Understanding the role of antibodies in murine infections with Heligmosomoides (polygyrus) bakeri: 35 years ago, now and 35 years ahead.

The rodent intestinal nematode H.p.bakeri has played an important role in the exploration of the host-parasite relationship of chronic nematode infections for over six decades, since the parasite was first isolated in the 1950s by Ehrenford. It soon became a popular laboratory model providing a tractable experimental system that is easy to maintain in the laboratory and far more cost-effective than other laboratory nematode-rodent model systems. Immunity to this parasite is complex, dependent on antibodies, but confounded by the parasite's potent immunosuppressive secretions that facilitate chronic survival in murine hosts. In this review, we remind readers of the state of knowledge in the 1970s, when the first volume of Parasite Immunology was published, focusing on the role of antibodies in protective immunity. We show how our understanding of the host-parasite relationship then developed over the following 35 years to date, we propose testable hypotheses for future researchers to tackle, and we speculate on how the new technologies will be applied to enable an increasingly refined understanding of the role of antibodies in host-protective immunity, and its evasion, to be achieved in the longer term.

Functional effects of CCL3L1 copy number.

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of CNV are not so well understood. Here, we present data exploring the functional consequences of CNV of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. The copy number of CCL3L1 was determined by the paralogue ratio test, and expression levels of macrophage inflammatory protein-1α (MIP-1α) and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of CNV of CCL3L1.

Treatment of severe, recalcitrant, chronic plaque psoriasis with fumaric acid esters: a prospective study.

BACKGROUND: Fumaric acid esters (FAE) are used in Germany as a first-line systemic treatment for chronic plaque psoriasis, with proven efficacy and low toxicity. Their use in the U.K. is variable, and they remain unlicensed. Consequently, efficacy and safety data from U.K. patients is limited and their place in the psoriasis treatment armamentarium is unclear. OBJECTIVES: To examine the efficacy and safety of FAE in a prospective cohort of U.K. patients with severe, treatment-recalcitrant, chronic plaque psoriasis. METHODS: A single-centre, open, nonrandomized, prospective study was performed in a regional referral centre for patients with severe psoriasis. Outcomes were measured by the Psoriasis Area and Severity Index (PASI), Dermatology Life Quality Index (DLQI), blood investigations and adverse events monitoring. RESULTS: Eighty patients were recruited. Fifty-nine per cent were taking a concomitant oral antipsoriatic agent; 20% achieved a PASI-50, 8% a PASI-75 and 4% a PASI-90 on intention-to-treat analysis at 3 months with an overall, statistically significant, reduction in PASI from 13.9 + or - 9.0 to 11.3 + or - 9.2 (P < 0.0001). At 3 months, lymphopenia was seen in 33% of the cohort with significantly lower counts in patients responsive to FAE (P = 0.008). In addition, by 3 months, 36% of concomitant antipsoriatic medication had been stopped and 25% of doses had been reduced without loss of disease control. Side-effects (most commonly diarrhoea, abdominal pain and flushing) were reported by 74% of patients resulting in cessation of FAE in 36%. CONCLUSIONS: FAE is a useful alternative treatment option in patients with severe, treatment-resistant, chronic plaque psoriasis and can allow dose reduction, and subsequent cessation, of other, potentially more toxic agents.

Fc-receptors and immunity to malaria: from models to vaccines.

The complexity and number of antigens (Ags) seen during an immune response has hampered the development of malaria vaccines. Antibodies (Abs) play an important role in immunity to malaria and their passive administration is effective at controlling the disease. Abs represent approximately 25% of all proteins undergoing clinical trials, and these 'smart biologicals' have undergone a major revival with the realization that Abs lie at the interface between innate and adaptive immunity. At least 18 Abs have FDA approval for clinical use and approximately 150 are in clinical trials, the majority for the treatment of cancer, allograft rejection or autoimmune disease. Despite these triumphs none are in development for malaria, principally because they are perceived as being too expensive for a disease mainly afflicting poor and marginalized populations. Although unlikely, at least in the foreseeable future, that Ab-based prophylaxis will be made available to the millions of people at risk from malaria, they may be incorporated into current vaccine approaches, since Abs act as correlates of protection in studies aimed at defining the best Ags to include in vaccines. Abs may also form the basis for novel vaccination strategies by targeting Ags to appropriate antigen presenting cells. Therefore, to develop the most efficacious vaccines it will be necessary to fully understand which Abs and Fc-receptors (FcRs) are best engaged for a positive outcome.

When is a malaria immune complex not an immune complex?

Immune complexes (ICs) are believed to play an important role in malaria pathology, and an interesting article by Mibei et al. recently published by Parasite Immunology suggests that IgG4 and IgE are particularly important. However, researchers should be aware of potential pitfalls to current assays aimed at measuring plasma ICs and correlating these to deposition in tissues.

