Hiroko Asahi

The immunobiology of Th1 polarization in high‐pathology schistosomiasis

Summary:  Schistosomiasis is a serious global helminthic disease, in which the main immunopathology consists of a granulomatous and fibrosing reaction against tissue‐trapped parasite eggs. The severity of this inflammatory process, the product of a CD4+ T‐cell‐mediated immune response against parasite egg antigens, is, however, markedly uneven, both in human patients and among mouse strains in an experimental model. Severe schistosomiasis is associated with persistently elevated pro‐inflammatory T‐helper‐1 (Th1)‐type cytokines, whereas milder pathology is present when Th2 cytokines dominate. This scenario is supported by the pronounced pathology resulting from the obliteration of pathways that facilitate Th2 differentiation and by the development of more intense lesions in mouse strains that fail to downregulate the Th1 response. Genetically prone high‐pathology mice have a higher proportion of CD4+ T cells in lymph nodes and granulomas, in which the Th1 phenotype is driven by interleukin‐12; they also develop a dominant repertoire against peptide 234–246 of the major Sm‐p40 egg antigen, utilizing a strikingly restricted T‐cell receptor structure that involves Vα11.3β8. In turn, low‐pathology mice exhibit enhanced CD4+ T‐cell apoptosis, which contributes to limit pathology. The definition of distinctive immune profiles associated with polar forms of schistosomiasis opens opportunities for targeted immuno‐intervention in individuals suffering from or at risk of severe disease.

Schistosome infection stimulates host CD4(+) T helper cell and B-cell responses against a novel egg antigen, thioredoxin peroxidase.

ABSTRACT Egg granuloma formation during schistosome infections is mediated by CD4+ T helper (Th) cells sensitized to egg antigens; however, most of the relevant sensitizing egg antigens are still unknown. Here we show that schistosome thioredoxin peroxidase (TPx)-1 is a novel T- and B-cell egg antigen in schistosome-infected mice. CD4+ Th cell responses to fractionated egg components identified a significant response against a 26-kDa antigen; a partial amino acid sequence of this antigen was found to be identical to that of Schistosoma mansoni TPx-1. The native TPx-1 elicited significant proliferative responses as well as gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-5 secretion in CD4+ cells from 8.5-week-infected CBA and C57BL/6 mice. By comparison, recombinant TPx-1 elicited a smaller, more type 1-polarized response, with significant production of IFN-γ and IL-2, less IL-5, and essentially no IL-4. In C57BL/6 mice the responses to TPx-1 were relatively more prominent than that directed against the major egg antigen, Sm-p40, whereas in CBA mice the reverse was true. B-cell responses were also monitored in infected C57BL/6, C3H, CBA, and BALB/c mice. All strains had significant antibody levels against the TPx-1 protein, but the most significant antibody production ensued following parasite oviposition. TPx-1 was localized in eggs and shown to be secreted by eggs. The identification of egg antigens is important to understand the specific basis of granuloma formation in schistosome infections and may prove to be useful in strategies to ameliorate pathological responses.

Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.

Arginine‐dependent generation of reactive nitrogen intermediates is instrumental in the in vitro killing of protoscoleces of Echinococcus multilocularis by activated macrophages

The interaction between protoscoleces of Echinococcus multilocularis and activated murine macrophages was examined in this study. Marked protoscolicidal activity was displayed by peritoneal macrophages (PM) activated with interferon‐γ (IFN‐γ), or IFN‐y plus lipopolysaccharide. Pretreatment of the parasites with heat‐inactivated specific murine infection serum, but not with normal serum rendered them more susceptible to PM killing. NG‐monomethyl‐Larginine, a competitive inhibitor of L‐arginine completely inhibited the killing activity of activated PM. while reconstitution of arginine‐free medium with L‐arginine restored the killing properties of the activated PM. The results show that activated PM have the ability to kill E. multilocularis protoscoleces in vitro and suggest that reactive nitrogen intermediates (RNl) play an important role in the mechanism. An oxygen‐mediated mechanism did not appear to play a role because scavengers of reactive oxygen species did not reduce the killing activity. The arginine‐dependent killing mechanism was enhanced by superoxide dismutase (SOD), probably because SOD might prolong the effect of nitric oxide. Secretion of RNI by activated macrophages may be capable of a significant role in preventing of the dissemination of E. multilocularis infection in vivo.

