Sublethal Exposure to Plasma-Activated Water Influences the Morphological Characteristics, Phagocytic Ability, and Virulence of Acanthamoeba castellanii.

PURPOSE: This study aimed to examine the ultrastructure, cytotoxicity, phagocytosis, and antioxidant responses of Acanthamoeba castellanii trophozoites exposed to sublethal plasma-activated water. METHODS: Trophozoites were exposed to a sublethal treatment of PAW and compared to untreated viable trophozoites via adhesion assays on macrophage monolayers, osmo- and thermotolerance tests. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. RESULTS: In PAW-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Moreover, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by PAW-treated trophozoites than untreated cells. Activities of superoxide dismutase and catalase and catalase were significantly greater in the treated trophozoites, and the glutathione and glutathione/glutathione disulfide were significantly lower in the PAW-treated cells. Exposure to PAW also significantly increased the malondialdehyde level and total antioxidant capacity. Treatment with PAW led to significantly higher expression of virulent genes like MBP, CP3, and SEP. CONCLUSION: PAW is a double-edged sword against A. castellanii. PAW is an effective antiamoebic agent in proper usage, whereas its sublethal exposure may reduce its effectiveness and increase amoebas' pathogenicity. An agent's adequate concentration and exposure time are essential to achieve optimum results.

Exposure to sublethal concentrations of chlorine enhances the cytotoxicity of Acanthamoeba castellanii.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections in humans and animals. Many studies are being conducted to find effective compounds against amoebae, but their sublethal concentration effects on surviving amoebae seem to have been overlooked. Chlorine is a common disinfection agent commonly added to public water facilities and supplies. In this study, the cytopathic and phagocytic properties of Acanthamoeba castellanii trophozoites following exposure to sublethal concentrations of chlorine were examined. Two hours of exposure to 5 ppm hypochlorite calcium was considered the sublethal concentration for A. castellanii trophozoites. To compare the pathogenic potential of treated and untreated Acanthamoeba trophozoites, cytotoxicity, adhesion assays in RAW 264.7 macrophages, osmo, and thermotolerance tests were carried out. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the mRNA expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. In all the experiments, untreated trophozoites were considered the control. In comparison to untreated trophozoites, in chlorine-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Besides, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by chlorine-treated trophozoites than untreated cells. The treated and untreated cells had significantly different glutathione and glutathione/glutathione disulfide ratios. Antioxidant enzyme activities, total antioxidant capacity, and malondialdehyde levels were increased significantly in chlorine-treated cells. Quantifying mRNA expression in chlorine-treated trophozoites revealed that virulence genes were upregulated. Chlorine can form resistance and virulent amoebae if it is not used at a proper concentration and exposure time. Identification of stress responses, their mechanisms in Acanthamoeba, and their relation to amoeba virulence would give us a better perception of their pathophysiology.

Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.

The opportunistic protist Acanthamoeba, which interacts with other microbes such as bacteria, fungi, and viruses, shows significant similarity in cellular and functional aspects to human macrophages. Intracellular survival of microbes in this microbivorous amoebal host may be a crucial step for initiation of infection in higher eukaryotic cells. Therefore, Acanthamoeba-microbe adaptations are considered an evolutionary model of macrophage-pathogen interactions. This paper reviews Acanthamoeba as an emerging human pathogen and different ecological interactions between Acanthamoeba and microbes that may serve as environmental training grounds and a genetic melting pot for the evolution, persistence, and transmission of potential human pathogens.

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection.

INTRODUCTION: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. AIMS: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. METHODOLOGY: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). RESULTS: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. CONCLUSIONS: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection.

Acanthamoeba, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. Acanthamoeba feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that Acanthamoeba acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within Acanthamoeba following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in clinical isolates of Acanthamoeba, whereas Legionella, adenovirus, mimivirus, and unidentified bacteria (Candidatus) were often identified in environmental Acanthamoeba. Increasing resistance of Acanthamoeba associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in Acanthamoeba, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of Acanthamoeba infections.

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Acanthamoeba proteases contribute to macrophage activation through PAR1 , but not PAR2.

