In Vitro Assay of Endotoxin by the Inhibition of Macrophage Migration

A quantitative in vitro technique was used to compare the ability of different endotoxins to inhibit the migration of macrophages from explants of rabbit spleen cultured in a coagulated plasma medium. The order of potency was different from that observed in chick embryo assays, and in assays with mice, of the same endotoxins. In general, however, the sensitivity of the macrophage inhibition test was comparable to that of other bioassay methods. A highly purified endotoxin from Salmonella enteritidis (Ribi) in a concentration of 0.004 mug/ml regularly inhibited macrophage migration. The in vitro method was used to detect a progressive loss of biological activity in fractions obtained during acid hydrolysis of the purified endotoxin. The selective toxicity of very low concentrations of endotoxin for mammalian macrophages was important in estimating the degree of specificity of the reaction. The pattern of cellular response in explant cultures made it possible to differentiate endotoxic damage from the specific cytotoxic action of antigen associated with delayed hypersensitivity.     

Inhibition by Interferon Preparations of a Solid Malignant Tumour and Pulmonary Metastases in Mice

WE have reported the efficacy of exogenous interferon preparations in inhibiting the development of several virus-induced leukaemias of mice. These are the Friend^1,2 and Rauscher leukaemias³ as well as the spontaneously appearing lymphoid leukaemia of AKR mice⁴. Comparable interferon preparations also inhibited the growth of transplantable ascitic tumours (of viral and non-viral origin) and markedly increased the survival of tumour inoculated mice^5,6. We thought it would be interesting to determine the effect of interferon on the growth of a solid metastasizing tumour.     

Macrophage Migration Inhibition by Serum from Desensitized Animals Previously Sensitized with Tubercle Bacilli

MIGRATION of peritoneal exudate cells removed from guinea-pigs or mice exhibiting delayed hypersensitivity is inhibited by specific antigen^1–3. This in vitro macrophage migration inhibition has been regarded as a useful immunological test for delayed skin hypersensitivity^4,5. Studies of the mechanism of this phenomenon revealed that, in contact with specific antigen, lymphocytes from sensitized animals released into the medium a specific substance (migration inhibitory factor; MIF) capable of inhibiting the migration of normal macrophages^6,7. When injected intradermally into normal guinea-pigs, MIF elicits inflammatory reactions characterized by induration, erythema and mononuclear cell infiltration⁸.     

Inhibition of Macrophage Migration Inhibitory Factor Ameliorates Ocular Pseudomonas aeruginosa-Induced Keratitis

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.     

Inhibition of Bacteria by Lactulose Preparations

Lactulose syrups were similar to sucrose syrups in water activity-lowering effects but were more inhibitory toward test microorganisms. Heat-treated commercial lactulose syrups were most inhibitory, whereas non-heat-treated pure lactulose was only slightly more inhibitory than sucrose.     

Macrophage Migration Inhibitory Factor Inhibition Is Deleterious for High-Fat Diet-Induced Cardiac Dysfunction

Aims

Development of metabolic syndrome is associated with impaired cardiac performance, mitochondrial dysfunction and pro-inflammatory cytokine increase, such as the macrophage migration inhibitory factor MIF. Depending on conditions, MIF may exert both beneficial and deleterious effects on the myocardium. Therefore, we tested whether pharmacological inhibition of MIF prevented or worsened metabolic syndrome-induced myocardial dysfunction.

Methods and Results

C57BL/6J mice were fed for ten weeks with 60% fat-enriched diet (HFD) or normal diet (ND). MIF inhibition was obtained by injecting mice twice a week with ISO-1, for three consecutive weeks. Then, triglycerides, cholesterol, fat mass, glucose intolerance, insulin resistance, ex vivo cardiac contractility, animal energetic substrate utilization assessed by indirect calorimetry and mitochondrial respiration and biogenesis were evaluated. HFD led to fat mass increase, dyslipidemia, glucose intolerance and insulin resistance. ISO-1 did not alter these parameters. However, MIF inhibition was responsible for HFD-induced cardiac dysfunction worsening. Mouse capacity to increase oxygen consumption in response to exercise was reduced in HFD compared to ND, and further diminished in ISO-1-treated HFD group. Mitochondrial respiration was reduced in HFD mice, treated or not with ISO-1. Compared to ND, mitochondrial biogenesis signaling was upregulated in the HFD as demonstrated by mitochondrial DNA amount and PGC-1α expression. However, this increase in biogenesis was blocked by ISO-1 treatment.

