Immunopathogenesis of human schistosomiasis.

Schistosomiasis continues to be a significant cause of parasitic morbidity and mortality worldwide. This review considers the basic features of the pathology and clinical outcomes of hepatointestinal and genitourinary schistosomiasis, presents an overview of the numerous studies on animal models that have clarified many of the immunopathological features, and provides insight into our current understanding of the immunopathogenesis and genetic control of human schistosomiasis. In murine schistosomiasis, pathology is induced by a CD4(+) Th2 driven granulomatous response directed against schistosome eggs lodged in the host liver. The Th2 cytokines IL-4 and IL-13 drive this response, whereas IL-10, IL13Ralpha2, IFN-gamma and a subset of regulatory T-cells act to limit schistosome induced pathology. A variety of cell types including hepatic stellate cells, alternatively activated macrophages and regulatory T-cells have also been implicated in the pathogenesis of schistosomiasis. Current knowledge suggests the immunopathogenic mechanisms underlying human schistosomiasis are likely to be similar. The review also considers the future development of anti-pathology schistosome vaccines. As fibrosis is an important feature of many other diseases such as Crohn's disease and sarcoidosis, a comprehensive understanding of the cellular and molecular mechanisms involved in schistosomiasis may also ultimately contribute to the development an effective disease intervention strategy for other granulofibrotic diseases.

Comparative real-time PCR and enzyme analysis of selected gender-associated molecules in Schistosoma japonicum.

Schistosomes are complex parasitic helminths with discrete life-cycle stages, adapted for survival in their mammalian and snail hosts and the external aquatic environment. Recently, we described the fabrication and use of a microarray to investigate gender-specific transcription in Schistosoma japonicum. To address transcriptional differences, 8 gender-associated gene transcripts identified previously by the microarray analysis were selected for further study. First, differential transcription patterns were investigated in 4 developmental stages using real-time PCR. Subsequently, we undertook functional analysis of a subset of 4 transcripts encoding metabolic enzymes, so as to correlate gender-associated transcript levels with enzyme activity in protein extracts from adult worms. The 8 characterized molecules serve as a basis for further investigation of differential gene expression during the schistosome life-cycle and for studying the sexual dimorphism of adult worms. Continual refinement and annotation of the microarray used in the current study should support future work on these aspects.

Oral vaccination of mice with recombinant Schistosoma japonicum proteins induces specific anti-parasite antibodies and damage to adult worms after a challenge infection.

Mucosal immunisation by the oral route represents a cheap and simple method for delivering protective antigens to a host against gastrointestinal and respiratory pathogens. In the case of schistosome (bloodfluke) worms, 2 life-cycle stages may be exposed to the host's mucosa; the larval schistosomulum is exposed to the respiratory mucosa and, depending on the species, the egg may come into contact with the intestinal or urinogenital mucosa. Both IgA and some Isotypes of IgG have been implicated in protective immunity against schistosomiasis in humans and in experimental animal models. We have used a novel approach to determine whether schistosome-specific antibodies and protective immunity could be generated in mice by oral administration of bacterial lysates containing recombinant Schistosoma japonicum proteins. The mice produced specific antibodies to paramyosin and GST26, 2 important vaccine candidates for schistosomiasis, but there was no significant reduction in worm burdens in groups of mice immunised with either protein. Significantly, however, transmission electron microscopy revealed damage to the teguments of adult female and male S. japonicum worms obtained from mice vaccinated with recombinant paramyosin; there was also extensive damage to the tegument of male worms recovered from mice vaccinated with recombinant GST26. Our observations that oral vaccination with bacterial lysates containing recombinant proteins induced particular classes and subclasses of circulating antibodies with resultant damage to the surface of adult worms may have important implications for the future development of oral vaccines against a systemic infection such as schistosomiasis.

Immunolocalisation of the glutathione S-transferases, GST-26 and GST-28, within adult Schistosoma japonicum.

This paper describes the first ultrastructural immunolocalisation study of the 26-kDa and 28-kDa glutathione S-transferases within adult Schistosoma japonicum (GST-26 and GST-28). Polyclonal antibodies raised against GST-28 (in mice) and against GST-26 (in rabbits) were used to examine the distribution of the proteins within adult parasites. Both proteins were localised within the parenchymal region of the male parasite. Additionally, both proteins were present within parenchymal cells located between the vitelline glands of female parasites. There were no detectable levels of GST-26 or GST-28 on the surface or within the tegument matrix of either the male or female worms. Possible functions for GST-26 and GST-28 within S. japonicum and their significance as vaccine target molecules are addressed.

Ultrastructural analysis of the adult Schistosoma japonicum by lectin cytochemistry.

Eight lectin probes were used to detect a range of carbohydrate residues in the tegument matrix of Schistosoma japonicum. In addition, other areas of the parasite, such as the gut, vitelline glands and flame cells, were examined for carbohydrate residues. Some minor differences in the carbohydrate residue composition between tegument orientations and between the sexes were identified. Differences between the distribution of carbohydrate residues of S. japonicum examined in this study and previous reports of Schistosoma mansoni were also noted. This study further illustrates the high level of complexity within the tegument of the adult S. japonicum and has demonstrated differences between this species and the widely studied S. mansoni.

Immunolocalization of schistosome proteins.

