Recruitment of hepatic macrophages from monocytes is independent of IL-4Rα but is associated with ablation of resident macrophages in schistosomiasis.

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL-4 receptor α-chain (IL-4Rα)-signalling in the dynamics of liver Mφ responses. We observed that IL-4Rα signalling was dispensable for the recruitment of Ly6Chi monocytes and for their conversion into F4/80hi CD64hi CD11bhi Mφ. Moreover, while IL-4Rα provided an AAMφ phenotype to liver F4/80hi CD64hi CD11bhi Mφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80hi CD64hi CD11blo Mφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169+ cells in naive mice resulted in the accumulation of F4/80hi CD64hi CD11bhi Mφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80hi CD64hi CD11bhi Mφ with lyz2-dependent IL-4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.

Helminth-induced IL-4 expands bystander memory CD8+ T cells for early control of viral infection.

Infection with parasitic helminths can imprint the immune system to modulate bystander inflammatory processes. Bystander or virtual memory CD8+ T cells (TVM) are non-conventional T cells displaying memory properties that can be generated through responsiveness to interleukin (IL)-4. However, it is not clear if helminth-induced type 2 immunity functionally affects the TVM compartment. Here, we show that helminths expand CD44hiCD62LhiCXCR3hiCD49dlo TVM cells through direct IL-4 signaling in CD8+ T cells. Importantly, helminth-mediated conditioning of TVM cells provided enhanced control of acute respiratory infection with the murid gammaherpesvirus 4 (MuHV-4). This enhanced control of MuHV-4 infection could further be explained by an increase in antigen-specific CD8+ T cell effector responses in the lung and was directly dependent on IL-4 signaling. These results demonstrate that IL-4 during helminth infection can non-specifically condition CD8+ T cells, leading to a subsequently raised antigen-specific CD8+ T cell activation that enhances control of viral infection.

Type I interferon is required for T helper (Th) 2 induction by dendritic cells.

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN-I) in enabling this process. An IFN-I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN-I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1-/- mice were incapable of initiating Th2 responses in vivo These data demonstrate for the first time that the influence of IFN-I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.

Experimental hookworm infection and gluten microchallenge promote tolerance in celiac disease.

BACKGROUND: Celiac disease (CeD) is a common gluten-sensitive autoimmune enteropathy. A gluten-free diet is an effective treatment, but compliance is demanding; hence, new treatment strategies for CeD are required. OBJECTIVE: Parasitic helminths hold promise for treating inflammatory disorders, so we examined the influence of experimental hookworm infection on the predicted outcomes of escalating gluten challenges in CeD subjects. METHODS: A 52-week study was conducted involving 12 adults with diet-managed CeD. Subjects were inoculated with 20 Necator americanus larvae, and escalating gluten challenges consumed as pasta were subsequently administered: (1) 10 to 50 mg for 12 weeks (microchallenge); (2) 25 mg daily + 1 g twice weekly for 12 weeks (GC-1g); and (3) 3 g daily (60-75 straws of spaghetti) for 2 weeks (GC-3g). Symptomatic, serologic, and histological outcomes evaluated gluten toxicity. Regulatory and inflammatory T cell populations in blood and mucosa were examined. RESULTS: Two gluten-intolerant subjects were withdrawn after microchallenge. Ten completed GC-1g, 8 of whom enrolled in and completed GC-3g. PRIMARY OUTCOMES: median villous height-to-crypt depth ratios (2.60-2.63; P = .98) did not decrease as predicted after GC-1g, and the mean IgA-tissue transglutaminase titers declined, contrary to the predicted rise after GC-3g. SECONDARY OUTCOMES: quality of life scores improved (46.3-40.6; P = .05); celiac symptom indices (24.3-24.3; P = .53), intra-epithelial lymphocyte percentages (32.5-35.0; P = .47), and Marsh scores were unchanged by gluten challenge. Intestinal T cells expressing IFNγ were reduced following hookworm infection (23.9%-11.5%; P = .04), with corresponding increases in CD4(+) Foxp3(+) regulatory T cells (0.19%-1.12%; P = .001). CONCLUSIONS: Necator americanus and gluten microchallenge promoted tolerance and stabilized or improved all tested indices of gluten toxicity in CeD subjects.

Genome of the human hookworm Necator americanus.

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.

Lipid core peptide targeting the cathepsin D hemoglobinase of Schistosoma mansoni as a component of a schistosomiasis vaccine.

