Expression of measles virus antigens in Streptococcus gordonii.

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.

Laboratory diagnosis of Toscana virus infection by enzyme immunoassay with recombinant viral nucleoprotein.

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.

Diagnostic potential of Toscana virus N protein expressed in Escherichia coli.

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.

Natural antiviral activity of mouse macrophages against encephalomyocarditis virus.

Resident mouse peritoneal cells (PC) express a significant antiviral activity against encephalomyocarditis virus (EMCV) in vitro, as judged by decreased virus yield from infected mouse embryo fibroblasts (MEF). This natural antiviral activity of PC was not due either to enhanced lysis of virus-infected cells, as these were protected from lysis rather than destructed by PC, or to interferon (IFN) production, as no direct correlation between IFN and anti-EMCV activity was found. Among PC, macrophages (M phi) appear to be responsible for the anti-EMCV activity, which was indeed attributable to a Thy 1.2-negative, adherent mononuclear cell. Moreover, M phi-defective C3H/HeJ mice showed a significant impairment of anti-EMCV activity, whereas M phi of mice defective for natural killer (NK) activity (bg/bg, SJL/J) or for mature T cells (nu/nu) possessed an intact antiviral capacity.

Macrophage antitumor activity in vitro. Comparative analysis of cytolytic, cytostatic, and cytotoxic activities of mouse macrophages and human monocytes.

Mouse peritoneal M phi and human blood monocytes were assayed for their antitumor activity in vitro with a cytolysis, a cytostasis and a cytotoxicity test performed in parallel. Both natural and stimulus-induced M phi antitumor capacities were assessed. Results indicate that natural cytolytic activity of unstimulated M phi is generally unable to restrict final tumor cell growth, since it is not coupled with cytostatic capacity. In contrast, exposure of M phi in vitro to either MAF or IFN-beta, besides augmenting M phi cytolytic capacity, induced a very significant cytostatic activity and thus efficiently restricted the survival of tumor cells.

Interferon decreases production of hydrogen peroxide by macrophages: correlation with reduction of suppressive capacity and of anti-microbial activity.

Mouse peritoneal macrophages (M phi) expressed enhanced tumoricidal activity upon in vitro stimulation either with the lymphokine M phi-activating factor (MAF) or with fibroblast interferon (IFN-beta). In contrast, M phi suppressive activity on lymphoproliferation was not affected by MAF pretreatment, but was drastically reduced or abolished by IFN-beta. Catalase, the enzyme involved in the destruction of hydrogen peroxide (H2O2), did significantly decrease M phi suppressive capacity but had no effect on M phi tumoricidal activity. Analysis of the phagocytosis-dependent H2O2 production by IFN-beta-treated M phi demonstrated a strong impairment of the oxygen metabolite release, which strictly paralleled the decreased M phi suppressive capacity. On the other hand, MAF did not modify H2O2 release by M phi. Studies on M phi antibacterial activity against Salmonella typhimurium, a function thought to depend upon H2O2 production, showed that exposure of M phi to IFN-beta significantly impaired their bactericidal and bacteriostatic capacity, again in close correlation with the decrease in H2O2 production. Thus, IFN-beta appears as modulating both suppressive and antibacterial capacities of M phi through reduction of their oxygen metabolism, whereas regulation of M phi anti-tumour activity is possibly controlled by different mechanisms.

Dissociation between macrophage tumoricidal capacity and suppressive activity: analysis with macrophage-defective mouse strains.

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.

Macrophage activation by interferon: dissociation between tumoricidal capacity and suppressive activity.

Resident peritoneal macrophages (M phi) from untreated mice and inflammatory M phi induced by a sterile irritant were able to exert significant suppressive activity on lymphocyte functions. M phi-mediated suppression was evident in lymphocyte proliferation and in a proliferation-independent lymphocyte response (i.e. lymphokine production). The lymphokine macrophage-activating factor (MAF), which enhances tumoricidal capacity of inflammatory but not of resident M phi in vitro, was ineffective in modulating suppressive activity of both resident and inflammatory M phi. In contrast, a significant effect on M phi-mediated suppression was observed upon treatment with IFN-beta. In fact, suppression of lymphoproliferation and of lymphokine production by either resident or inflammatory M phi was significantly decreased or abolished by IFN-beta. Like MAF, IFN-beta was able to increase M phi cytotoxicity against tumor cells. Such as effect, however, was evident in both resident and inflammatory M phi, thus confirming different M phi activation mechanisms for MAF and IFN-beta. These data indicate that IFN-beta acts mainly on mature M phi, which thus appear as the major M phi type involved in suppression. The contrasting effects of IFN-beta on M phi suppression and on M phi-mediated cytotoxicity strongly suggest a dissociation between the 2 induction mechanisms of suppression and cytotoxicity. The in vivo relevance of these two IFN activities was demonstrated by treating mice with the potent IFN inducer polyinosinic polycytidylic acid (poly(I).poly(C). M phi from poly(I).poly(C)-primed mice simultaneously showed enhanced tumoricidal activity and complete abolishment of suppressive capacity.

Natural killer activity of gut mucosal lymphoid cells in mice.

Intraepithelial lymphoid cells (IEL) obtained from the mucosa of mouse small intestine were tested for natural killer (NK) activity in a 20-h 51Cr-release assay against YAC-1 and RLfemale1 tumor cells. It was found that IEL possess a strong NK activity, higher than spleen cells, whereas lymphocytes from Peyer's patches (PPL) did not show any NK activity. The cytotoxic activity of IEL could be boosted by interferon (IFN-beta ) treatment, but no NK activity could be induced in PPL by in vitro IFN-beta treatment. The characteristics of the NK effector cells present in the mouse intestine strictly resemble those of NK cells in the spleen. In fact, intestinal NK activity is not affected by depletion of adherent cells and only partially reduced by anti-Thy-1.2 antibodies plus complement. Moreover, the age dependency of NK cytotoxicity is identical for IEL and spleen. Finally, NK-insensitive target cells are not lysed by IEL.