AIM: Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is known to affect virulence. Herein, the ability of Acanthamoeba trophozoite proteases, of either the laboratory Neff strain or a recently isolated clinical strain, to stimulate IL-12 and IL-6 and to activate protease-activated receptors, PAR1 and PAR2 expressed on murine macrophages, was investigated. METHOD AND RESULTS: Using selected protease inhibitors, leupeptin and E64, we showed that Acanthamoeba proteases can stimulate IL-12 and IL-6 by murine macrophages. Subsequently, using specific antagonists to inhibit PAR1 , and bone marrow-derived macrophages from PAR2 gene-deficient mice, we demonstrate that PAR1 , but not PAR2 contributes to macrophage IL-12 production in response to Acanthamoeba. In contrast, Acanthamoeba-induced IL-6 production is PAR1 and PAR2 independent. CONCLUSION: This study shows for the first time the involvement of PARs, expressed on macrophages, in the response to Acanthamoeba trophozoites and might provide useful insight into Acanthamoeba infections and their future treatments.

Structural and functional studies of histidine biosynthesis in Acanthamoeba spp. demonstrates a novel molecular arrangement and target for antimicrobials.

Acanthamoeba is normally free-living, but sometimes facultative and occasionally opportunistic parasites. Current therapies are, by necessity, arduous and yet poorly effective due to their inabilities to kill cyst stages or in some cases to actually induce encystation. Acanthamoeba can therefore survive as cysts and cause disease recurrence. Herein, in pursuit of better therapies and to understand the biochemistry of this understudied organism, we characterize its histidine biosynthesis pathway and explore the potential of targeting this with antimicrobials. We demonstrate that Acanthamoeba is a histidine autotroph, but with the ability to scavenge preformed histidine. It is able to grow in defined media lacking this amino acid, but is inhibited by 3-amino-1,2,4-triazole (3AT) that targets Imidazoleglycerol-Phosphate Dehydratase (IGPD) the rate limiting step of histidine biosynthesis. The structure of Acanthamoeba IGPD has also been determined in complex with 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate [(R)-C348], a recently described novel inhibitor of Arabidopsis thaliana IGPD. This compound inhibited the growth of four Acanthamoeba species, having a 50% inhibitory concentration (IC50) ranging from 250-526 nM. This effect could be ablated by the addition of 1 mM exogenous free histidine, but importantly not by physiological concentrations found in mammalian tissues. The ability of 3AT and (R)-C348 to restrict the growth of four strains of Acanthamoeba spp. including a recently isolated clinical strain, while not inducing encystment, demonstrates the potential therapeutic utility of targeting the histidine biosynthesis pathway in Acanthamoeba.

Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.

Characterisation of sterol biosynthesis and validation of 14α-demethylase as a drug target in Acanthamoeba.

The soil amoebae Acanthamoeba causes Acanthamoeba keratitis, a severe sight-threatening infection of the eye and the almost universally fatal granulomatous amoebic encephalitis. More effective treatments are required. Sterol biosynthesis has been effectively targeted in numerous fungi using azole compounds that inhibit the cytochrome P450 enzyme sterol 14α-demethylase. Herein, using Gas Chromatography Mass Spectrometry (GCMS), we demonstrate that the major sterol of Acanthamoeba castellanii is ergosterol and identify novel putative precursors and intermediate sterols in its production. Unlike previously reported, we find no evidence of 7-dehydrostigmasterol or any other phytosterol in Acanthamoeba. Of five azoles tested, we demonstrate that tioconazole and voriconazole have the greatest overall inhibition for all isolates of Acanthamoeba castellanii and Acanthamoeba polyphaga tested. While miconazole and sulconazole have intermediate activity econazole is least effective. Through GCMS, we demonstrate that voriconazole inhibits 14α-demethylase as treatment inhibits the production of ergosterol, but results in the accumulation of the lanosterol substrate. These data provide the most complete description of sterol metabolism in Acanthamoeba, provide a putative framework for their further study and validate 14α-demethylase as the target for azoles in Acanthamoeba.

Acanthamoeba Activates Macrophages Predominantly through Toll-Like Receptor 4- and MyD88-Dependent Mechanisms To Induce Interleukin-12 (IL-12) and IL-6.

