protein, which in the corresponding ncp biotype is present in its uncleaved form, NS23. Previously, we have shown that cp but not ncp BVDV induces the formation of alpha/beta interferons in bovine macrophages. In this study, we demonstrate that ncp BVDV inhibits the induction of apoptosis and the expression of interferon alpha/beta by poly(IC), a synthetic double-stranded RNA (dsRNA). Inhibition was observed only in cells which had been infected with ncp BVDV at least 12 h prior to the addition of dsRNA, which indicates that expression of viral proteins is necessary for the ncp virus to inhibit the effects of poly(IC). Additional experiments using transfected poly(IC) showed that ncp BVDV interfered with the intracellular action of dsRNA rather than with its uptake into the cells. Infected cells were not resistant to induction of apoptosis by actinomycin D or staurosporine, which suggests that ncp BVDV may specifically interfere with signaling through dsRNA. Interference with the innate antiviral host responses may explain the successful establishment of persistent infection by ncp BVDV in fetuses early in their development.

Champedak (Artocarpus integer) lectin-M is a lectin with high specificity and affinity for the core-mannosyl residues of the N-linked oligosaccharides of glycoproteins. We have studied the interaction of the champedak seed lectin with human serum glycoproteins that were resolved by 2-dimensional (2-D) gel electrophoresis. The lectin demonstrated strong interaction with haptoglobin beta chain, orosomucoid, alpha1-antitrypsin, alpha2-HS glycoprotein, transferrin, hemopexin, alpha1B-glycoprotein, and the heavy chains of IgA, IgM and IgG of the human serum. With exceptions of the heavy chains of the immunoglobulins and alpha1B-glycoprotein, all the other lectin-M-probed glycopeptides are acute-phase proteins. The use of champedak lectin-M to probe for serum glycoproteins that were separated in a 2-D gel electrophoresis and Western blotting technique may be conveniently applied to analyse the acute-phase and humoral immune responses simultaneously. Subjecting human serum to immobilised-lectin-M affinity chromatography was able to isolate intact haptoglobin, alpha1-antitrypsin, alpha1B-glycoprotein, hemopexin and IgA.

Despite the wide usage of immunomodulating preparates including interferon inducers in medical practice little is known about their mechanism of action. We investigated some theoretical aspects of action of potent interferon inducer--cycloferon (10-carboxymethyl-9-acridanone), such as intracellular localization, ability to DNA binding and cytokine expression stimulation. This preparate has been found to be localized in nuclei of monocytic cells U-937, T- and B-lymphocytes and HeLa cells. In Hep-2 line cycloferon was bound to cells from non-adhesive subpopulation and was not detected in cells of monolayer. Human fibroblasts did not bind the substance. Interaction with double-stranded but not with single-stranded DNA occurred at pH lower than 4.7 regardless of the GC-contain. As shown by dot-hybridization cycloferon stimulated the transcription of interferon-alpha gene in U-937 cells 29-44-fold compared to the control but did not affect the transcription of tumor necrosis factor and interleukin-2 genes. Our data allow to propose that some specific receptor exists in cell with affinity to cycloferon.

