Immune responses directed at egg proteins during experimental infection with the liver fluke Fasciola hepatica.

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. Unlike the well characterized schistosomes, only the adult and juvenile stages of F. hepatica are implicated in disease, whereas the freely voided egg is not thought to contribute to host-parasite interactions. We investigated specific immune responses to soluble F. hepatica egg proteins (SFHEP), during a 14-week experimental infection, demonstrating significant increases in anti-SFHEP IgG1 (P = 0.001), transforming growth factor beta-1 (P = 0.008) and IL-10 (P < 0.001) titres at the onset of egg production. Western blot analysis of soluble SFHEP demonstrates that protein bands migrating at 61.6, 54.8 and 44 kDa become sero-reactive before the appearance of eggs within host faeces. Therefore, expression of some egg-associated proteins indicates progression to chronic disease. Antigenic bands were investigated through mass spectrometry, identifying a protein disulphide isomerase (PDI) (61.6 kDa), an enolase and ferritin-related proteins (54.8 kDa), and a cocktail of dehydrogenases (44 kDa). Biochemical analysis of egg secretions reveals proteolytic activity, which increases over time, indicating that proteases may be continually secreted during the course of egg maturation. The implications of egg-specific immune responses and proteolytic secretions are further discussed.

Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Analysing proteomic data.

The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.

The detection of antibodies against Schistosoma mansoni soluble egg antigens (SEA) and CEF6 in ELISA, before and after chemotherapy.

Circulating IgG antibody reactivity and excreted egg counts were investigated in 489 Kenyans given chemotherapy for schistosomiasis mansoni. Antibody reactivity was measured in ELISA, using either unfractionated aqueous soluble constituents of Schistosoma mansoni eggs (SEA) or CEF6 (a soluble fraction of S. mansoni eggs containing two cationic antigens) as the antigen source. Antibody reactivity for each antigen source was strongly associated with egg counts, both pre- and post-treatment. Approximately 6 months after chemotherapy, egg counts were zero in 84% of the subjects. The mean optical densities (OD) measured in the post-treatment ELISA were 60% (CEF6) or 45% (SEA) lower than the pre-treatment values, the reduction in the OD with CEF6 as antigen source being significantly greater than that observed with SEA (P <0.001). The usefulness of an assay for antibody reactivity in monitoring the effects of the treatment of schistosomiasis is discussed.

Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans.

Using ELISA we provide direct evidence that the midgut defensins of the blood-sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post-transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS-stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans.

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.

Periodate-sensitive immunological cross-reactivity between keyhole limpet haemocyanin (KLH) and serodiagnostic Schistosoma mansoni egg antigens.

Both CEF6, a cation-exchange fraction of soluble Schistosoma mansoni egg antigens (SEA), composed of the 2 antigens, alpha-1 and omega-1, and haemocyanin from the keyhole limpet, Megathura crenulata, have shown potential for immunodiagnosis of human schistosomiasis. Possible cross-reactivity between antigens in SEA and keyhole limpet haemocyanin (KLH) was explored by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with KLH, SEA, CEF6, alpha-1, omega-1, or egg antigen k5. Both immunoassays revealed a high degree of serological cross-reactivity between the schistosome egg antigens and KLH, much of it due to sodium periodate-sensitive epitopes. Cross-reactivity with schistosome antigens with proven diagnostic efficacy may thus, in part, explain the usefulness of KLH for the diagnosis of human schistosomiasis mansoni.

Diagnosis of schistosomiasis: antibody detection, with notes on parasitological and antigen detection methods.

Schistosomiasis remains a serious world-wide public health problem with a still unfulfilled need for routine cost-effective methods of diagnosis. Such methods are required not only for people in endemic areas, but increasingly for tourists who may have become infected during visits to such places. This article reviews the wide range of immunoassays and antigenic preparations that have been shown to have potential for diagnosis of schistosomiasis by the indirect method of antibody detection. Antigens in native form derived from cercariae, adult worms and eggs are considered, as well as schistosome antigens produced by recombinant DNA technology and the schistosome cross-reactive antigen, keyhole limpet haemocyanin (KLH). Respective advantages and disadvantages of antibody detection, circulating antigen detection and parasitological methods of diagnosis are analysed. It is suggested that due to the relative insensitivity of both parasitology and antigen detection, antibody detection methods could find increasing use in situations of low infection intensity.

Efficacy of treatment of murine Schistosoma mansoni infections with praziquantel and oxamniquine correlates with infection intensity: role of host antibody.

The reduction in worm burden obtained by treatment of Schistosoma mansoni with praziquantel and oxamniquine was greater in mice with heavy infections than in relatively lightly infected animals. The reduction in worm burden achieved by each drug correlated with the size of the pre-treatment worm burden (r2 = 0.82 and 0.81 for praziquantel and oxamniquine, respectively). Intensity of infection did not affect the degree of tegumental damage and drug-induced antigen exposure on worms recovered soon after treatment with praziquantel. However, praziquantel-treated worms from mice with heavy infections had significantly more murine antibody attached to the treated-worm surface than worms from praziquantel-treated lightly infected mice. Heavily infected mice had greater levels of circulating anti-worm antibodies than lighter infected mice. The correlation between infection intensity and cure rates achieved by praziquantel and oxamniquine may thus be a reflection of the higher titres of relevant antibody in heavily infected mice mediating death of drug-treated worms.

Supplemental oxygen and dependent positioning as adjunctive measures to improve forefoot tissue oxygenation.

Fifty-seven patients with resting pain or tissue necrosis were found to have a forefoot transcutaneous tissue oxygen (tcPO2) level less than 30 mm Hg. The adjunctive measures of foot dependency (36 cm below heart level) and nasal oxygen of 3 L/min were evaluated in these patients. In general, the improvement in tcPO2 levels with these adjunctive measures was not related to basal levels of forefoot or arm tcPO2. One can expect an increase in tcPO2 level of approximately 22 mm Hg while employing the dependent position and an additional benefit of 12 mm Hg with the administration of nasal oxygen. Of the 35 patients with a basal forefoot tcPO2 level of less than 10 mm Hg, 11 did not respond to these adjunctive measures.

Suction curettage: therapeutic effectiveness in dysfunctional uterine bleeding.

A group of 60 patients between the ages of 30 and 45 were randomly selected to have either a suction curettage or dilatation and curettage. The criteria used for patient selection was that they had dysfunctional uterine bleeding, a normal gynecologic examination, and were receiving no hormonal therapy. These patients were followed up for at least 3 months. Although the number of patients is relatively small, the results showed that, therapeutically, suction curettage was equal to dilatation and curettage in alleviating dysfunctional uterine bleeding.