Acanthamoeba castellanii is a ubiquitous free-living amoeba with a worldwide distribution that can occasionally infect humans, causing particularly severe infections in immunocompromised individuals. Dissecting the immunology of Acanthamoeba infections has been considered problematic due to the very low incidence of disease, despite the high exposure rates. While macrophages are acknowledged as playing a significant role in Acanthamoeba infections, little is known about how this facultative parasite influences macrophage activity. Therefore, in this study we investigated the effects of Acanthamoeba on the activation of resting macrophages. Consequently, murine bone marrow-derived macrophages were cocultured with trophozoites of either the laboratory Neff strain or a clinical isolate of A. castellaniiIn vitro real-time imaging demonstrated that trophozoites of both strains often established evanescent contact with macrophages. Both Acanthamoeba strains induced a proinflammatory macrophage phenotype characterized by the significant production of interleukin-12 (IL-12) and IL-6. However, macrophages cocultured with the clinical isolate of Acanthamoeba produced significantly less IL-12 and IL-6 than the Neff strain. The utilization of macrophages derived from MyD88-, TRIF-, Toll-like receptor 2 (TLR2)-, TLR4-, and TLR2/4-deficient mice indicated that Acanthamoeba-induced proinflammatory cytokine production was through MyD88-dependent, TRIF-independent, TLR4-induced events. This study shows for the first time the involvement of TLRs expressed on macrophages in the recognition of and response to Acanthamoeba trophozoites.

Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

Use of nanoscale mechanical stimulation for control and manipulation of cell behaviour.

The ability to control cell behaviour, cell fate and simulate reliable tissue models in vitro remains a significant challenge yet is crucial for various applications of high throughput screening e.g. drug discovery. Mechanotransduction (the ability of cells to convert mechanical forces in their environment to biochemical signalling) represents an alternative mechanism to attain this control with such studies developing techniques to reproducibly control the mechanical environment in techniques which have potential to be scaled. In this review, the use of techniques such as finite element modelling and precision interferometric measurement are examined to provide context for a novel technique based on nanoscale vibration, also known as "nanokicking". Studies have shown this stimulus to alter cellular responses in both endothelial and mesenchymal stem cells (MSCs), particularly in increased proliferation rate and induced osteogenesis respectively. Endothelial cell lines were exposed to nanoscale vibration amplitudes across a frequency range of 1-100 Hz, and MSCs primarily at 1 kHz. This technique provides significant potential benefits over existing technologies, as cellular responses can be initiated without the use of expensive engineering techniques and/or chemical induction factors. Due to the reproducible and scalable nature of the apparatus it is conceivable that nanokicking could be used for controlling cell behaviour within a wide array of high throughput procedures in the research environment, within drug discovery, and for clinical/therapeutic applications. STATEMENT OF SIGNIFICANCE: The results discussed within this article summarise the potential benefits of using nanoscale vibration protocols for controlling cell behaviour. There is a significant need for reliable tissue models within the clinical and pharma industries, and the control of cell behaviour and stem cell differentiation would be highly beneficial. The full potential of this method of controlling cell behaviour has not yet been realised.

Biochemical and immunological characterization of Toxoplasma gondii macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 µmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

Toxoplasma gondii HLA-B*0702-restricted GRA7(20-28) peptide with adjuvants and a universal helper T cell epitope elicits CD8(+) T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice.

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Selective inhibition and augmentation of alternative macrophage activation by progesterone.

Progesterone is the female sex hormone necessary for the maintenance of pregnancy, and is known to modulate macrophage activation. However, studies have concentrated exclusively on the ability of progesterone to negatively regulate the innate and classical pathways of activation, associated with nitric oxide (NO) and interleukin (IL)-12 production. Our aim was to examine the ability of progesterone to modulate alternative macrophage activation. Bone marrow cells were isolated and differentiated from male BALB/c mice, exposed to varying concentrations of progesterone and stimulated with lipopolysaccharide (LPS) (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. Our present study demonstrates that progesterone not only down-regulates inducible nitric oxide synthase 2 (iNOS) activity in macrophages but also arginase activity, in a dose-dependent manner, independent of the stimuli, whether it is induced by LPS (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. The ability of progesterone to down-modulate IL-4-induced cell surface expression of the mannose receptor further suggested a negative regulation of alternative macrophage activation by this hormone. Analysis of mRNA expression, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), of genes associated with innate and alternative macrophage activation revealed that progesterone down-regulated LPS-induced macrophage nos2, argI and p40 (IL-12/IL-23) expression and IL-4-induced argI, mrc-1 and fizz1 expression. However, progesterone up-regulated IL-4-induced macrophage expression of ym1, while dectin-1 expression remained unaltered. Following treatment of macrophages with LPS and IL-4 in combination a similar pattern was observed, with the exception that progesterone up-regulated macrophage expression of fizz1 as well as ym1 and did not modify mrc-1 expression. Our data demonstrate for the first time that a hormone has the ability to regulate selectively the expression of different genes associated with alternative macrophage activation.

Drug target identification, validation, characterisation and exploitation for treatment of Acanthamoeba (species) infections.