This review discusses some of the recent advances in the characterization of potential vaccine molecules against Schistosoma japonicum, utilizing microscopy and immunocytochemistry methods. Microscopy has demonstrated the stage-specific expression of the muscle protein paramyosin onto the parasite surface, an important consideration as a vaccine target. Other potential vaccine component proteins examined include glutathione S-transferase (GST) and fatty acid binding protein (FABP); although not associated with the adult parasite surface, their localization to internal structures such as lipid droplets and regions of the female reproductive system have provided valuable insights into the biology of the parasite. Localization of the transport protein SGTP (schistosome glucose transporter protein) has demonstrated that the protein is more prevalent in the juvenile stages of the parasite development. This further highlights the diversity of the parasite life cycle. Using both light microscopy and transmission electron microscopy, the localization of a number of schistosome proteins has demonstrated the functions and significance of these proteins within the parasite. Molecular localization studies are crucial in understanding how and when a vaccine may work against the organism and may provide insights into which can be used in the design of future vaccines.

Biology of the schistosome lung-stage schistosomulum.

Past and more recent research has examined the ultrastructure, metabolism, cell biology, genomics and post-genomics of schistosome schistosomula. These areas are considered and discussed in this review with particular emphasis on (1) the early migration phases through the host, (2) interaction of the host immune response with the parasite surface, (3) glucose uptake mechanisms, and (4) defining the transcriptional profiles of lung-stage schistosomula compared with other developmental stages using microarrays. The microarray profiling studies suggest caution is required when considering the use of schistosomes obtained by in vitro means for molecular or biochemical studies.

Transcriptome profiling of lung schistosomula,in vitro cultured schistosomula and adult Schistosoma japonicum.

The schistosomulum is the main target of vaccine-induced protective immunity; however, most studies have utilized schistosomula produced by mechanical transformation of infective larvae followed by in vitro culture rather than larvae isolated directly from the lungs of infected mammals. Using transmission electron microscopy, we demonstrated that there was little difference in the ultrastructure of Schistosoma japonicum schistosomula obtained by the two methods. However, significant differences in gene expression profiles were apparent when we used an oligonucleotide microarray to compare the gene expression profiles of schistosomula obtained in vivo from lung tissue with those maintained in vitro, and with adult worms of S. japonicum. It is likely that host environmental factors, which cannot be reliably reproduced in vitro, do influence the growth, development and overall biology of schistosomes. Thus caution is urged when using in vitro-cultured schistosomes and mechanically transformed/cultured schistosomula in molecular, biochemical and immunological studies.

Microarrays: new tools to unravel parasite transcriptomes.

The ability to monitor the expression levels of thousands of genes in a single microarray experiment is a huge progression from conventional Northern blot analysis or PCR-based techniques. Microarrays can play a pivotal role in the mass screening of genes in a wide range of fields including parasitology. The relatively few parasites that can be readily cultured or isolated from a host, as compared with cell lines or tissue sources, makes microarray technology ideal for maximizing experimental results from a limiting source of starting material. Khan et al. (1999 a) commented in an early review of microarray technology " With this system in place, one can anticipate a time when data from thousands of gene expression experiments will be available for meta-analysis........leading to more robust results and subtle conclusions". Now in 2005, microarrays represent a very powerful resource that can play an important role in the characterization and annotation of the transcriptomes of many parasites of medical and veterinary importance.

The Role of Microscopy in the Investigation of Paramyosin as a Vaccine Candidate against Schistosoma japonicum.

There has been growing interest in paramyosin as a vaccine component to combat schistosomiasis. Immunological and molecular techniques have been used in the past to investigate the effectiveness of a paramyosin vaccine as an anti-schistosomal treatment. However, recent localization studies at ultrastructural and morphological levels have highlighted a number of questions concerning the role of paramyosin within schistosome parasites. Debates about how a non-surface protein such as paramyosin might provide protection against schistosome infections have recently been addressed by microscopy results. Immunolocalization studies have indicated multiple functions of paramyosin within the parasite and provided insights into how a vaccine may target the parasite, as discussed here by Geoffrey Gobert.

Improved ultrastructure of the desmosome-intermediate filament complex in MCF-7 breast cancer cells.

We report on the application of variations to the traditional TEM processing methods, which provide improved clarity of the desmosome-cytoskeleton complex of MCF-7 human breast cancer cells. A more comprehensive understanding of the ultrastructure is presented, which in the past has been demonstrated in diagrammatic form based on numerous electron micrographs. Ultrastructural analysis shows that intermediate filament bundles do not terminate at the desmosome structure, but instead are continuous into the cytoplasm. Furthermore only a minor proportion of individual filaments are in actual contact with the desmosome plaque. Intermediate filaments were also observed throughout the cytoplasm and to the surface of the nuclear membrane. Extraction protocols allowed clear identification of other cellular features such as nuclear pores, which are approximately 80-85 nm in diameter, and were best viewed in sections cut tangentially to the nuclear surface.

Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum.

This paper describes the first localization study of the 14.7 kDa fatty acid-binding protein in Schistosoma japonicum (SjFABPc) using transmission electron microscopy. A polyclonal antibody raised against recombinant Sj-FABPc was used in combination with a colloidal gold marker to determine the distribution of the protein within adult parasites. Sj-FABPc was localized within lipid droplets below the subtegumental region of the male parasite. Additionally, Sj-FABPc was present in the vitelline droplets of the vitelline glands of female parasites. There were no detectable levels of Sj-FABPc on the surface or within the tegument of male or female parasites. Possible functions of Sj-FABPc within S. japonicum and the relevance of these immunolocalization findings in light of the recent reports that the homologue Sm-FABPc is an important anti-S. mansoni vaccine target molecule are also discussed.

Schistosoma japonicum: immunolocalization of paramyosin during development.

This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy.

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.