Analysis of egg antigens inducing hepatic lesions in schistosome infection

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best-studied egg component is the Sm-p40 antigen. Sm-p40 elicits a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are characterized by severe egg-induced immunopathology. Two additional described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Other components, including moieties with molecular weights of 25 kDa (Sm-p25), 150/166 kDa (Sm-p155/166), and 29 kDa (Sm-GST29), are also found to stimulate specific T cells. These findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the hosts genetic background. A better knowledge of the principal immunogenic egg components is necessary to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

Schistosoma mansoni Phosphoenolpyruvate Carboxykinase, a Novel Egg Antigen: Immunological Properties of the Recombinant Protein and Identification of a T-Cell Epitope

ABSTRACT In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4+ T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4+ Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4+ Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4+ Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4+ Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis.

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the hosts genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

BackgroundIn 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.MethodsThe prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.ResultsAll successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00).ConclusionThe pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.

Plasmodium falciparum: Chemically defined medium for continuous intraerythrocytic growth using lipids and recombinant albumin.

Dioleoylphosphatidylcholine and other phosphatidylcholines containing different fatty acid moieties were found to increase the ability of nonesterified fatty acids (NEFA) to sustain continuous intraerythrocytic growth of Plasmodium falciparum in the presence of specific proteins. Other phospholipids, including phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid, were beneficial to parasite growth. Different combinations and concentrations of NEFA tested in the presence of phospholipids and bovine albumin had variable effects on parasite growth. The most effective combination for promoting parasite growth consisted of 30 microg/ml cis-9-octadecenoic acid (oleic acid) plus 15 microg/ml hexadecanoic acid (palmitic acid). Recombinant human albumin could replace bovine or human albumin in culture media enriched with structurally defined lipids. This study therefore established a chemically defined culture medium suitable for sustaining the growth of P. falciparum.

Perturbation of copper homeostasis is instrumental in early developmental arrest of intraerythrocytic Plasmodium falciparum

BackgroundMalaria continues to be a devastating disease. The elucidation of factors inducing asexual growth versus arrest of Plasmodium falciparum can provide information about the development of the parasite, and may help in the search for novel malaria medication. Based on information from genome-wide transcriptome profiling of different developmental stages of P. falciparum, we investigated the critical importance of copper homeostasis in the developmental succession of P. falciparum with regard to three aspects of copper function. These were:1) inhibition of copper-binding proteins, 2) copper-ion chelation, and 3) down-regulated expression of genes encoding copper-binding proteins associated with a specific growth-promoting factor.ResultsInhibition of copper-binding proteins with tetrathiomolybdate (TTM) caused cessation of growth of the parasite. TTM arrested the parasite irreversibly during the trophozoite to schizont stage progression. Target molecules for TTM may be present in P. falciparum. The involvement of copper ions in developmental arrest was also investigated by copper-ion chelating methods, which indicated a critical function of reduced copper ions (Cu1+) in the parasite during the early developmental stage. Copper ions, not only in the parasite but also in host cells, were targets of the chelators. Chelation of Cu1+caused blockage of trophozoite progression from the ring stage. Profound growth arrest was detected in parasites cultured in a chemically defined medium containing hexadecanoic acid alone as a growth-promoting factor. This developmental arrest was associated with down-regulated expression of genes encoding copper-binding proteins. Cis-9-octadecenoic acid completely prevented the down-regulation of gene expression and developmental arrest that were observed with the use of hexadecanoic acid.ConclusionsThe critical importance of copper homeostasis in early developmental stages of P. falciparum was confirmed. Perturbation of copper homeostasis induced profound and early developmental arrest of P. falciparum. These findings should help to elucidate the mechanisms behind the development of P. falciparum, and may be applied in the development of effective antimalarial strategies.