New more efficacious antimicrobials as required for the treatment of Acanthamoeba infections as those currently available require arduous treatment regimes, are not always effective and are poorly active against the cystic stages. Herein, we review potential drug targets including tubulin, alternative oxidase, amino acid biosynthesis and myosin. In addition, we review the literature for current missing tools and resources for the identification, validation and development of new antimicrobials for this organism. Additional targets should come to light through a concerted genome sequencing effort.

IL-33 receptor (T1/ST2) signalling is necessary to prevent the development of encephalitis in mice infected with Toxoplasma gondii.

T1/ST2 is an immunoregulatory protein of the IL-1 receptor family that has recently been reported as being a component of the IL-33 receptor. IL-33 is a newly described cytokine known to amplify the Th2 response and reduce production of Th1 cytokines. The function of T1/ST2 during Toxoplasma gondii infection is as yet undescribed. Given the requirement of a balanced type 1/type 2 response for effective control of parasite number and immunopathology, it is likely that T1/ST2 may play a part in aiding this process. Accordingly, we have shown that T1/ST2 mRNA transcripts are upregulated in the brains of mice infected with T. gondii and that mice deficient in T1/ST2 demonstrated increased susceptibility to infection with T. gondii that correlated with increased pathology and greater parasite burden in the brains. Real-time PCR analysis of cerebral cytokine levels revealed increased mRNA levels of iNOS, IFN-gamma and TNF-alpha in infected T1/ST2(-/-) mice. These effects were independent of changes in IL-10 production. This study provides the first evidence of a specific role for IL-33 receptor signalling in the brain as well as highlighting the requirement of this mechanism in limiting infection with an intracellular parasite.

Acanthamoeba alternative oxidase genes: identification, characterisation and potential as antimicrobial targets.

Alternative oxidase (AOX) is a mitochondrial protein that acts as an alternative terminal oxidase to the conventional cytochrome oxidases. It is present in certain prokaryotes, plants, fungi and some protozoa but absent in mammals. AOX activity has previously been described in Acanthamoeba, although no genetic evidence has been reported. Herein, two AOX (AcAOX) genes designated isoforms A and B, were obtained from Acanthamoeba castellanii by a combination of degenerate PCR from cDNA and a series of 5' and 3' rapid amplification of cDNA ends. The corresponding genomic sequences of these AcAOXs were also obtained. Each gene spans six exons over a region of 1607 and 1619bp, respectively. Isoforms A and B have open reading frames of 1113 and 1125bp, respectively. Each encodes a protein with a predicted molecular weight of 42kDa. Each AcAOX protein has a predicted cleavable mitochondrial targeting sequence. The full-length AcAOX is functionally active as it complements hemL-deficient Escherichia coli and inhibited by the inhibitor of AOX, salicylhydroxamic acid (SHAM). SHAM is effective against A. castellanii and Acanthamoeba polyphaga only when used in conjunction with antimycin A, an inhibitor of the conventional cytochrome respiratory pathway. Transcripts for AcAOX are increased during the encystment process, indicating a possible role for alternative respiration during stress.

Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors.

Macrophage function has been demonstrated to be subject to modulation by progesterone. However, as this steroid hormone can act through the glucocorticoid receptor as well as the progesterone receptor, the mechanism of action has not been precisely characterized. To determine the mode of action, we compared the ability of progesterone, norgestrel (a synthetic progesterone-receptor-specific agonist) and dexamethasone (a synthetic glucocorticoid receptor agonist) to modulate macrophage function following stimulation of the Toll-like receptor-4 (TLR-4) ligand lipopolysaccharide (LPS). The results demonstrate that following stimulation of TLR-4 with LPS and cotreatment with either progesterone or dexamethasone, but not norgestrel, there is a significant reduction in nitric oxide (NO) production, indicating that this progesterone-mediated effect is through ligation of the glucocorticoid receptor. In contrast, LPS-induced interleukin-12 (IL-12) production could be downregulated by all three steroids, indicating that ligation by progesterone of either the glucocorticoid or the progesterone receptors or both could mediate this effect. While progesterone downmodulated NO-mediated killing of Leishmania donovani by activated macrophages in vitro, most probably via the glucocorticoid receptor, it had little effect on Toxoplasma gondii growth in these cells. This would suggest that progesterone-mediated increased susceptibility to T. gondii during pregnancy is more likely to be related to the ability of the hormone to downregulate IL-12 production and a type-1 response utilizing the progesterone as well as the glucocorticoid receptors.