Heterogeneous interspecific interactions in a host-parasite system.

Macroparasites of vertebrates usually occur in multi-species communities, producing infections whose outcome in individual hosts or host populations may depend on the dynamics of interactions amongst the different component species. Within a single co-infection, competition can occur between conspecific and heterospecific parasite individuals, either directly or via the host's physiological and immune responses. We studied a natural single-host, multi-parasite model infection system (polystomes in the anuran Xenopus laevis victorianus) in which the parasite species show total interspecific competitive exclusion as adults in host individuals. Multi-species infection experiments indicated that competitive outcomes were dependent on infection species composition and strongly influenced by the intraspecific genetic identity of the interacting organisms. Our results also demonstrate the special importance of temporal heterogeneity (the sequence of infection by different species) in competition and co-existence between parasite species and predict that developmental plasticity in inferior competitors, and the induction of species-specific host resistance, will partition the within-host-individual habitat over time. We emphasise that such local (within-host) context-dependent processes are likely to be a fundamental determinant of population dynamics in multi-species parasite assemblages.

Monocyte chemoattractant protein-2 is a potent agonist of CCR2B.

The binding and functional activity of the CC chemokines monocyte chemoattractant protein-1 (MCP-1), MCP-2, and MCP-3 have been characterized using Chinese hamster ovary DXB-11 cells transfected with the chemokine receptor CCR2B. Receptor binding studies demonstrated that 125I-labeled MCP-1 bound to a single class of high-affinity receptors with a Kd of 0.14 (0.07-0.32) nM. In competition studies MCP-1, MCP-2, and MCP-3 completely inhibited 125I-labeled MCP-1 binding with Ki values of 0.3 (0.16-0.46), 8.8 (3.4-26), and 12.2 (0.6-22) nM, respectively. In calcium mobilization studies, MCP-1 and MCP-3 induced robust elevations in intracellular calcium concentrations, whereas MCP-2 was only weakly active. In contrast, using changes in extracellular acidification rate as a functional readout, all three chemokines were identified as potent agonists of CCR2B. These data demonstrate that MCP-2, in addition to MCP-1 and MCP-3, is a potent agonist of CCR2B and furthermore that MCP-2 activates either different or a subset of the signaling pathways activated by MCP-1 and MCP-3.

Fc receptors and immunity to parasites.

Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.

Characterization of platelet-activating factor-induced elevation of cytosolic free calcium concentration in eosinophils.

In order to evaluate the role of calcium in the activation processes in eosinophils induced by platelet-activating factor (PAF), we investigated the changes in free cytoplasmatic Ca2+ concentration using fura-2. PAF causes a rapid and transitory rise of the intracellular free calcium ion concentration [( Ca2+]i) in purified guinea pig eosinophils of approx. 1000 nM above a basal level of 120.7 +/- 36.5 nM (n = 10). The effect was dose-related with a maximum rise at 1000 nM PAF and an EC50 of 17.4 nM and specifically inhibited by the PAF antagonist WEB 2086 with an IC50 of 95.5 nM. WEB 2086 did not affect either the leukotriene B4- or the fMet-Leu-Phe-induced elevation of [Ca2+]i. The response to PAF was dependent on external Ca2+ as it was significantly inhibited by EGTA (85.6 +/- 5.4%) and Ni2+ (95.8 +/- 2.1%) but not by the dihydropyridine antagonist nimodipine. We conclude that Ca2+ entry via receptor-operated Ca2+ channels may be involved in PAF-induced degranulation of eosinophils.

The effects of gamma radiation on the development of Heligmosomoides polygyrus bakeri in mice.

The development of a Heligmosomoides polygyrus bakeri (H. polygyrus) primary infection in its definitive host was severely effected by a wide range of gamma radiation doses (10-400 Gy). Male worms were more susceptible to gamma radiation than female worms. A dose of 400 Gy prevented the development of L3 larvae to mature female worms and 200 Gy abrogated the maturation of males. At 300 Gy, a dose known to stimulate high levels of protective immunity, male worms were unable to moult to the L4 stage and females failed to develop into morphologically normal adults. An experiment to select for a radiation resistant parasite line provided data on the cumulative effects of gamma rays on successive parasite generations. Parasite fitness data demonstrated that worm development, at the level of embryogenesis, was far more sensitive to radiation damage than either post embryonic development or adult worm fecundity. The parasite line died out on the 14th generation of selection after receiving an accumulated dose of 420 Gy. It is concluded that gamma radiation profoundly alters the developmental biology of H. polygyrus in a dose-dependent manner, with maximal sensitivity exhibited during embryogenesis.

The effect of gamma-radiation and heat shock on protein synthesis and antioxidant enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus.