Nmi was initially identified through a yeast two-hybrid interaction with N-Myc but it also interacts with c-Myc, Max, Fos, and several other transcription factors, including signal transducer and activator of transcription (Stat) proteins. Nmi is an interferon (IFN)-inducible protein with 25% amino acid identity to the IFN-inducible protein IFP 35. We have found that this homology consists of a novel domain of approximately 90-92 amino acids (aa) that is repeated in tandem in each protein. This region, termed Nmi/IFP 35 domain (NID), is important for subcellular localization of Nmi. Full-length Nmi protein or deletion constructs containing a single NID are localized to the cytoplasm, but amino-terminal Nmi fragments of up to 92 aa containing neither NID are nuclear. Fusion of the amino-terminal end of Nmi to pyruvate kinase, an exclusively cytoplasmic protein, results in a cytoplasmic fusion protein, suggesting that the amino-terminal end of Nmi does not contain a classic nuclear localization signal (NLS). Fusion of the amino-terminal end of Nmi to green fluorescent protein (GFP), which is normally found in both nuclear and cytoplasmic compartments, does not alter GFP distribution, whereas fusion of a single NID to GFP targets the fusion to the cytoplasm. Fusion of a nuclear localization signal (NLS) to full-length Nmi or NID repeats targets the hybrid to the nucleus, suggesting that a strong NLS is dominant to the cytoplasmic localization function of NID. NID may mediate cytoplasmic localization of the full-length Nmi protein through NID-NID protein interactions as demonstrated by yeast two-hybrid assay, immunoprecipitation, and the presence of Nmi in a high molecular weight protein complex. These results suggest that Nmi is composed of a modular structure with an amino-terminal domain that when separated from the rest of the protein is nuclear. The carboxy-terminal two thirds of the protein is composed of two NID that mediate cytoplasmic localization of the full-length protein.

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed., (Copyright 1999 Academic Press.)

Initially described in 1989 as interferon-gamma (IFN-gamma) inducing factor (IGIF), interleukin-18 (IL-18) is a novel pro-inflammatory cytokine that is clearly more than an inducer of IFN-gamma. The cytokine possesses several biological properties such as activation of nuclear factor-kappaB (NF-kappaB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL-1 receptor-activating kinase (IRAK), leading to translocation of NF-kappaB. This property and others support the concept that IL-18 is related to the IL-1 family. Indeed, one of the IL-18 receptor chains is the IL-1 receptor-related protein, a member of the IL-1R family. In addition, IL-18 is structurally similar to IL-1beta and like IL-1beta is first synthesized as a leaderless precursor requiring the IL-1beta converting enzyme for cleavage into an active molecule. The biology of IL-18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented.

We used differential display analysis to identify mRNAs that accumulate to enhanced levels in human cytomegalovirus-infected cells as compared with mock-infected cells. RNAs were compared at 8 hr after infection of primary human fibroblasts. Fifty-seven partial cDNA clones were isolated, representing about 26 differentially expressed mRNAs. Eleven of the mRNAs were virus-coded, and 15 were of cellular origin. Six of the partial cDNA sequences have not been reported previously. All of the cellular mRNAs identified in the screen are induced by interferon alpha. The induction in virus-infected cells, however, does not involve the action of interferon or other small signaling molecules. Neutralizing antibodies that block virus infection also block the induction. These RNAs accumulate after infection with virus that has been inactivated by treatment with UV light, indicating that the inducer is present in virions. We conclude that human cytomegalovirus induces interferon-responsive mRNAs.

Mycobacterium avium is an intracellular microorganism that infects and multiplies within macrophages. Cell-mediated immunity plays an important role in host defense. Two cytokines, interleukin 12 (IL-12) and interferon-gamma-inducing factor (IGIF), produced mainly by macrophages, are critical for the development of type 1 helper T (Th1) cell/interferon-gamma (IFN-gamma) responses and cell-mediated immunity. In this report, by using a mouse model of disseminated M. avium infection, we demonstrate that genetically susceptible BALB/c mice show decreased expression of IL-12 and IGIF in association with a diminished IFN-gamma/Th1 response. Conversely, resistant DBA/2 mice exhibited increased expression of IL-12, IGIF, and IFN-gamma. In both strains of infected mice, the level of IL-4/Th2 response was similar. These results suggest that decreased expression of IL-12 and IGIF leads to a diminished Th1 response without reciprocal enhanced Th2 responses in susceptible mice.

Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

Interleukin-12 (IL-12), known to be a strong inducer of interferon-gamma (IFN-gamma), plays a vital role in activating the immune surveillance system against intracellular pathogens and malignant tumors. The authors have found that cancer patients showing marked IFN-gamma induction after inoculation with BCG-CWS (the cell wall skeleton from Bacille Calmette-Guérin) have a good prognosis. The present study was undertaken to determine whether the level of IL-12 is increased prior to, or along with, IFN-gamma induction in the serum of patients inoculated with BCG-CWS. Unexpectedly, we found no detectable amount of IL-12 in the serum throughout the entire time course. This suggests that a novel IFN-gamma inducing factor (IGIF) or another unknown IFN-gamma inducer may be working in place of IL-12 in the BCG-CWS system.

Protein B had a much higher affinity for human IgA than Jacalin, increasing the sensitivity and specificity of the measurement of total human IgA. Protein B, used as a capturing agent, greatly enhanced the measurement of antigen-specific IgA as compared to alpha chain-specific antibodies.

Distinguishing reactive mesothelial cells from adenocarcinoma cells in serous effusions on the basis of morphological criteria alone is often difficult. Interest has therefore been focused on identifying reliable methods to supplement the conventional cytological techniques. Plant lectins have been reported as diagnostic markers for malignant cells. We studied 51 aspirated samples of benign and malignant effusions using horseradish-peroxidase-conjugated jackfruit lectin. No significant difference was observed between the cells of pleural and peritoneal fluids. The reactively proliferated mesothelial cells of benign effusions showed a predominance of mild staining while moderate and intense staining was predominant in malignant effusions. Intense and irregular lectin binding was observed in macrophages irrespective of the cause of effusion. The lectin staining method therefore appears to have some clinical significance as an additional diagnostic aid for use in effusion cytology.

Binding capacity of serum IgA to jacalin in 22 patients with IgA nephropathy, 14 patients with diffuse mesangial proliferative glomerulonephritis (non-IgA nephropathy) and 20 age-matched healthy adults was examined by enzyme-linked immunoassay (ELISA) using jacalin-coated microplates. In contrast to previous findings, the binding capacity of serum IgA to jacalin in patients with IgA nephropathy measured by ELISA using jacalin-coated microplates was significantly higher than that in healthy adults. The ratio of serum IgA levels measured by this method to those obtained by single radial immunodiffusion was significantly increased in patients with IgA nephropathy. It appeared that the capacity of serum IgA binding to jacalin was marked in these patients. It is concluded that the binding capacity of serum IgA to jacalin is not ubiquitously impaired in all patients with IgA nephropathy.

We report the purification and characterization of a unique DNA-binding protein termed TH3 that interacts with the positive regulatory domain (PRD) I and PRDIII domains of the interferon (IFN) beta promoter. In cells treated with poly rl:rC and cycloheximide, appearance of TH3 DNA-binding activity was inversely proportional to the disappearance of a constitutive complex TH1 and coincided temporally with induction of IFN-beta gene transcription. The TH3 DNA-binding protein is a small 14-kDa polypeptide that appears to be derived from the TH1 complex; TH1 in turn is related to interferon regulatory factor (IRF) 2 by immunological cross-reactivity. The TH3 protein appeared to lack the epitope required for recognition by anti-IRF-2 antisera; however, a short microsequence obtained for TH3 overlapped a sequence from the IRF-2 protein. Although TH3 binds to multimers of the AAGTGA hexamer and to PRDI, the TH3 protein alone had a predominantly neutral phenotype on PRDI-dependent transcription in vitro and lacked the negative transcriptional effect attributed to IRF-2. These results raise the possibility that specific proteolysis of a negative regulatory protein involved in silencing the IFN-beta promoter may be an important event leading to transcriptional activation of the interferon gene.