The self-adjuvanting lipid core peptide (LCP) system offers a safe alternative vaccine delivery strategy, eliminating the need for additional adjuvants such as CpG Alum. In this study, we adopted the LCP as a scaffold for an epitope located on the surface of the cathepsin D hemoglobinase (Sm-CatD) of the human blood fluke Schistosoma mansoni. Sm-CatD plays a pivotal role in digestion of the fluke's bloodmeal and has been shown to be efficacious as a subunit vaccine in a murine model of human schistosomiasis. Using molecular modeling we showed that S. mansoni cathepsin D possesses a predicted surface exposed α-helix (A263K) that corresponds to an immunodominant helix and target of enzyme-neutralizing antibodies against Necator americanus APR-1 (Na-APR-1), the orthologous protease and vaccine antigen from blood-feeding hookworms. The A263K epitope was engineered as two peptide variants, one of which was flanked at both termini with a coil maintaining sequence, thereby promoting the helical characteristics of the native A263K epitope. Some of the peptides were fused to a self-adjuvanting lipid core scaffold to generate LCPs. Mice were vaccinated with unadjuvanted peptides, peptides formulated with Freund's adjuvants, or LCPs. Antibodies generated to LCPs recognized native Sm-CatD within a soluble adult schistosome extract, and almost completely abolished its enzymatic activity in vitro. Using immunohistochemistry we showed that anti-LCP antibodies bound to the native Sm-CatD protein in the esophagus and anterior regions of the gastrodermis of adult flukes. Vaccines offer an alternative control strategy in the fight against schistosomiasis, and further development of LCPs containing multiple epitopes from this and other vaccine antigens should become a research priority.

Peptide-based subunit vaccine against hookworm infection.

Hookworms infect more people than HIV and malaria combined, predominantly in third world countries. Treatment of infection with chemotherapy can have limited efficacy and re-infections after treatment are common. Heavy infection often leads to debilitating diseases. All these factors suggest an urgent need for development of vaccine. In an attempt to develop a vaccine targeting the major human hookworm, Necator americanus, a B-cell peptide epitope was chosen from the apical enzyme in the hemoglobin digestion cascade, the aspartic protease Na-APR-1. The A(291)Y alpha helical epitope is known to induce neutralizing antibodies that inhibit the enzymatic activity of Na-APR-1, thus reducing the capacity for hookworms to digest hemoglobin and obtain nutrients. A(291)Y was engineered such that it was flanked on both termini by a coil-promoting sequence to maintain native conformation, and subsequently incorporated into a Lipid Core Peptide (LCP) self-adjuvanting system. While A(291)Y alone or the chimeric epitope with or without Freund's adjuvants induced negligible IgG responses, the LCP construct incorporating the chimeric peptide induced a strong IgG response in mice. Antibodies produced were able to bind to and completely inhibit the enzymatic activity of Na-APR-1. The results presented show that the new chimeric LCP construct can induce effective enzyme-neutralising antibodies in mice, without the help of any additional toxic adjuvants. This approach offers promise for the development of vaccines against helminth parasites of humans and their livestock and companion animals.

Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.

Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia.

The first autochthonous Leishmania infection in Australia was reported by Rose et al. (2004) and the parasite was characterised as a unique species. The host was the red kangaroo (Macropus rufus) but the transmitting vector was unknown. To incriminate the biological vector, insect trapping by a variety of methods was undertaken at two field sites of known Leishmania transmission. Collected sand flies were identified to species level and were screened for Leishmania DNA using a semi-quantitative real-time PCR. Collections revealed four species of sand fly, with a predominance of the reptile biter Sergentomyia queenslandi (Hill). However, no Leishmania-positive flies were detected. Therefore, alternative vectors were investigated for infection, giving startling results. Screening revealed that an undescribed species of day-feeding midge, subgenus Forcipomyia (Lasiohelea) Kieffer, had a prevalence of up to 15% for Leishmania DNA, with high parasitemia in some individuals. Manual gut dissections confirmed the presence of promastigotes and in some midges material similar to promastigote secretory gel, including parasites with metacyclic-like morphology. Parasites were cultured from infected midges and sequence analysis of the Leishmania RNA polymerase subunit II gene confirmed infections were identical to the original isolated Leishmania sp. Phylogenetic analysis revealed the closest known species to be Leishmania enriettii, with this and the Australian species confirmed as members of Leishmania sensu stricto. Collectively the results strongly suggest that the day-feeding midge (F. (Lasiohelea) sp. 1) is a potential biological vector of Leishmania in northern Australia, which is to our knowledge the first evidence of a vector other than a phlebotomine sand fly anywhere in the world. These findings have considerable implications in the understanding of the Leishmania life cycle worldwide.