Protein synthesis and antioxidant enzyme activities were investigated in gamma-irradiated (300 Gy) and heat shocked (42 degrees C) larval stages of the gastrointestinal parasite, Heligmosomoides polygyrus bakeri (H. polygyrus). No qualitative or quantitative differences were observed in the incorporation of (35S)-methionine into somatic proteins of unirradiated or irradiated exsheathed third-stage (L3) larvae at either 37 degrees C or 42 degrees C. The rate of protein synthesis doubled in L3 stages maintained at 42 degrees C compared with 37 degrees C, irrespective of whether the larvae had been irradiated or not. The composition of excretory/secretory (ES) proteins varied between unirradiated and irradiated exsheathed L3 larvae maintained under identical conditions. Prominent heat-inducible proteins of 26 and 17 kDa were synthesised and excreted at 42 degrees C by both unirradiated and irradiated L3 stages. No major differences in protein synthesis could be detected between unirradiated and irradiated fourth-stage (L4) larvae. Temperature elevation significantly reduced protein synthesis in L4 stages, most notably in unirradiated parasites. Heat-inducible proteins were not detected in response to either irradiation or temperature elevation in L4 larvae. Immune sera recognised a similar spectrum of antigens in both unirradiated and irradiated L4 somatic and ES preparations and reacted with antigens from irradiated L4 parasites with less intensity than with antigens from unirradiated L4 larvae. Catalase was the only antioxidant enzyme examined with activity that changed significantly in irradiated parasites, being reduced to approximately 36% of normal levels in irradiated L4 stages. No significant difference existed between irradiated and unirradiated parasites in the levels of activity of superoxide dismutase and glutathione reductase.

Variation in attractiveness of human subjects to malaria mosquitoes (Diptera: Culicidae) in The Gambia.

During experimental hut trials to assess the efficacy of insecticide-treated bednets against malaria mosquitoes, we observed that human subjects varied consistently in their attractiveness to mosquitoes. Attractiveness was assessed by estimating the numbers of wild Anopheles gambiae Giles mosquitoes entering a hut in which a man was sleeping, and the numbers of human-bloodfed An. gambiae sensu lato collected from each hut each morning. Five trials were carried out at Wali Kunda in rural Gambia during 2.5 yr. During each 6-wk trial a man slept under a bednet in each of the six huts. Morning collections of mosquitoes from the room, enclosed verandas, and window traps of each hut provided estimates of the number of mosquitoes that had entered during the night. Blood meals were analyzed using an ELISA technique to identify those mosquitoes feeding on humans. Specimens were collected by field workers, not the subjects; therefore, sampling was independent of the subjects' ability to catch mosquitoes. Moreover, the trials were designed to measure the relative attractiveness of individual sleepers to mosquitoes, allowing for other sources of variation (i.e., among huts, bednets, nights, and day of the week). Attractiveness of men to mosquitoes differed significantly among individuals as indicated by the consistent differences between the numbers of mosquitoes entering each man's hut and the numbers feeding on each man. However, the two measures of attractiveness were apparently independent of each other: subjects who attracted consistently high numbers of vectors into their hut did not necessarily have high numbers of mosquitoes feeding on them. These findings support the view that some individuals within a community are at greater risk from mosquito-borne pathogens than others.

Metabolic labelling of wild and laboratory subspecies of the trichostrongyle nematode Heligmosomoides polygyrus.

The immunological, biochemical and taxonomic relationship between wild and laboratory subspecies of Heligmosomoides polygyrus was studied by metabolically labelling parasite proteins with [35S]-methionine. Much variability, both in content and synthesis of proteins was observed between the two subspecies. Laboratory female worms had a higher protein content and incorporated more radioactive label into somatic proteins than their wild counterparts. Incorporation of radioactivity into excretory/secretory (ES) proteins, predicted to contain important antigens, demonstrates a major reduction in synthesis of proteins with molecular weights 66, 55, 43, 41, 40, 39, 37, 28 and 16 kDa by laboratory females. These differences in protein synthesis might explain the differing infectivities of the two subspecies when passaged in inbred laboratory (Mus musculus) and wild (Apodemus sylvaticus) mice.

Characterization of immunoglobulin binding by schistosomes.

Although controversial, schistosomes are believed to cloak themselves in antibody through non-specific interactions with the immunoglobulin (Ig) molecule. The acquisition of host Ig by the schistosome may mask its foreign status and/or interfere with Fc-dependent functions. We report experiments aimed at characterizing the interaction between Ig-Fc and paramyosin, a schistosome Fc-receptor previously reported to bind human IgG. We show that certain Ig classes, in particular murine IgG2b and IgG3, are not only able to bind recombinant paramyosin, but also associate with other parasite proteins. The Fc region of IgG contains four hydrophobic patches, two of which are known to interact with distinct molecules: one in the Cgamma2-Cgamma3 interdomain region bound by protein G, mannose binding lectin (MBL), and the neonatal Fc-receptor (FcRn), and one at the top of the Cgamma2 domain bound by phagocytic FcgammaRs and C1q. We provisionally discounted the involvement of these regions, since IgG binding by paramyosin did not inhibit FcgammaR-mediated NADPH respiratory bursts, and protein G was unable to block IgG binding to paramyosin. Given their apparent low affinity, we postulate hydrogen bonding between reactive residues in a hydrophobic patch at the bottom of the Cgamma3 domain and negatively charged Glu or Asp amino acids in paramyosin.

Cytokines induce lymphocyte migration in vitro by direct, receptor-specific mechanisms.

Several cytokines have been found to induce human peripheral blood lymphocyte (PBL) migration in vitro. The mechanisms involved are unclear, therefore experiments were carried out to determine whether PBL migration in response to selected cytokines is due to a direct effect, or to the generation of nonspecific secondary mediators, and whether migration is receptor specific. Purified human PBL were incubated with the lymphocyte chemotactic cytokines interleukin (IL)-1 alpha and IL-8, followed after 30-60 min by addition of specific neutralizing monoclonal antibody. Supernatants of these mixtures were shown by subsequent assay to be devoid of PBL attractant activity, whereas positive control supernatants containing no antibody induced dilution-related migration. Addition of antibody to the high affinity IL-2 receptor abolished the potent attractant effect of IL-2 on PBL, but had little effect on responses to IL-1 alpha and IL-8. These results demonstrate that the in vitro locomotor responses of human PBL to selected cytokines are due to direct, receptor-specific effects and are not dependent upon the generation of secondary mediator(s).

The role of adult worms in suppressing functional protective immunity to Heligmosomoides polygyrus bakeri challenge infections.

Adult Heligmosomoides polygyrus bakeri, radiolabelled with [35S]-methionine were successfully transferred to naive NIH mice by oral gavage. Adult worms and radiolabel could be detected up to 45 days post-infection. Adult worms gavaged into immune NIH mice, immunized with a drug abbreviated larval infection, were rejected within 45 days. These adults worms were unable to ablate the development of a functional protective response to a larval challenge infection in the NIH strain. In fact 50 adult worms were sufficient to significantly immunize NIH mice against a larval challenge infection. However, adult worms were able to suppress the development of a functional protective response in an outbred CFLP strain. Although a protective immune response could not be elicited to a challenge infection in CBA mice, the presence of gavaged adult worms was shown to increase the susceptibility of mice to a challenge infection. For all mouse strains, no significant difference in levels of L4 antigen-specific serum IgG, IgG1, IgG2a, and IgA existed between immune mice and groups of mice immunosuppressed by adult worms. Levels of L4 antigen-specific serum IgG1 were significantly lower in the poorly immunizable CBA strain compared to CFLP and NIH strains. No correlation was found across mouse strains between the intensity of the antibody response and the mean worm burdens per animal group. In addition, no correlation was found between levels of L4 antigen-specific antibody within each mouse and the loss of worms by individual mice.

Irradiated larval vaccination and antibody responses evaluated in relation to the expression of immunity to Heligmosomoides polygyrus.

Infections induced in NIH mice by irradiated (300 Gy) larvae of Heligmosomoides polygyrus effectively stimulated immunity to challenge, whereas unirradiated larvae did not. Importantly, this difference was lost by the elimination of the adult worms arising from unirradiated sensitising infections by drug treatment prior to challenge. No difference in the level of parasite-specific serum and mucosal IgG, IgG1, IgG2a, or IgA was detected between immune mice sensitised either with drug-abbreviated unirradiated or irradiated larval infections and non-immune mice receiving two superimposed unirradiated infections. An enzyme-linked immunosorbent assay (ELISA) and immunoblotting data suggested that parasite-specific IgG1 was the predominant antibody class in both serum and intestinal perfusates. IgA exhibited differences in antigen specificity between the serum and the intestine. In serum, IgA responses were directed predominantly to L4 somatic antigens, whereas at the mucosal surface they were biased towards L4 excretory/secretory (ES) antigens. No correlation was found between the intensity of the serum or mucosal antibody responses and the mean worm burdens in groups of immune or non-immune mice. Moreover, no correlation was found between levels of parasite-specific serum or mucosal IgG, IgG1, IgG2a or IgA and the loss of worms in individual mice.

Cleavage of human IgE mediated by Schistosoma mansoni.

BACKGROUND: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. METHODS: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcepsilon receptors. RESULTS: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cepsilon2/Cepsilon3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcepsilonRII. CONCLUSIONS: An elastase-like protease in S. mansoni is able to render IgE non-functional.

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.