Jacalin [Artocarpus integrifolia (jack fruit) agglutinin] is made up of two types of chains, heavy and light, with M(r) values of 16,200 +/- 1200 and 2090 +/- 300 respectively (on the basis of gel-permeation chromatography under denaturing conditions). Its complete amino acid sequence was determined by manual degradation using a 4-dimethylaminoazobenzene 4'-isothiocyanate double-coupling method. Peptide fragments for sequence analysis were obtained by chemical cleavages of the heavy chain with CNBr, hydroxylamine hydrochloride and iodosobenzoic acid and enzymic cleavage with Staphylococcus aureus proteinase. The peptides were purified by a combination gel-permeation and reverse-phase chromatography. The light chains, being only 20 residues long, could be sequenced without fragmentation. Amino acid analyses and carboxypeptidase-Y-digestion C-terminal analyses of the subunits provided supportive evidence for their sequence. Computer-assisted alignment of the jacalin heavy-chain sequence failed to show sequence similarity to that of any lectin for which the complete sequence is known. Analyses of the sequence showed the presence of an internal repeat spanning residues 7-64 and 76-130. The internal repeat was found to be statistically significant.

The binding of Artocarpus integrifolia lectin (jacalin) to 4-methylumbelliferyl (Meumb)-glycosides, Gal alpha Meumb, Gal beta Meumb, GalNAc alpha Meumb, GalNAc beta-Meumb, and Gal beta 3GalNAc beta Meumb was examined by extrinsic fluorescence quenching titration and stopped flow spectrofluorimetry. The binding was characterized by 100% quenching of fluorescence of Meumb-glycosides. Their association constants range from 2.0 x 10(4) to 1.58 x 10(6) M-1 at 15 degrees C. Entropic contribution is the major stabilizing force for avid binding of Meumb-glycosides indicating the existence of a hydrophobic site that is complementary to their methylumbelliferyl group. The second order association rate constants for interaction of these sugars with lectin at 15 degrees C vary from 8.8 x 10(5) to 3.24 x 10(6) M-1 S-1, at pH 7.2. The first order dissociation rate constants range from 2.30 to 43.0 S-1 at 15 degrees C. Despite the differences in their association rate constants, the overall values of association constants for these saccharides are determined by their dissociation rate constants. The second order rate constant for the association of Meumb-glycosides follows a pattern consistent with the magnitude of the activation energies involved therin. Activation parameters for association of all ligands illustrate that the origin of the barrier between binding of jacalin to Meumb-glycosides is entropic, and the enthalpic contribution is small. A correlation between these parameters and the structure of the ligands on the association rates underscores the importance of steric factors in determining protein saccharide recognitions.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.

The resistance of polyribonucleotide inductors of interferon to blood ribonucleases was studied. Blood resistance of larifan and ridostin in the free and shielded state as well as that of the complexes of poly(I)-poly(C) and poly(G)-poly(C) were also investigated. A protective action of polylysine against the inductors was detected which, in case it had no effect on the biological activity of the drugs, could provide its recommendation as a compound for shielding the inductors.

We have developed a method for identifying IgG-complexed creatine kinase (CK; EC 2.7.3.2) (IgG-CK) and IgA-complexed CK (IgA-CK) in serum. We used immobilized Protein G to bind IgG-CK and immobilized jacalin to bind IgA-CK, leaving noncomplexed CK in solution. The noncomplexed CK and total CK were measured kinetically. The results are reported as CK bound to immobilized Protein G and CK bound to immobilized jacalin. We validated the method by using sera determined immunochemically to contain IgG-CK, IgA-CK, mitochondrial CK (CKmt), and free CK-BB. We demonstrated concomitant binding of CK and approximately 99% of IgG, and of CK and approximately 87% of IgA. For CK bound to immobilized Protein G and to immobilized jacalin, intra- and interassay precisions ranged from 2.5% to 9.6%, and detection limits were less than 9 318 U/L in 40 sera containing IgG-CK, and CK bound to immobilized jacalin ranged from 10 to 59 U/L in eight sera containing IgA-CK. These ranges represent the activities of immunoglobulin-bound CK in the sera. In 13 sera containing CKmt and in eight sera containing free CK-BB, the binding of CK was less than 9 U/L. Evidently, this method is useful for identifying IgG-CK and IgA-CK in serum.

