Tetanus toxin selectively impairs anti-tumoral but not anti-microbial macrophage-mediated effector functions

Abstract The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v- raf /v- myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-γ), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets.     

Immunoadjuvant effects of Candida albicans and its cell wall fractions in a mouse lymphoma model

Summary Chemical, ultrastructural, and immunoadjuvant properties of Candida albicans (CA) and of a number of its fractions have been characterized through the analysis of the antitumor activity of soluble and insoluble cell wall components.CD2F₁ mice were inoculated IP with Moloney virus-induced lymphoma LSTRA and treated with bis-1-chloroethyl-nitrosurea (BCNU) on day +5 after tumor challenge. A significant increase of the antitumor efficacy of BCNU treatment was found in mice inoculated with CA as immunoadjuvant on days –14 and +1 (–14/+1 schedule) with respect to tumor challenge.However, no significant difference in survival time was found between mice treated with BCNU alone and those treated with BCNU plus either soluble mannan or glucan-protein fractions extracted from CA and administered according to –14/+1 or –7/+1 schedules. On the other hand, mice treated with BCNU plus the insoluble glucan fraction (wall ghosts) given on days –14/+1 or even on day –7 only (i.e., without boosting after tumor challenge) survived longer than animals treated with BCNU alone.The immunoadjuvant effect of CA and of other classic immunoadjuvants, such as BCG and Corynebacterium parvum, was completely abolished by total-body irradiation (400 R) given 5 h before the first administration of the agent on day –7 prior to tumor challenge.These results indicate that: (a) the minimal structure required for the expression of the immunoadjuvant effect of CA is the insoluble, -glucan component of the cell wall; (b) the soluble components of CA cell wall (i.e., mannan and glucan-protein) per se do not show any detectable immunoadjuvant effect in the present animal-tumor system; they may, however, modulate this effect, as shown by the fact that whole CA, but not the insoluble -glucan, needs a boosting injection for the expression of its immunoadjuvant properties; (c) the immunoadjuvanticity of CA is radiosensitive.     

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.     

In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall.

Mannoprotein components from Candida albicans were investigated for their ability to induce production of tumor necrosis factor (TNF) by cultured splenocytes from naive or Candida-infected mice. Two chromatographically separated mannoproteins preparations, designated F1 and F2, were as able as the heat-inactivated Candida cells to induce the production of TNF from splenocytes of naive animals. In addition, they caused a significant augmentation of basic TNF secretion by splenocytes of Candida-infected animals. Experiments using plastic and/or nylon wool adherence, as well as treatments with antibodies depleting T or NK cells, consistently indicated that most if not all TNF was produced by splenic macrophages. In cultures of splenocytes from Candida-infected mice, mannoprotein addition also stimulated interferon-gamma (IFN-gamma) production by Thy 1.2 positive cells. Depletion of these cells or addition of anti-IFN-gamma antibodies abolished IFN production and reduced TNF secretion by adherent cells to the levels found in the cultures of mannoprotein-stimulated spleen cells from naive mice. These data add further evidence to the immunomodulatory properties possessed by some cell wall constituents of the human commensal microorganism C. albicans and suggest that IFN-gamma is endowed with a regulatory role in TNF production by mouse macrophages in vitro.     

Anti-cyclic citrullinated peptide antibody titer predicts time to rheumatoid arthritis onset in patients with undifferentiated arthritis: results from a 2-year prospective study

Introduction

The diagnostic, predictive and prognostic role of anti-cyclic citrullinated peptide (CCP) antibodies in rheumatoid arthritis (RA) patients is widely accepted. Moreover, detection of these antibodies in subjects presenting with undifferentiated arthritis (UA) is associated with a significant risk to develop the disease. On the other hand, clinical and prognostic significance of evaluating anti-CCP levels in subjects with inflammatory arthritis at disease onset has not been fully clarified. The goal of this prospective study is to analyze the value and prognostic significance of anti-CCP titer quantification in UA subjects.

Methods

Serial anti-CCP assays were measured in 192 consecutive patients presenting with UA lasting less than 12 weeks. Clinical and serological data and arthritis outcome were evaluated every 6 months until two years of follow-up.

Results

Anti-CCP positivity, at both low and high titer, and arthritis of hand joints significantly predicted RA at two years, risk increasing in subjects with high anti-CCP titers at baseline. Moreover, time to RA diagnosis was shorter in patients with high anti-CCP2 titers at enrollment with respect to those with low antibody concentration.

Conclusions

Presence of anti-CCP antibodies, at both low and high concentration, is significantly associated with RA development in subjects with recent onset UA. However, time interval from the onset of the first symptoms to the fulfilment of the classification criteria appears to be directly related to the initial anti-CCP level.     

Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii

The micronemal protein 2 (MIC2) of Toxoplasma gondii shares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to T. gondii micronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species. The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes of T. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional relationship that links these organelles with the endoplasmic reticulum of the parasite.