Antibody-mediated control of Trypanosoma vivax infection fails in the absence of tumour necrosis factor.

Trypanosoma vivax causes a wasting disease affecting livestock breeding and agriculture in developing countries of sub-Sahara Africa and South America. Being an extracellular parasite, control of T. vivax has been proposed to be mediated by host antibodies. However, the use of a comparative infection model of wild-type (WT) and tumour necrosis factor (TNF) knockout (TNF(-/-) ) mice shows that the latter is unable to control first-peak parasitaemia, despite the presence of specific antitrypanosome antibodies. In contrast, WT mice parasitaemia peak control coincides with a combined early onset of TNF production and induction of specific antibodies. TNF is mainly produced by liver-associated monocytes and neutrophils. In this study, no other correlation between cellular immunomodulations and peak parasitaemia control was observed, underscoring the importance of the role of TNF in the control of T. vivax infections.

Antibacterial activities of coagulase-negative staphylococci from bovine teat apex skin and their inhibitory effect on mastitis-related pathogens.

AIMS: To explore antibacterial activities of coagulase-negative staphylococci (CoNS) from teat apices of dairy cows towards mastitis-causing pathogens. METHODS AND RESULTS: Of 254 CoNS, 38 displayed bacteriocin-like activity after a first screening. Seven of these strains displayed activity against at least one mastitis-related pathogen (Streptococcus uberis, Streptococcus dysgalactiae and Staphylococcus aureus). Staphylococcus chromogenes L217 displayed the strongest inhibitory effect, being active against all tested mastitis-related pathogens and most tested CoNS. Based on cation exchange and reversed-phase chromatography, in addition to N-terminal Edman degradation and PCR, the antibacterial peptide was identified as a nukacin-type bacteriocin and named nukacin L217. Although staphylococcal bacteriocins are generally found in the cell-free supernatants of liquid cultures, Staph. chromogenes L217 only led to detectable activity when grown on agar medium. CONCLUSIONS: Bacteriocin-like activities are not uncommon among CoNS from teat apices and may inhibit mastitis-causing pathogens, as found for nukacin L217 production by Staph. chromogenes L217. SIGNIFICANCE AND IMPACT OF THE STUDY: Nukacin L217 is the first identified bacteriocin of the species Staph. chromogenes and displays unusual production kinetics, that is, requiring surface growth of its producer. The fact that nukacins are produced by different CoNS species suggests a role in the teat skin ecosystem.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Control of experimental Trypanosoma brucei infections occurs independently of lymphotoxin-alpha induction.

Trypanosome infections are marked by severe pathological features, including anemia, splenomegaly, and suppression of T-cell proliferation. We have used lymphotoxin-alpha-deficient (LT-alpha(-/-)) mice, as well as LT-alpha-tumor necrosis factor-double-deficient (LT-alpha(-/-) TNF(-/-)) mice, to analyze the contributions of these related cytokines in both induction of trypanosomosis-associated immunopathology and infection control. Moreover, as the cytokine-deficient mice used have no detectable lymph nodes and lack germinal-center formation upon immune stimulation, we have analyzed the functional importance of both the lymph nodes and spleen during experimental Trypanosoma brucei infections. First, we show that the absence of LT-alpha does not significantly alter early trypanosomosis development or pathology but does result in better control of late-stage parasitemia levels and slightly prolonged survival. This increased survival of infected LT-alpha(-/-) mice coincides with the appearance of increased chronic-stage anti-trypanosome immunoglobulin M (IgM)-IgG2a serum titers that are generated in the absence of functional peripheral lymphoid tissue and do not require germinal-center formation. Second, we show that splenectomized mice control their parasitemia to the same extent as fully immune-competent littermates. Finally, using LT-alpha(-/-) TNF(-/-) double-deficient mice, we show that in these mice T. brucei infections are very well controlled during the chronic infection stage and that infection-induced pathology is minimized. Together, these findings indicate that while increased IgM-IgG2a anti-trypanosome antibody titers (generated in the absence of LT-alpha, peripheral lymph nodes, and germinal-center formation) coincide with improved parasitemia control, it is TNF that has a major impact on trypanosomosis-associated immunopathology.

Distinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria.

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of lipopolysaccharide (LPS), beta-1,3-glucans, or N,N'-diacetylchitobiose present, respectively, on Gram-negative bacteria or yeast cell walls, CCF triggers the prophenoloxidase activating pathway. We report that CCF recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan constituent muramyl dipeptide as well as muramic acid. To identify the pattern recognition domains of CCF, deletion mutants were tested for their ability to reconstitute the prophenoloxidase cascade in E. foetida coelomic fluid depleted of endogenous CCF in the presence of LPS, beta-1,3-glucans, N,N'-diacetylchitobiose, and muramic acid. In addition, affinity chromatography of CCF peptides was performed on immobilized beta-1,3-glucans or N,N'-diacetylchitobiose. We found that the broad specificity of CCF for pathogen-associated molecular patterns results from the presence of two distinct pattern recognition domains. One domain, which shows homology with the polysaccharide and glucanase motifs of beta-1,3-glucanases and invertebrate defense molecules located in the central part of the CCF polypeptide chain, interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain mediates interactions of CCF with N,N'-diacetylchitobiose and muramic acid. These data provide evidence for the presence of spatially distinct carbohydrate recognition domains within this invertebrate defense molecule.

A conserved flagellar pocket exposed high mannose moiety is used by African trypanosomes as a host cytokine binding molecule.

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.

Attenuation of Trypanosoma brucei is associated with reduced immunosuppression and concomitant production of Th2 lymphokines.

Mechanisms regulating resistance to African trypanosomes were addressed by comparing the immune responses of mice infected with attenuated Trypanosoma brucei brucei lacking the phospholipase C gene (PLC-/-) and those of mice infected with wild-type (WT) parasites. Inhibition of concanavalin A (ConA)-induced T cell proliferation occurred in spleen and lymph nodes of PLC-/-- and WT-infected mice. Although suppressive cells were elicited in spleen and lymph nodes of WT-infected animals, such cells were not detected in lymph nodes of PLC-/--infected mice. PLC-/--infected mice had more interleukin-4 and -10 in their blood than did WT-infected mice. Correspondingly, PLC-/--infected mice had higher IgG1 antibody levels against variant surface glycoprotein than did WT-infected mice. These data indicate that attenuation of T. b. brucei correlates with the absence of cells suppressing ConA-induced T cell proliferation in the lymph nodes, with increased production of Th2 cytokines and a stronger IgG1 antibody response to trypanosome antigens.

Comparative analysis of antibody responses against HSP60, invariant surface glycoprotein 70, and variant surface glycoprotein reveals a complex antigen-specific pattern of immunoglobulin isotype switching during infection by Trypanosoma brucei.

During Trypanosoma brucei infections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimental T. brucei infection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.

The glycosyl-inositol-phosphate and dimyristoylglycerol moieties of the glycosylphosphatidylinositol anchor of the trypanosome variant-specific surface glycoprotein are distinct macrophage-activating factors.

The TNF-alpha-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C3H/HeJ mice and LPS-sensitive C3H/HeN mice. The variant-specific surface glycoprotein (VSG) was identified as the major TNF-alpha-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-alpha-inducing capacities, indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-alpha triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-alpha-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-alpha production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-alpha-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-gamma preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages toward IL-1alpha secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-alpha induction, the DMG compound of the mfVSG anchor contributes to IL-1 alpha induction and LPS sensitization.