Tilorone aza-analogues, derivatives of 4,7-phenanthroline and 1,8-diazafluorene, were examined as DNA-complexing agents by spectral and electrophoretic methods. The binding process includes at least two types of interactions: electrostatic and, possibly, intercalation. Complex formation with the denatured DNA was also observed, but its nature remained unsolved. Binding and thermodynamic parameters were determined. All ligands studied showed weak antiviral activity and essentially no interferon induction when assayed in vitro and in vivo. It was concluded that interferon induction by tilorone may involve specific cell receptors or intermediaries.

10-Carboxymethyl-9-acridanone (CMA) was shown to be a very potent interferon inducer in young and old Swiss albino as well as congenitally asplenic mice. This compound (molecular weight, 275) induced titers of interferon in plasma comparable to those obtained with the best viral inducers, attaining > 400,000 U/ml in mice in 2 to 3 h. CMA concentrations were highest in mouse plasma 1 h after intraperitoneal or oral delivery. Induction was dose dependent over a wide range. Intraperitoneal, subcutaneous, or intramuscular injections of CMA gave comparable ranges of interferon titers. Oral delivery by gavage gave lower titers (65,000 U/ml), but higher than those reported with other low-molecular-weight interferon inducers in mice. CMA injected into week-old hamsters (which usually produce iterferon poorly) induced levels of interferon as high as 6,400 U per ml of plasma in a dose-dependent fashion, but kinetics of interferon induction was less rapid than in mice. The mouse and hamster interferons induced by CMA had physical characteristics similar to those of virus-induced interferons of the homologous species. The unusually high yields of interferon obtainable with this chemical inducer suggest its use for further experimental antiviral or antitumor therapy.

Depletion of macrophages from human peripheral blood mononuclear cells (PBMC) caused a marked decrease in galactose oxidase and sodium periodate, but not a calcium ionophore, stimulated Interferon-gamma (IFN-gamma) production. Reconstitution of such depleted cultures with galactose oxidase treated macrophages, but not lymphocytes, restored IFN-gamma levels to those of control nonfractionated PBMC. Thus, galactose oxidase seemed to act on macrophages which in turn stimulated lymphocyte production of IFN-gamma. Unlike human cells which have terminal galactose residues on glycoproteins, murine cell glycoproteins terminate their oligosaccharide component in the order N-acetyl-neuraminic acid followed by D-galactose, N-acetyl-glucosamine, and glycoprotein. Galactose oxidase or sodium periodate only activated murine macrophages to stimulate lymphocyte IFN-gamma production after exposing D-galactose residues by the removal of the terminal N-acetyl-neuraminic acid residues with neuraminidase. Removal of such exposed terminal galactose residues with beta-galactosidase inhibited the effect of galactose oxidase on murine macrophages. Taken together, these results strongly suggest that oxidation of terminal galactose residues on macrophages is the initial site of action of galactose oxidase and sodium periodate. Studies with Boyden chambers have shown that galactose oxidase-treated macrophages released a soluble factor which stimulates lymphocyte production of IFN-gamma. Based on these findings, it appears that the oxidation of terminal galactose residues on the surface of macrophages leads to the induction and transmission of a soluble signal for lymphocyte production of IFN-gamma.

New choline and halogen derivatives of CMA (9-oxo-10-acridine acetic acid) were investigated as interferon (IFN) inducers in mice and in the mouse bone marrow-derived macrophage cultures. Two of the choline derivatives, DMCMA and CSCMA, were active IFN inducers presumably because they were hydrolyzed so as to release CMA. The halogen analogues of CMA were inactive or weak IFN inducers in vivo and in vitro. On the contrary, the Br and I derivatives of CMA were potent inhibitors of IFN induction by CMA in vitro. The behavior of the agonists and antagonists of CMA suggests that the induction of interferon may occur indirectly via a specific CMA-receptor complex.