Use of a single-nucleotide polymorphism genotyping system to demonstrate the unique epidemiology of methicillin-resistant Staphylococcus aureus in remote aboriginal communities.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.

Identification and characterization of Sarcoptes scabiei and Dermatophagoides pteronyssinus glutathione S-transferases: implication as a potential major allergen in crusted scabies.

The astigmatid mite Sarcoptes scabiei is the causative agent of scabies, a highly infectious parasitic disease of the skin. Although the mite causes marked hypersensitivity reactions, particularly in crusted (severe) scabies, little is known about the specific scabies mite molecules involved in such immunologic responses. We have identified six genes encoding scabies mite homologues of mu and delta-like glutathione S-transferases (GSTs) as well as novel house dust mite GSTs. A mu class S. scabiei GST was subcloned into a prokaryotic expression system. The purified recombinant protein rSsGST01 reacted strongly with IgE and IgG4 in sera from crusted scabies patients. This response was not observed with control antigens or with ordinary scabies and uninfested patient sera. In addition, the specific IgE response to rSsGST01 did not correlate with the total IgE level of the patient. These results suggest that GST may play a role in the pathophysiology associated with crusted scabies.

Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus.

To investigate whether genetic variants of A. fumigatus are found among clinical isolates, four isolates that were originally identified as poorly sporulating strains of Aspergillus fumigatus were subjected to molecular analysis. DNA sequence analysis of the alkaline protease genes of these isolates showed that each is genetically distinct and each shows substantial variation (7 to 11%) from the A. fumigatus nucleotide sequence. Subsequent morphological examination suggested that all of the isolates could be classified as Aspergillus viridinutans. To clarify the taxonomic status of these four clinical isolates and of two previously identified as atypical A. fumigatus isolates, partial beta-tubulin and 18S rRNA gene sequences were determined. Each of the six atypical strains had a unique beta-tubulin sequence, whereas the sequences of three standard isolates of A. fumigatus, which were included as controls, were identical to the published A. fumigatus beta-tubulin sequence. The very low level of DNA sequence variation detected in standard isolates of A. fumigatus compared with other isolates from members of Aspergillus section Fumigati suggests that it may be a relatively recently evolved species. The 18S rRNA gene of two of the atypical isolates differed from that of A. fumigatus at a single nucleotide position. Phylogenetic analyses do not support the classification of all of these isolates as A. viridinutans. Thus, some of these isolates represent new species which are potential opportunistic pathogens.

Acaricidal activity of Melaleuca alternifolia (tea tree) oil: in vitro sensitivity of sarcoptes scabiei var hominis to terpinen-4-ol.

OBJECTIVE: To compare the acaricidal activity of Melaleuca alternifolia (tea tree) oil (TTO) and some of its individual active components on the itch mite Sarcoptes scabiei var hominis. DESIGN: In vitro acaricide sensitivity assessment. SETTING: The Menzies School of Health Research laboratory, located near the Infectious Diseases Ward of the Royal Darwin Hospital, Australia, where patients are admitted and treated for crusted scabies. PARTICIPANTS: Scabies mites (S scabiei var hominis) were collected from a 20-year-old Aboriginal woman admitted to the Royal Darwin Hospital with crusted scabies. Interventions Within 3 hours of collection, scabies mites were placed in continuous direct contact with the TTO products and control acaricides and were observed at regular intervals. MAIN OUTCOME MEASURES: Percentage of mites dead at regular observation intervals between 5 minutes and 24 hours during continuous exposure to the TTO products and acaricides. RESULTS: The 5% TTO and active component terpinen-4-ol were highly effective in reducing mite survival times. Statistically significant differences in mite survival curves were observed for 5% TTO, 2.1% terpinen-4-ol, 5% permethrin, and ivermectin (100 microg/g of Emulsifying Ointment British Pharmacopoeia 88). In vivo effectiveness was also observed. CONCLUSIONS: Documentation of resistance against antiectoparasitic compounds is increasing. Reported S scabiei treatment failures with lindane, crotamiton, and benzyl benzoate, as well as likely emerging resistance to 5% permethrin and oral ivermectin, are of concern and advocate for the identification and development of novel acaricidal drugs. Tea tree oil is a membrane-active biocide extracted from the tree M alternifolia. It is a principal antimicrobial in a wide range of pharmaceuticals sold in Australia, with the main active component being oxygenated terpenoids. The results suggest that TTO has a potential role as a new topical acaricide and confirm terpinen-4-ol as the primary active component.