Conclusion

MIF inhibition achieved by ISO-1 was responsible for a reduction in HFD-induced mitochondrial biogenesis signaling that could explain majored cardiac dysfunction observed in HFD mice treated with MIF inhibitor.     

Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.     

Ubiquitin-Specific Proteases 25 Negatively Regulates Virus-Induced Type I Interferon Signaling

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.     

Selective Inhibition of Newcastle Disease Virus-Induced Glycoprotein Synthesis by d-Glucosamine Hydrochloride

d-Glucosamine selectively affected the synthesis of virus-induced glycoproteins in Newcastle disease virus-infected chick fibroblasts, but had no effect upon post-translational cleavage of the glycoprotein precursor for one of the two major glycoproteins of the mature virion. In response to glucosamine, at least one of the two virus-induced glycoproteins is formed in reduced quantity and the second is either not formed or has an electrophoretic mobility identical to that of another virus-induced protein.     

Cyclic AMP affects Macrophage Migration

ADENOSINE 3′,5′-cyclic monophosphate (cyclic AMP) has been shown to restore contact inhibition of transformed cells¹ and to enhance the aggregation of thyroid cells². I have studied the effects of exogenous and endogenous cyclic AMP on the centrifugal migration of guinea-pig macrophages from capillary tubes onto a glass surface. This test system is widely used for the measurement of a soluble lymphocyte product known as the migration inhibitory factor (MIF)^3,4.     

Inhibition of Plant Viruses by Human Gamma Interferon

In order to evaluate the inhibitory effect induced by gamma interferon (Hu g IFN) on plant viruses, pre-inoculation treatments (brushing the leaves) were carried out in two different plant-virus systems: D. stramonium* TMV and G. globosa* PVX. Results showed that Hu g IFN induced a higher inhibitory effect (IP = 90%) in D. stramonium* TMV system (Table 1). Comparing the antiviral effect of the three IFNs: gamma, alpha and beta-like interferons, it was verified that Hu g IFN was more effective than the other two (Table 2). Hu g IFN was also used for post-inoculation treatments (incubation of tobacco leaf-discs) and using different dilutions a dose response curve could be obtained (Fig. 1). Hu g IFN inhibitory effect was confirmed by the neutralization of its inhibitory effect using monoclonal antibody (Table 3). Results suggest that although the three IFNs differ in their composition, they present similarities in their biological activities probably triggering an antiviral state in plants.     

Comparison of In Vitro and In Vivo Labeling of Virus-Induced L-Cell Interferon

Preparations of NDV_uv-induced L-cell interferon were labeled in vitro with ¹²⁵I and ³H gas, or in vivo through incorporation of amino acids-³H during synthesis. Prior to purification, more than 90% of the interferon titer was lost during in vitro labeling by either procedure, whereas 34% of the initial activity of in vivo-labeled material was preserved during preparatory handling. Purification by carboxymethyl-Sephadex chromatography and electrophoresis in polyacrylamide gels was about 100-fold, and electrophoretic profiles revealed close concordance between isotopes and interferon titers in all instances. Noninterferon proteins from control cells, although less extensively labeled with tritium during synthesis than proteins from interferon-producing cells and released in lesser amounts, also contained components of identical electrophoretic mobility and distribution in acrylamide gels as interferon. The highest specific activity (6 x 10⁶ U/mg protein) but lowest cpm per interferon unit ratio (0.3) were exhibited by in vivo-labeled interferon. The advantage of better isotope incorporation through in vitro labeling techniques was largely offset by extensive losses in interferon activity.     

Skin Reaction and Macrophage Migration Inhibition Tests for Polysaccharides from Aspergillus fumigatus and Candida albicans

Guinea pigs sensitized with purified galactomannan from Aspergillus fumigatus and mannan from Candida albicans, each containing negligible quantities of nitrogen, were examined for their immunological responses against the corresponding polysaccharides with respect to the delayed-type skin reaction and the macrophage migration inhibition phenomenon. In both cases, the delayed-type skin reaction test and the macrophage migration inhibition test showed positive results. The reactivity was stronger in animals sensitized with polysaccharides in Freund's complete adjuvant than those sensitized with the same polysaccharides in Freund's incomplete adjuvant. Polysaccharides chemically modified by partial acid degradation or by periodate oxidation were found to be completely incapable of eliciting such immune responses. These results are also discussed in relation to the antigenic determinant of the polysaccharides in such immune responses and the precipitin reaction previously observed by us and other investigators.     

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2_∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2_∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2_∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.     

A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication.