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Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4+ T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4+ T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation. [ABSTRACT FROM AUTHOR]

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC5024h = 2.8 µM) being more sensitive than WB-C6 (IC5024h = 6.2 µM). The effect was significantly more potent compared to MTZ (IC5024h = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis. [ABSTRACT FROM AUTHOR]

Abstract: Malaria vaccines, comprised of irradiated Plasmodium falciparum sporozoites or a synthetic peptide containing T and B cell epitopes of the circumsporozoite protein (CSP), elicit multifunctional cytotoxic and non-cytotoxic CD4+ T cells in immunised volunteers. Both lytic and non-lytic CD4+T cell clones recognised a series of overlapping epitopes within a ‘universal’ T cell epitope EYLNKIQNSLSTEWSPCSVT of CSP (NF54 isolate) that was presented in the context of multiple DR molecules. Lytic activity directly correlated with T cell receptor (TCR) functional avidity as measured by stimulation indices and recognition of naturally occurring variant peptides. CD4+ T cell-mediated cytotoxicity was contact-dependent and did not require de novo synthesis of cytotoxic mediators, suggesting a granule-mediated mechanism. Live cell imaging of the interaction of effector and target cells demonstrated that CD4+ cytotoxic T cells recognise target cells with their leading edge, reorient their cytotoxic granules towards the zone of contact, and form a stable immunological synapse. CTL attacks induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies in target cells. Together, these findings suggest that CD4+ CTLs trigger target cell apoptosis via classical perforin/granzyme-mediated cytotoxicity, similar to CD8+ CTLs, and these multifunctional sporozoite- and peptide-induced CD4+ T cells have the potential to play a direct role as effector cells in targeting the exoerythrocytic forms within the liver. [Copyright &y& Elsevier]

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified., (© 2024 Bellinzona et al.; Published by Cold Spring Harbor Laboratory Press.)

Cystatins are important regulators of papain-like cysteine proteases. In the protozoan parasite Giardia intestinalis, papain-like cysteine proteases play an essential role in the parasite's biology and pathogenicity. Here, we characterized a cysteine protease inhibitor of G. intestinalis that belongs to type-I-cystatins. The parasite cystatin is shown to be a strong inhibitor of papain (K i  ≈ 0.3 nm) and three parasite cysteine proteases (CP14019, CP16160 and CP16779, K i  ≈ 0.9-5.8 nm), but a weaker inhibitor of human cathepsin B (K i  ≈ 79.9 nm). The protein localizes mainly in the cytoplasm. Together, these data suggest that cystatin of G. intestinalis plays a role in the regulation of cysteine protease activities in the parasite and, possibly, in the interaction with the host., (© 2019 Federation of European Biochemical Societies.)

Possibilities to study the biology of human protozoan parasites and their interaction with the host remain severely limited, either because of non-existent or inappropriate animal models or because parasites cannot even be cultured in vitro due to strict human-host specificity or physiology. Here we discuss the prospects of using induced pluripotent stem cell (iPSC)-derived culture systems including organoids as a strategy to address many of these experimental bottlenecks. iPSCs already allow the generation of differentiated cell cultures for many human organs, and these cells and derivatives are amenable to reverse genetics in combination with advanced tools for genetic manipulation. We present examples of blood, neuron, liver, and intestine-dwelling protozoa, i.e. Plasmodium falciparum, Toxoplasma gondii and Giardia duodenalis, where iPSCs or organoids would allow addressing questions of cell and developmental biology, immunology, and pharmacology in unprecedented ways. Starting points and resources for iPSC experimentation are briefly discussed., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Mast cells (MCs) are potent inflammatory cells that are distributed throughout mucosal barrier tissues and respond rapidly to pathogenic stimuli. During helminth infections, MCs play an important role as late-stage effectors. However, it is currently unknown whether MCs contribute to the early innate events that determine the priming of adaptive immunity. MC-deficient mouse strains and mice treated with the MC stabilizing agent cromolyn sodium had dramatically reduced Th2 priming and type 2 cytokine production and harbored increased parasite burdens following infection with gastrointestinal helminths (Heligmosomoides polygyrus bakeri and Trichuris muris). In addition, early production of the tissue-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) was significantly diminished in MC-deficient mice and resulted in decreased numbers of infection-elicited IL-25-dependent (Lin(-)CD45(-))CD34(+)Sca-1(+) progenitors, which produced type 2 cytokines and could be differentiated into mast cells ex vivo. Finally, repair of MC deficiency increased production of IL-25, IL-33, and TSLP, restored progenitor cell numbers and Th2 priming, and reduced parasite burden. Our data reveal an innate IgE-independent role for MCs in orchestrating type 2 immune responses via the regulation of IL-25, IL-33, and TSLP.

The cystatin superfamily comprises several groups of protease inhibitors. In this chapter we will focus on I25 family members, which consist predominantly of the type 2 cystatins. Recently, a wealth of information on these molecules and their activities has been described. Parasite cystatins are shown to have dual functions via interaction with both parasite and host proteases. Thereby, parasite cystatins are not only essentially involved in the regulation of physiological processes during parasite development, but also represent important pathogenicity factors. Interestingly, some studies indicate that parasite cystatins evolved exceptional immuno-modulatory properties. these capacities could be exploited to interfere with unwanted immune responses in unrelated human inflammatory diseases. We highlight the different biological roles of parasite cystatins and the anticipated future developments.

Parasitic nematodes can downregulate the immune response of their hosts through the induction of immunoregulatory cytokines such as interleukin-10 (IL-10). To define the underlying mechanisms, we measured in vitro the production of IL-10 in macrophages in response to cystatin from Acanthocheilonema viteae, an immunomodulatory protein of filarial nematodes, and developed mathematical models of IL-10 regulation. IL-10 expression requires stimulation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and p38, and we propose that a negative feedback mechanism, acting at the signalling level, is responsible for transient IL-10 production that can be followed by a sustained plateau. Specifically, a model with negative feedback on the ERK pathway via secreted IL-10 accounts for the experimental data. Accordingly, the model predicts sustained phospho-p38 dynamics, whereas ERK activation changes from transient to sustained when the concentration of immunomodulatory protein of Acanthocheilonema viteae increases. We show that IL-10 can regulate its own production in an autocrine fashion, and that ERK and p38 control IL-10 amplitude, duration and steady state. We also show that p38 affects ERK via secreted IL-10 (autocrine crosstalk). These findings demonstrate how convergent signalling pathways may differentially control kinetic properties of the IL-10 signal.

Helminth infections are commonly associated with a Th2 immune response, yet only a few parasite molecules involved in triggering such immune responses have been identified. Here, we describe the Th2-skewing property of calreticulin of Heligmosomoides polygyrus (HpCRT). HpCRT is a secreted protein most abundantly expressed by tissue invasive larvae (L4). Native HpCRT purified from adult worm extract (nHpCRT) stimulated robust IL-4 release from CD4(+) T cells of H. polygyrus infected mice. Interestingly, CD4(+) T cells also produced significant amounts of IL-10 while IFN-gamma was not detectable. Likewise, immunization with recombinant HpCRT (rHpCRT) without extrinsic adjuvant led predominantly to a specific IL-4 production implying the innate ability of HpCRT to drive Th2 responses. The triggering of a Th2-skewed immune response to rHpCRT is corroborated by the induction of HpCRT-specific IgG1 and IgE antibodies. Furthermore, rHpCRT bound to scavenger receptor type A (SR-A) on dendritic cells, and interaction of HpCRT with SR-A led to internalization of HpCRT that could be partially blocked by competition with SR-A ligands as well as with an anti-SR-A monoclonal antibody. Hence, our data imply that nematode calreticulin interacts with a mammalian scavenger receptor and at the same time induces a Th2 response.

Plasmodium sporozoites traverse Kupffer cells on their way into the liver. Sporozoite contact does not elicit a respiratory burst in these hepatic macrophages and blocks the formation of reactive oxygen species in response to secondary stimuli via elevation of the intracellular cAMP concentration. Here we show that increasing the cAMP level with dibutyryl cyclic adenosine monophosphate (db-cAMP) or isobutylmethylxanthine (IBMX) also modulates cytokine secretion in murine Kupffer cells towards an overall anti-inflammatory profile. Stimulation of Plasmodium yoelii sporozoite-exposed Kupffer cells with lipopolysaccharide or IFN-gamma reveals down-modulation of TNF-alpha, IL-6 and MCP-1, and up-regulation of IL-10. Prerequisite for this shift of the cytokine profile are parasite viability and contact with Kupffer cells, but not invasion. Whilst sporozoite-exposed Kupffer cells become TUNEL-positive and exhibit other signs of apoptotic death such as membrane blebbing, nuclear condensation and fragmentation, sporozoites remain intact and appear to transform to early exo-erythrocytic forms in Kupffer cell cultures. Together, the in vitro data indicate that Plasmodium possesses mechanisms to render Kupffer cells insensitive to pro-inflammatory stimuli and eventually eliminates these macrophages by forcing them into programmed cell death.

Malaria sporozoites must cross at least two cell barriers to reach their initial site of replication in the mammalian host. After transmission into the skin by an infected mosquito, they migrate towards small dermal capillaries, traverse the vascular endothelial layer, and rapidly home to the liver. To infect hepatocytes, the parasites must cross the sinusoidal cell layer, composed of specialized highly fenestrated sinusoidal endothelia and Kupffer cells, the resident macrophages of the liver (Fig. 1). The exact route Plasmodium sporozoites take to hepatocytes has been subject of controversial discussions for many years. Recent cell biological, microscopic, and genetic approaches have considerably enhanced our understanding of the initial events leading to the establishment of a malaria infection in the liver.

In order to identify secretory proteins as possible new vaccine candidates, a cDNA-library from E. tenella sporozoites was generated in yeast and was used to select secreted and surface proteins. Herein 191 clones were isolated and analysis of the nucleic acid sequences revealed 162 deduced open reading frames with a prediction for signal peptides. These sequences are characterized by high redundancy, comprising 25 unique protein fragments with a high degree of stage specificity. Only three sequences showed identical homology to already known E. tenella proteins. The majority, 16 fragments, revealed homology to known or hypothetical proteins, and six fragments had no sequence homologues in protein databases. In order to obtain optimised conditions for a DNA vaccination trial in chickens, with which our selected new sequences could be tested, we performed variant DNA immunisations with the well-characterized E. tenella antigen SO7. The cDNA of the SO7 antigen was subcloned into two different eucaryotic expression vectors, i.e. pcDNA3 and pVR1012. In addition, the SO7 sequence was fused to the stabilizing sequence of the enhanced green fluorescence protein (EGFP). All SO7 constructs induced a SO7 specific immune response after intramuscular application and no significant differences were found on using constructs with or without the EGFP fusion or with different vector systems. Full-length open reading frames from six selected Eimeria sequences were introduced into the eucaryotic expression vector pcDNA3. Subsequent immunisation trials revealed a decrease in parasite excretion for three constructs after challenge infection in comparison to the control animals. Our approach represents a rapid screening to identify and test putative new vaccine candidates from E. tenella sporozoites that could also be adopted to other apicomplexan parasites.

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.